WO2016054298A1 - Magnetogenetics - Google Patents

Abstract

The present invention is directed generally to host cells with artificial endosymbionts, particularly magneto-endosymbionts, where the endosymbiont can be controlled and/or regulated by external means. The present invention provides artificial endosymbionts that secretes a polypeptide, nucleic acid, lipid, carbohydrate, or other factor into the host cell in a regulatable manner. In some embodiments, the polypeptide, nucleic acid, lipid, carbohydrate, or other factor secreted into the host cell by the magneto-endosymbiont imparts a phenotype to the host cell. The secreted polypeptide may be a selectable marker, a reporter protein, a transcription factor, a signal pathway protein, a receptor, a growth factor, or an effector molecule.

Description

[0001 ] The present invention relates generally to modifying eukaryotic cells with artificial endosymbionts such that function of the eukaryotic cell can be modulated. The artificial endosymbionts are designed to communicate with the host cell such that this interaction directs function of the host ceil.

BACKGROUND

[0002] Mitochondria, chloroplasts and other membrane bound organelles add heritable functionalities, such as photosynthesis, to eukaryotic cells. Such organelles (identified byheir vestigial circular DNA) are believed to be endosymbioticaily derived.

0003] Bacteria exist with a wide range of functionalities not found in various eukaryotic celts. For example, magnetotactic bacteria (MTB) can orient and swim along a geomagnetic field (Blakemore, "Magnetotactic bacteria," Science 24: 377-379, 1975). These magnetotactic bacteria produce magnetic structures called magnetosomes that are composed of magnetite (Fe₃O₄) or greigite (Fe₃S₄) enclosed by a lipid membrane. A targe number of MTB species have been identified since their initial discovery. In addition to the range of available functionalities, bacteria are readily modified by genetic and recombinant techniques, allowing for introduction of new functionalities.

[0004] In contrast, stable modifications of eukaryotic cells require extensive genetic engineering of the eukaryotic cell, for example by homologous or site specific recombination. It is desirable to find alternative methods of engineering eukaryotic cells to introduce new functionalities into the cells.

SUMMARY

[0005] The present invention is directed to host cells that have been altered or reprogrammed by transfer of chemical information from artificial endosymbionts to the host cell. In one aspect, the present invention relates to changing the phenotype and/or genotype of host cells, n a regulated manner, by introducing into the host cell artificial endosymbionts that can secrete within the host cell polypeptides, nucleic acids, lipids, carbohydrates, amino acids, therapeutic agents, or other factors in response to specific signals, /.«., regulated secretion, to affect host cell function. In some embodiments, the artificial endosymbionts comprise magnetic-endosymbionts, which are used to deliver factors into the host cell in a controlled (Le., regulated) manner. In some embodiments, the magneto-endosymbiont secretes a polypeptide into the host cell. In some embodiments, the secreted protein is a heterologous polypeptide to the magneto-endosymbiont. In some embodiments, the secreted polypeptide from the magneto-endosymbiont causes a phenotypic change in the host cell. In some embodiments, the magneto-endosymbiont secretes a nucleic acid into the host cell. In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, the nucleic acid secreted from the magneto-endosymbiont causes a phenotypic change in the host cell.

(0006] In some embodiments, the artificial endosymbiont can be subjected to any appropriate treatment, e.g., physical or biochemical, that that can control the levels of the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s). In some embodiments, for magneto-endosymbionts, the treatment comprises magnetic hyperthermia, eg., by application of an alternating magnetic field. In some embodiments, the treatment comprises a small molecule that controls the level of the secreted polypeptide^), nucleic acid(s), Hpid(s), carbohydrate(s), amino acid(s), or other factor(s). In some embodiments, the treatment comprises x-ray, ultrasound, light, radiofrequencies, or other electromagnetic signals that act on the endosymbiont

[0007] In some embodiments, the artificial endosymbionts secrete into the host cell potypeptide(s), nucleic acid(s), lipid(s), carbohydrate^), amino acid(s), or other factoids) mat contain regions that direct delivery to specific cellular compartments or organelles of the host cell. In some embodiments, the artificial endosymbionts secrete into the host cell

polypeptide^), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) that contain regions to direct their delivery to the cytoplasm. In some embodiments, the artificial endosymbionts secrete into the host cell polypeptide^), nucleic acid(s), lipid(s),

carbohydrate(s), amino acid(s), or other factors) that contain regions that direct delivery to the nucleus. In some embodiments, the artificial endosymbionts secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factors) mat contain regions that direct delivery to the mitochondria. In some embodiments, the magneto- endosymbionts of the invention secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factors) that contain regions to direct delivery to a specific intracellular organelle such as mitochondria, lysosomes, and

endoplasmic reticulum. In some embodiments, the artificial endosymbionts secrete into the amino acid(s), or other factors) that contain regions to direct their delivery to the plasma membrane specifically for expression on the surface of the host cell, and these can confer function on the host cell or serve as a marker on the host cell. In some embodiments, the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) acts at the level of the nucleic acid or chromosomes of the host cell. In some embodiments, the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factoids) changes the phenotype of the host cell.

[0008] In some embodiments, the host cell can comprise any appropriate eukaryotic cell for modification with the artificial endosymbiont. In some embodiments, the cell can comprise a plant or animal cell, particularly a mammalian cell. In some embodiments, the host cell is a pluripotent, oligopotent, or unipotent cell (e.g., stem cell) and the method induces the cell to differentiate into a desired ceil. In some embodiments, the method uses a treatment, e.g., magnetic hyperthermia, to induce a magneto-endosymbiont to secrete polypeptides), nucleic acid(s), or other factor(s) that contribute to the differentiation of the host stem cell into a desired cell. In some embodiments, the magneto-endosymbiont secretes a Yamanaka factor (e.g., Oct4, Oct3, Sox2, Klf4, c-Myc, NANOG, and/or Lin28) into the host cell so the host cell becomes an induced pluripotent stem cell ("iPS"). In some embodiments, the iPS cell is induced to differentiate into a desired cell by other polypeptides or nucleic acids or factors secreted by the artificial endosymbiont.

[0009] In some embodiments, the host cells and methods of the invention are used to make medically and industrially important recombinant peptides/proteins that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications. In some

embodiments, the magneto-endosymbionts and described methods are used to introduce into host cells phenotypes that require the introduction of multiple factors and/or multiple genes. In some embodiments, the magneto-endosymbiont introduces multiple phenotypes into the host cell. In some embodiments, the magneto-endosymbiont is capable of imparting these multiple phenotypes to the host cell at different desired times. The multiple phenotypes may each be caused by single or multiple polypeptides or factors or nucleic acids secreted from the magneto-endosymbiont into the host cell.

[0010] In some embodiments, the host cells and methods of the invention are used to produce natural or recombinant biologies, e.g., therapeutic peptides/polypeptides, which will be useful for the in situ treatment of diseases. In some embodiments, the therapeutic peptides/protein comprises a neurotransmitter expressed in neurons to treat neurological diseases; or a wild- type gene in target tissues to correct a genetic defect in heritable diseases, e.g., enzymes such as a glucosidase, a-galactosidase A and 6-glucocerebrosidase. In some embodiments, the artificial endosymbionts and methods disclosed herein are used to introduce into host cells phenotypes mat require the introduction of multiple factors and/or multiple genes. In some embodiments, the artificial endosymbiont introduces multiple phenotypes into the host cell. In some embodiments, the artificial endosymbiont is capable of imparting these multiple phenotypes to the host cell at different desired times. The multiple phenotypes may each be caused by single or multiple polypeptides or factors or nucleic acids secreted from the magneto-endosymbiont into the host cell.

[0011] In some embodiments, the eukaryotic host cell is a mammalian cell. In some embodiments, the host cell is a human, mouse, rat, canine, primate, or rodent cell. In some embodiments, the host cell is a fibroblast cell, epithelial cell, keratinocyte, hepatocyte, neuron, immune cell, adipocyte, endothelial cell or other differentiated cell. In some embodiments, the host cell is a stem cell, p!uripotent ES cell, pluripotent iPS cell, a muhipotent mesenchymal stem cell, multipotent hematopoietic stem cell, or other pluripotent stem cell. In some embodiments, the host cell is a progenitor cell, such as for example, a neural progenitor cell, an angioblast, an osteoblast, a chondroblast, a pancreatic progenitor cell, or an epidermal progenitor cell. In some embodiments, the host cell is a solid tumor cell or a hematopoietic cancer cell. In some embodiments, the host cell is from a carcinoma, sarcoma, leukemia, lymphoma, or glioma. In some embodiments, the host cell is obtained from a prostate cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, liver cancer, or the NCI 60 panel of cancer cell lines.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 A, IB, 1C and ID depict MRI images of 231BR cells containing AMB-1 in mice. FIG. 1A shows an image of 10⁴231 BR cells containing AMB-1. FIG. IB shows an image of 10³231 BR cells containing AMB-1. FIG. 1C shows an image of 10²231 BR cells containing AMB-1. FIG. 1 D shows an image of single 231 BR cells containing AMB-1 in a mouse. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention is directed to host cells that contain an artificial endosymbiont, particularly a magneto-endosymbiont, where the artificial endosymbiont can secrete into the host cell a polypeptide, nucleic acid, or other factor in a regulated manner.

[0016] Before various embodiments of the present invention are further described, it is to be understood mat this disclosure is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purposes of describing particular embodiments only, and is not intended to be limiting.

[0017] It is also to be noted that as used in the present disclosure and in the appended claims, the singular terms "a", "an", and "the" include plural referents unless context clearlyndicates otherwise. Similarly, the word "or" is intended to include "and" (and vice versa) unless the context clearly indicates otherwise. In addition, the use of "or" means "and/or" unless stated otherwise.

[0018] In addition, the words "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting. Where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

[0019] Numerical limitations given with respect to concentrations or levels of a substance are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 10 ug, it is intended that the concentration be understood to be at least

approximately or about 10 ug.

[0020] The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.

Definitions

[0021] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings. [0022] As used herein, the term "polynucleotide'* or "nucleic acid' refers to two or more nucleosides that are covalently linked together. The polynucleotide may be wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of V deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2' deoxyribonucleosides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or may include bom single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (/.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and/or synthetic nucleobases, such as, for example , inosine, xanthine, hypoxanthine, etc. Preferably, such modified or synthetic nucleobases will be encoding nucleobases.

[0023] As used herein, the term "protein", "polypeptide," and "peptide" are used

interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0024] As used herein, the term "cellular life cycle" refers to series of events involving the growth, replication, and division of a eukaryotic cell. It is typically divided into stages mat include G<¾ in which the cell is quiescent, Gi and G₂, in which the cell increases in size, S, in which the cell duplicates its DNA, and M, in which the cell undergoes mitosis and divides.

[0025] As used herein, the term "daughter cell" refers to cells that are formed by the division of a cell.

[0026] As used herein, the term "essential molecule" refers to a molecule required by a cell for growth or survival.

[0027] As used herein, the term "genetically modified" refers to altering the DNA of a cell so that a desired property or characteristic of the cell is changed.

[0028] As used herein, the term "operably linked" refers to a situation in which two or more polynucleotide sequences or genes are positioned to permit their ordinary functionality. For example, a promoter is operably linked to a coding sequence if it is capable of controlling the expression of the sequence. In some embodiments, promoter transcriptional regulatory [0029] As used herein, the term "control region" or "control sequence" refers to a segment of nucleic acid that directs and regulates expression of a nucleic acid, such as a gene that encodes a protein. A control region may include a promoter, operator, enhancer(s), activation binding site(s), attenuators), and other sequences involved in regulation of expression. A "regulatable control region" refers to a control region in which expression of the nucleic acid can be controlled by signals or agents, particularly by external signals or agents, which can be chemical, biological, or physical. In some embodiments, regulatable control regions include control regions that can be controlled by biological molecules, chemical agents,

electromagnetic radiation, acoustic radiation, photo-illumination, and the like. An exemplary regulatable control region is the heat shock control region in which expression can be controlled by hyperthermia. It is to be understood that regulated expression from a control region generally involves one or more accessory molecules (e.g., repressor protein), which in some embodiments, such as a cell, can be endogenous or heterologous. In the embodiments herein, regulatable includes the capability of inhibiting or inducing expression by the control region.

[0030] As used herein, the term "localization signal" refers to a molecule or portion of a molecule that directs or mediates localization of another molecule, e.g., protein or nucleic acid, to a particular region of a cell, e.g., of a host cell. In some embodiments, the localization signal is a "targeting sequence" which refers to a sequence, either a polypeptide or polynucleotide sequence, that directs or mediates localization of biomolecules to a particular region in a cell when the biomolecule is attached to (e.g., fused to) the sequence.

[0031] As used herein, the term "treatment" or "treating" in the context of a "control region" refers to contacting or exposing the target (e.g., cell, tissue or organism) to a signal or agent that controls (e.g., induces) expression from the control region.

[0032] As used herein, the term "heterologous" when used in reference to a nucleic acid or polypeptide refers to a nucleic acid or polypeptide not normally present in nature.

Accordingly, a heterologous nucleic acid or polypeptide in reference to a host cell refers to a nucleic acid or polypeptide not naturally present in the given host cell. For example, a nucleic acid molecule containing a non-host nucleic acid encoding a polypeptide operably linked to a host nucleic acid comprising a promoter is considered to be a heterologous nucleic acid molecule. Conversely, a heterologous nucleic acid molecule can comprise an endogenous structural gene operably linked with a non-host (exogenous) promoter. Similarly, a peptide or polypeptide encoded by a non-host nucleic acid molecule, or an endogenous polypeptide fused to a non-host polypeptide is a heterologous peptide or polypeptide.

[0033] As used herein, the term "magnetic bacteria" refers to prokaryotic cells that mineralize iron or other metals into magnetosomes, which are intracellular structures comprising magnetic iron enveloped by a lipid membrane.

[0034] As used herein, the term "artificial endosymbiont" refers to a single celled organism, particularly bacteria, naturally occurring or modified, that is or has been introduced into a host cell through human intervention. In some embodiments, artificial endosymbiont is capable of secreting into the host cell polypeptide(s), nucleic acid(s), lipid(s),

carbohydrate(s), amino acid(s), and/or other factor(s). The secreted factor may act in the cytoplasm, nucleus, organelle or other sites in the host cell. As described herein, this secretion comprises communication between the artificial endosymbiont, which can result in a phenotypic or epigenetic change of the host cell.

[0035] As used herein, the term "magneto-endosymbiont" refers to magnetic bacteria, naturally occurring or modified, that is or has been introduced into a host cell through human intervention. In some embodiments, the magneto-endosymbiont is capable of secreting into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), and/or other factor(s). The secreted factor may act in the cytoplasm, nucleus, organelle or other sites in the host cell. As described herein, this secretion comprises communication between the magneto-endosymbiont and the host cell, which can result in a phenotypic or epigenetic change of the host cell.

[0036] As used herein, the term "magneto-genetics" refers to modulation of complex cellular functions in targeted single cells, or groups of cells that contain magneto-endosymbionts.

[0037] As used herein, the term "epigenetic changes" or "epigenetic modification" refers to modulation of simple or complex cellular functions, genetic functions in targeted single cells, or groups of cells without changes to the DNA sequence of the cell, e.g., host cell of the artificial endosymbiont.

[0038] As used herein, the term "magnetosome" refers to particles of magnetite (i.e., Fe30₄) or greigite (Fe3S₄) enclosed by a sheath or membrane. In some embodiments, the particles can be individual particles or chains of particles. [0040] As used herein, the term "mammal" refers to warm-blooded vertebrate animals all of which are characterized by hair on the skin and, in the female, milk producing mammary glands.

[0041] As used herein, the term "phenotype" refers to an observable characteristic or characteristics at any level - physical, morphological, biochemical, or molecular - of a cell, tissue, or organism.

[0042] As used herein, the term "secrete" refers to the passing of molecules or signals from one side of a membrane to the other side. Accordingly, in some embodiments, the term "secrete" or "secretion** refers to transport of a molecule from the interior of a bacterium to its exterior, such as for example, periplasrnic space or extracellular environment {e.g. , internal environment of a host cell).

[0043] As used herein, the term "magnetic hyperthermia" refers to use of or treatment with a magnetic field to induce hyperthermia in a target containing magnetic particles, such as a magnetosome. In some embodiments, the magnetic field applied for inducing hyperthermia is an alternating magnetic field.

Artificial Endosymbionts

[0044] Artificial endosymbionts of die present invention comprise a single celled organism, particularly bacteria, that are capable of surviving in a eukaryotic cell and maintain copy number such that the functionality {e.g., phenotype) introduced by the single-celled organism is observed in daughter cells of the eukaryotic cell. In the embodiments herein, the artificial endosymbiont secretes into the host cell a polypeptide(s), nucleic acid(s), other molecule(s), and/or other factor(s) in a regulated manner. In some embodiments, the polypeptide and/or nucleic acid are recombinant and heterologous to the artificial endosymbiont.

[0045] In some embodiments, the artificial endosymbiont (e.g., magneto-bacteria) is heritable to the daughter cells of the eukaryotic host cell. In some embodiments, the artificial endosymbiont is maintained in host daughter cells through at least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the host cell. In some embodiments, the artificial endosymbiont can be maintained in the host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions. [0046] In some embodiments, the introduction of functionality (e.g., phenotype) involves expression of a gene in the single-celled organism. In some embodiments, the functionality involves expression of one or more genes or set of genes in the single-celled organism. In some embodiments, the functionality involves expression of a protein in the single-celled organism. In some embodiments the functionality involves expression of a set of proteins in the single-celled organism. In some embodiments, the functionality involves expression of a gene or gene product that is transferred to the host to express the phenotype.

[0047] In some embodiments, the host cell maintains the phenotype for at least 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In some embodiments, the single celled organism can stably maintain the phenotype in host daughter cells through at least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the host cell. In some embodiments, the single celled organism can stably maintain phenotype in the host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions.

[0048] In some embodiments, the single-celled artificial embodiments are a-Proteobacteria, such as magnetic-bacteria further described below. A large number of a-proteobacterial genomes that cover all of the main groups within a-proteobacteria have been sequenced, providing information that identifies unique sets of genes or proteins that are distinctive characteristics of various higher taxonomic groups (e.g., families, orders, etc.) within <x- proteobacteria. (Gupta, supra).

[0049] In some embodiments, single-celled organisms useful as artificial endosymbionts include, by way of example and not limitation, Anabaena, Nostoc, Diazotroph,

Cyanobacteria, Trichodesmium, Beijerinckia, Clostridium, Green sulfur bacteria,

Azotobacteraceae, Rhizobia, Frankia, flavobacteria, Methanosarcinales, aerobic halophilic Archaea of the order Halobacteriales, the fermentative anerobyves of the order

Halanaerobiales (low G+C brand of the Firmicutes), the red aerobic Salinibacter

(Bacteroidetes branch), Marinobacter, Halomonas, Dermacoccus, Kocuria,

Micromonospora, Streptomyces, Williamsia, Tskamurella, Alteromonas, Colwel!ia,

Glaciecola, Pseudoalteromonas, Shewanella, Polaribacter, Pseudomonas, Psychrobacter, Athrobacter, Frigoribacterium, Subtercola, Microbacterium, Rhodoccu, Bacillus,

Bacteroides, Propionibacterium, Fusobacterium, Klebsiella, lecithinase-positive Clostridia, Veillonella, Listeria, Fusobacteria, Chromatiaceae, Chlorobiceae, Rhodospirillaceae, 0050] In some embodiments, the single celled organism for use as artificial endosymbionts clude, by way of example and not limitation, M. frigidum, M. burtonii, C. symbiosum, C.sychrerythraea, P. haloplanktis, Halorubrum lacusprofundi, Vibrio salmonicida,hotobacterium profundum, S. violacea, S. frigidimarina, Psychrobacter sp. 273-4, S.

enthica, Psychromonas sp. CNPT3, Moritella sp., Desulfotalea Psychrophila,

xiguobacterium 255-15, Flavobacterium psychrophilum, Psychroflexus torquis,olaribacter filamentous, P. irgensii, Renibacterium salmoninarum, Leifsonia-relatedHSC20-cl, Acidithiobctcillus ferrooxidans, Thermoplasma acidophilum, Picrophilus rridus, Sulfolobus tokodaii, and Ferroplasma acidarmanus.

051] In some embodiments, single-celled organisms useful as artificial endosymbionts are ose known to be intracellular pathogens or intracellular endosymbionts. In somembodiments, single celled organism is an intracellular pathogen characterized by genomic lands containing virulence genes encoding, for example, adherence factors that allow the tracellular pathogen to attach to target eukaryotic cells, and trigger phagocytosis of the tracellular pathogen (Juhas, M. et al., FEMS Microbiol Rev. 33:376-393, 2009). Many rulence factors utilize type III or type IV secretion systems. Some virulence factors are creted into the eukaryotic host cell and alter membrane traffic within the target eukaryoticell, some virulence factors interact with host proteins involved in apoptosis. (Dubreuil, R. et ., Cell Logis. 1 :120-124, 2011).

052] In some embodiments, the single celled organism useful as artificial endosymbionts, clude by way of example and not limitation, endosymbionts found in insects such as uchnera, Wigglesworthia, and Wolhachia; the methanogenic endosymbionts of anaerobic liates; the nitrogen-fixing symbionts in the diatom Rhopalodia; the chemosyntheticndosymbiont consortia of gutless tubeworms (Olavius or Jnanidrillus); the cyanobacterialndosymbionts of sponges; the endosymbionts of all five extant classes of Echinodermata; e Rhizobia endosymbionts of plants; various endosymbiotic algae; the Legionella-like Xacteria endosymbionts of Ameoba proteus; numerous Salmonella sp., Mycobacteriumuberculosis, Legionella pneumophila, etc. reside in membrane-bound vacuoles often termedymbiosomes, while some species, such as Blochmannia, the rickettsia, Shigella,nteroinvasive Escherichia coli, and Listeria (e.g., Listeria monocytogenes), have the abilityo inhabit the cytosol. 0053] In some embodiments, the single-celled organism useful as artificial endosymbiont is haracterized by a secretory system acquired by phagocytosis to evade the endocytic pathway nd allow the single celled organism to persist in the host cell. In some embodiments, the ngle-celled organism is characterized by the Dot-Icm Type IV secretory system, which is mployed by many intracellular bacteria to evade the endocytic pathway and persist in the ost cell. This system has been well-studied in L pneumophila and consists of the proteins: DotA through DotP, DotU, DotV, IcmF, IcmQ through IcmT, IcmV, IcmW and IcmX. In hotorhabdus lumirtescens, the luminescent endosymbiont of nematodes, the genes encoding TX-like toxins, proteases, type III secretion system and iron uptake systems were shown to upport intracellular stability and replication. The gene bacA and the regulatory system vrRS are essential for maintenance of symbiosis between Rhtzobia and plants as well as the urvival of Brucella abortus in mammalian cells. The PrfA regulon enables some Listeria pecies, e.g., Listeria monocytogenes, to escape the phagesome and inhabit the cytosol. The esired cellular location (e.g., symbiosome or cytosol) of the intracellular MTB will dictate which genes are required to be expressed in the MTB (either directly from the genome or hrough a stable vector) for survival and proliferation in the host environment The ndogenous plasm id pMGT is highly stable in MTB and a number of other broad range ectors (including those of IncQ, IncP, pBBRl, etc.) are capable of stable replication in MTB. hus, any of the foregoing single-celled organisms can be used as an artificial endosymbiont. 0054] In some embodiments, the single-ceiled organisms are genetically modified. In some mbodiments, the bacteria are genetically modified to improve weir survivability in ukaryotic host cells, and/or to reduce the toxicity of the single-ceiled organism to the ukaryotic cell, and/or to provide the eukaryotic cell with a useful phenotype. Such modifications can be directed modifications, random mutagenesis, introduction of eterologous genes, or a combination thereof. For example, molecular biology tools have een developed for genetic manipulations of MTB in AMB and M gryphiswaldense strain MSR-1 (reviewed in Jogler, C. and Schtiler, D., in "Magnetoreception and Magnetosomes in acteria," p 134-138, New York, Springer (2007), incorporated herein by reference).

0055] In some embodiments, the single-celled organism is genetically modified to express nd secrete polypeptide(s), nucleic acid(s), Hpid(s), carbohydrates), amino acid(s), or other actor(s) in a regulated manner into the host cell, as further described herein. In some mbodiments, recombinant transport pathways are engineered in whole or in part into the rtificial endosymbiont for the delivery of target protein(s), nucleic actd(s), carbohydrate(s), pid(s), other molecule(s), and/or other factor(s) to the host cells. In some embodiments, the rget protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or otheractor(s) are directed to exert an effect on the host cells by acting on cellular componentsresent in the cytoplasm, nucleus, mitochondria, organelle, or other sites in or around the hostell.

0056] In some embodiments, the single-celled organisms are also genetically modified for able association of the single-celled organism with the host cell and/or selection of the hostell containing the single celled organism. Natural colonization of a host by the symbiontsan follow the following stages: 1) transmission, 2) entry, 3) countering of host defense, 4)ositioning, 5) providing advantage to the host, 6) surviving in host environment, and 7) gulation. Accordingly, in some embodiments, the single-celled organism is genetically odified to affect one or more of the foregoing stages, particularly providing advantage to e host, survivability in the host environment, and regulation. In some embodiments, the ngle celled organism is genetically modified to create mutual nutritional dependence iotrophy) between the single-celled organism and the eukaryotic cell. In some

mbodiments, the single-celled organism comprises at least one deletion or inactivation of aene encoding an enzyme for synthesizing an essential molecule, thereby resulting in absencef enzyme or expression of inactive enzyme, wherein said essential molecule is produced by e eukaryotic host cell. An essential molecule can include, but is not limited to, an aminocid, a vitamin, a cofactor, and a nucleotide. For instance, biotrophy can be accomplished bynocking-out the ability of the single-celled organism to make an amino acid, which will thene derived from the host. An exemplary target is the metabolic pathway for synthesis of ycine, which is highly abundant in mammalian cells and a terminal product in bacterialmino acid biogenesis. For example, in some embodiments, the gene encoding the enzymeerine hydroxymethyltransferase, which converts serine into glycine at the terminus of the 3-hosphoglycerate biosynthetic pathway, is mutated (e.g., deletion, insertion, or substitution) eliminate presence of the enzyme or produce inactive enzyme. In some embodiments, the ngle-celled organism is an AMB in which the gene amb2339 (which encodes the enzymeerine hydroxymethyltransferase) is genetically modified.

0057] In some embodiments, genes encoding antibiotic resistance are inserted into theenome of the single-celled organism, and the eukaryotic host cell cultured in mediaontaining the antibiotic will require the single-celled organism for survival. In thembodiments herein, various antibiotic resistance genes can be introduced into the single- celled organism, such as neomycin resistance gene, hygromycin B resistance gene, and puromycin resistance gene. Neomycin resistance is conferred by either one of two aminoglycoside phosphotransferase genes, which also provide resistance against geneticin (G418), a commonly used antibiotic for eukaryotes. Hygromycin B resistance is conferred by a kinase that inactivates hygromycin B by phosphorylation. Puromycin is a commonly used antibiotic for mammalian cell culture, and resistance is conferred by the pac gene encoding puromycin N-acetyl-transferase. External control of the antibiotic concentration allows intracellular regulation of the copy number of the single-ceiled organism. Any other system where resistance or tolerance to an external factor is achieved by chemical modification of this factor can also be employed.

[0058] In some embodiments, the single-celled organism is genetically modified with other selection genes {e.g., negative or positive selection genes), such as bacteriostatic gene(s), siderophore gene(s), metabolic requirement gene(s), suicide gene(s), life cycle regulation gene(s), transporter gene(s), and escape from the phagosome gene(s).

[0059] In some embodiments, the single-celled organisms are randomly mutated and subsequently screened for enhanced integration within the host cell. Random mutation can be accomplished by treatment with mutagenic compounds, exposure to UV -light or other methods known to those skilled in the art.

[0060] In some embodiments, the single-celled organism is genetically modified so that its cell cycle is coordinated with the cell cycle of the eukaryotic host cell so that copy number of the single-celled organism can be maintained at a sufficient level to impart the phenotype to daughter cells. In some embodiments, the genes localize artificial endosymbionts to specific subcellular locations. In some embodiments, the genes provide enhanced or blocked entry of the artificial endosymbionts to specific host cells. In some embodiments, the gene suppresses or alters the host immune system response to the artificial endosymbiont or genes and proteins expressed from it.

[0061] In some embodiments, transgenetic modification(s) are made to counter eukaryotic cell defenses using genes from various parasites or endosymbionts. In some embodiments, the population of the single-celled organisms in the eukaryotic host cell is regulated though a balance of intrinsic use of host mechanisms (nutrient availability, control of reproduction, etc.) and antibiotic concentration.

agneto-Endosymbionts 0062] As described above, in some embodiments, the artificial endosymbiont or single cell rganism is an MTB characterized by the presence of magnetic particles, such as those ontaining magnetite (Fe₃O₄) or greigite (Fe₃S₄), enclosed by a sheath or membrane. In some mbodiments, the single-celled organism is an MTB that produces magnetic particles upon ulturing of the eukaryotic host cell. In some embodiments, magneto-endosymbionts are agnetic bacteria capable of surviving in a eukaryotic cell, where the magneto-endosymbiont ecretes into the host cell a polypeptide(s), nucleic acid(s), other molecule(s), and/or otheractor(s) in a constitutive or regulated manner, particularly in a regulated manner. In some mbodiments, the polypeptide and/or nucleic acid are recombinant and heterologous to the agneto-endosymbiont.

0063] In some embodiments, the magneto-endosymbiont introduces a phenotype into the ost cell through secretion from the magneto-endosymbiont into the host cell. In some mbodiments, this phenotype introduced by the magneto-endosymbiont is maintained in aughter cells. In some embodiments, the host cell maintains the phenotype for at least 1 day, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In some embodiments, e magneto-endosymbiont can stably maintain the phenotype in host daughter cells through least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the ost cell. In some embodiments, the magneto-endosymbiont can stably maintain phenotype in e host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 visions.

0064] In some embodiments, the magneto-endosymbiont comprises magnetotactic bacteriaMTB). MTB are a diverse group of bacteria that belong to different subgroups of the roteobacteria and Nitrospirae phylum, and are mostly represented within the a- roteobacteria. Many MTB have a Gram-negative cell wall structure (inner membrane, eriplasm, and outer membrane). Many MTB inhabit water bodies or sediments with vertical hemical concentration gradient, predominantly at the oxic-anoxic interface thus are ategorized as microaerophiles, anaerobic, facultative aerobic or some combination of thehree. Many MTB are chemoorganoheterotrophic and some strains can also grow

hemolithoautotrophically (Bazylinski et al., Nature Rev Microbiol. 2:217-230, 2004;

Williams et al., Appl Environ Microbiol. 72(2): 1322-9, 2006). MTB contain magnetosomes, which are intracellular structures comprising magnetic iron crystals enveloped by a hospholipid bilayer membrane (Gorby et al., J Bacteriol. 170(2): 834-41, 1988). In some embodiments, the transport pathways of MTB can deliver endogenous and/or heterologous proteins and/or nucleic acids to the host cell. In some embodiments, recombinant transport pathways are engineered in whole or in part into the magneto-endosymbiont for the delivery of target protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or other factor(s) to the host cells. In some embodiments, the target protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or other factor(s) are directed to exert an effect on the host cells by acting on cellular components present in the cytoplasm, nucleus, mitochondria, organelle, or other site in or around the host cell.

[0065] A large number of MTB species are known to those of ordinary skill in the art (see, e.g., Blakemore, "Magnetotactic bacteria," Science 24: 377-379, 1975; Faivre et al.,

"Magnetotactic bacteria and magnetosomes," Chem Rev. 108:4875-4898, 2008; incorporated herein by reference). MTB have been identified in different subgroups of the Proteobacteria and the Nitrospira phylum with most of the phylotypes grouping in a-Proteobacteria.

Culturable MTB strains assigned as a-Proteobacteria by 16S rRNA sequence similarity include the strains Magnetospirillum magnetotactium (formerly Aquasprillium

magnetotactium), M. gryphiswaldense, M. magneticum strain AMB-1, M. polymorphum, Magnetosprillum sp. MSM-4 and MSM-6, Magnetococcus marinns, marine vibrio strains MV-1 and MV-2, a marine spirillum strain MMS-1 and Magnetococcus sp. strain MC-1, as well as others.

[0066] In some embodiments, the magneto-endosymbiont can introduce multiple factors into the host cell without the need for integration of each individual gene into a host cell chromosome. Instead, the desired genes are introduced into the magneto-endosymbiont, and when the magneto-endosymbiont is introduced into the host cell the gene products of interest are introduced into the host cell. Expression of the gene products in the bacterium, and thus their introduction into the host cell, can be done in a regulated (controlled) or unregulated (uncontrolled, e.g., constitutive) manner. Magneto-endosymbionts may contain numerous constitutive or regulated genes either in operon cassettes, or as individual genes, allowing complete genetic programs to be transiently, or permanently, transferred to host cells.

Magneto-endosymbionts can utilize operon structures to express multiple genes from a single control region. By incorporating the operon structure, sets of genes can be engineered into the magneto-endosymbiont for coordinated expression, and groups of operons can be used to express sets of genes at different desired times. [0067] In some embodiments, the magneto-endosymbiont circumvents the need for integration of engineered genes into the host genome for long-term expression and provides necessary spatiotemporal control or removal of the genes at desired times. Use of the magneto-endosymbiont also enables selective ablation of (a) the engineered magneto- endosymbiont, (b) magneto-endosymbiont and its host cell, or (c) magneto-endosymbiont, its host cell, and surrounding tissues.

[0068] In some embodiments, the magneto-endosymbiont can be used to reprogram and differentiate host cells, thus directing cell fates and function in the body. As described in more detail below, in some embodiments, gene expression and viability of the artificial endosymbionts, particularly magneto-endosymbionts, and the cells that contain them, can be controlled by a treatment with an agent that regulates expression of the gene of interest. In some embodiments, as further described below, the treatment can be with a chemical, biological, or physical agent. In some embodiments, the chemical or biological agent induces expression of the gene. In some embodiments, the physical agent can comprise, among others, thermal, acoustic (e.g., ultrasound), electromagnetic (e.g., infrared-thermal, visible light, radio-frequency, X-ray, etc.), and magnetic radiation. The treatment can be applied locally (e.g., focused), or applied to the organism as a whole.

[0069] For example, as further described herein, low frequency alternating magnetic fields (AMF) applied to the entire body can be absorbed by the magnetic structures in the magneto- endosymbiont and subsequently dissipated as heat. This conversion of the alternating magnetic field into heat raises the temperature in the magneto-endosymbiont, and optionally the host cell and further optionally in surrounding tissues. When heat shock control regions are linked to the target genes in the magneto-endosymbiont, this system allows direct, noninvasive control of expression in the body via magnetic hyperthermia. In some embodiments, expression in the endosymbiont can be controlled with hyperthermia induced by focused ultrasound. In some embodiments, the role of host cells with magneto- endosymbionts in tissue regeneration can be followed using MRI, optical imaging, ultra sound, or nuclear medicine imaging methods such as positron emission tomography (PET) or single photon emission tomography (SPECT). In some embodiments, magneto- endosymbionts are used to control stem cells while they reside in target tissues of the body. In some embodiments, magneto-endosymbiont can be used to deliver, control, and finally silence large sets of developmental genes in host cells resulting in differentiation of host cells into desired cell types at desired locations in the body. In some embodiments, magneto- endosymbionts are used to control cells to rebuild damaged tissues while they reside in target organs and tissues of the body. In some embodiments, magneto-endosymbionts are used to control stem cells to restore function to damaged tissues while they reside in target organs and tissues of the body. In some embodiments, magneto-endosymbionts are used to control stem cells to become cardiomyocytes, hepatocytes, beta cells, or other tissue specific cell types while they reside in target tissues and organs of the body.

[0070] Unlike passive magnetic particles, magneto-endosymbionts have the ability to self- replicate inside the host cells, and are thus retained over numerous cell divisions and maintain their magnetic properties through continued biogenesis of magnetosomes.

[0071] In some embodiments, the artificial endosymbionts, particularly the magneto- endosymbiont, is designed to target specific cell types and enter these cells to become endosymbionts. For example, internalins such as InlA or InlB can be coded into the endosymbiont that facilitate entry into human epithelial cells, hepatocytes, fibroblasts, and epithelioid cells by interacting with surface proteins on these cells and inducing

internalization; other means to accomplish this are known to those skilled in the art, one In some embodiments, the magneto-endosymbiont can be delivered systemically to an animal and localize to, and infect, a specific cell of a target organ or tissue. In some embodiments, the magneto-endosymbiont is directed to infect cells of the heart. In some embodiments, the magneto-endosymbiont is directed to infect damaged cells for directed repair. In some embodiments, the magneto-endosymbiont is directed to cells of the liver. In these embodiments the magneto-endosymbiont is designed for systemic delivery and targeted infection of host cells at a distance from the site of introduction into a host organism.

[0072] In some embodiments, a natural endosymbiont or an intracellular parasite is genetically modified to produce magnetosomes. Endosymbionts of insects such as Buchnera, Wigglesworthia, and Wolbachia; the methanogenic endosymbionts of anaerobic ciliates; the nitrogen-fixing symbionts in the diatom Rhopalodia; the chemosynthetic endosymbiont consortia of gutless tubeworms (Olavius or Inanidrillus); the cyanobacterial endosymbionts of sponges; the endosymbionts of all five extant classes of Echinodermata; the Rhizobia endosymbionts of plants; various endosymbiotic algae; the Legionella-like X bacteria endosymbionts of Ameoba proteus, numerous Salmonella sp., Mycobacterium tuberculosis, Legionella pneumophila belonging to a-proteobacteria are genetically engineered to produce magnetosomes. In some embodiments, a pre-existing organelle can be genetically modified to express one or more magnetosome genes to produce an artificial endosymbiont. For instance, mitochondria, plastids, hydrogenosomes, apicoplasts or other organelles, which harbor their own genetic material, can be genetically altered. Bacteria modified to produce magnetosomes can include Francisella tularensis, Listeria monocytogenes, Salmonella typhi, Brucella, Legionella, Mycobacterium, Nocardia, Rhodococcus equi, Yersinia, Neisseria meningitidis, Chlamydia, Rickettsia, Coxiella and the like. Methods for engineering cells to express magnetosomes are described in various publications, for example patent publication no. US2009/0311194, incorporated herein by reference).

Secretion Systems

[0073] In the present invention, secretion of protein(s), nucleic acid(s), other molecule(s) and other factor(s) from the artificial endosymbiont, particularly magneto-endosymbiont, can make use of the endogenous secretion systems of the artificial endosymbiont. In some embodiments, the artificial endosymbiont can be engineered with heterologous secretion systems, or portions thereof, for directed secretion of these target molecules. In some embodiments, these transport systems of the invention are used to transport medically and industrially important genes, recombinant peptides/proteins, and/or other factors that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications. However, although various secretion systems are known in the art and described in the present disclosure, it is to be understood that practice of the present invention does not require knowledge or understanding of a specific secretion system. Generally, the signals or sequences that cause secretion of polypeptides or other factors are known for many single celled organisms; they can also be found on known secreted proteins or nucleic acids, or identified based on similarities to sequences found on secreted proteins or nucleic acids. These secretion signals can then be used, particularly as fusions, to direct a target molecule for secretion in the artificial endosymbiont into the host cell without prior knowledge of the specific transport system that directs secretion using the particular secretion signal.

[0074] Various secretion systems can be used in the artificial endosymbionts of the disclosure, e.g., a magnetic endosymbiont. For example, the a-proteobacteria have transport pathways that include ABC transporter-based pathways including the type I secretion system (T1SS), type II secretion systems (T2SS), type III secretion systems (T3SS), type IV secretion systems (T4SS), type V secretion systems (T5SS), type VI secretion systems

(T6SS), type VII secretion systems (T7SS), and other transport systems that are known in the art. In some embodiments, these transport systems of a-proteobacteria are used to transport proteins, nucleic acids, and other factors from the magneto-endosymbiont into the host cell. In Gram-negative bacteria, secreted proteins are exported across the inner and outer membrane in a single step via the T1SS, T3SS, T4SS, and T6SS pathways. Proteins are exported into the periplasmic space across the inner membrane via Sec or two-arginine (Tat) pathways. Proteins are transported across the outer membrane from the periplasmic space by T2SS, T5SS or less commonly by Tl SS or T4SS.

[0075] T1SS consists of three proteins; an inner membrane protein with a cytoplasmic ATPase domain operating as an ATP-binding cassette (ABC) transporter (Escherichia coli HlyB), a periplasmic adaptor (also known as membrane fusion protein, MFP; E. coli HlyD), and an outer membrane channel protein of the TolC family (E. coli TolC) (Delepelaire, P., "Type I secretion in gram-negative bacteria," Biochim Biophys Acta 1694: 149-161, 2004). These proteins form a pore in the periplasm through which an unfolded protein may be translocated. T1SS protein substrates typically contain carboxy-terminal, glycine-and aspartate-rich repeats known as repeat-in-toxin (RTX) (Linhartova, I. et al., "RTX proteins: a highly diverse family secreted by a common mechanism," FEMS Microbiol. Rev." 34:1076- 1 1 12, 2010; incorporated herein by reference) and are often located close to ABC and MFP genes. Due to its simplicity, T1SS has been used to transport heterologous proteins. Several studies have shown the utility of T1SS to transport exogenous proteins to the extracellular medium (reviewed in Delepelaire, P., supra; Reed et al., "Biotechnological applications of bacterial protein secretion: from therapeutics to biofuel production," Res Microbiol. 164:675- 682, 2013, which is incorporated herein by reference). Low et al. found that cyclodextrin glucanotransferase secretion could be improved by overexpression of the E. coli - haemolysin transporter (Low et al., "An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production," J Ind. Microbiol. Biotechnol. 38:1587-1597, 2011; incorporated herein by reference). Su and coworkers also used E. coli a-haemolysin secretion systems to secrete protein (Su et al., "Extracellular overexpression of recombinant

Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21 (DE3)," Microb Cell Fact. 1 1:8, 2012; incorporated herein by reference). By overexpressing HlyBD, two strain-specific components of T1SS, they showed that the recombinant T1SS secreted a high level (1.5 g per liter) of protein from an E. coli. The E. coli hemolysin transporter is also known to secrete other heterologous T1SS substrates expressed in E. coli, including exotoxins Cya of Bordatella. pertussis (Sebo et al., "Repeat sequences in the B. pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the E. coli alpha-haemolysin translocator," Mol Microbiol. 9: 999-1009, 1993;

incorporated herein by reference); LtkA of Aggregatibacter actinomycetemcomitans (Lally et al., "Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene, Delineation of unique features and comparison to homologous toxins," J Biol Chem. 264: 15451-15456, 1989; incorporated herein by reference); PaxA of Pasteurella aerogenes (Kuhnert et al., "Characterization of PaxA and its operon: a cohemolytic RTXtoxin determined from pathogenic Pasteurella aerogenes," Infect lmmun. 68:6-12, 2000; incorporated herein by reference); and FrpA of Neisseria meningitidis (Thompson et al., "The RTX cytotoxin-related FrpA protein of Neisseria miningitidis is secreted extracellularly by meningococci and by HlyBD+ Escherichia coli." Infect. Immun. 61 :2906-2911, 1993; incorporated herein by reference). The assembly of the T1SS complex, best exemplified by E. coli hemolysin (Hly) secretion system, is nucleotide-independent, and the translocation of HlyA (the T1SS substrate) requires ATP hydrolysis catalyzed by HlyB (Thanabalu et al., "Substrate-induced assembly of a contiguous channel for the protein export from E. coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore, EMBO J. 17:6487-6496, 1998; incorporated herein by reference). HlyA is a member of the RTX (repeats in toxin) protein family and contains glycine-rich peptide repeats in the C-terminal domain, which have the consensus sequence GGXGXD (X represents any amino acid) and are important for the binding of Ca²⁺ ions. This triggers folding of HlyA in the extracellular medium, which in turn generates the biologically active form of the toxin. The Tl SS substrates contain a translocation signal at the C-terminus (last 27 to 218-amino acids fragment of HlyA), and a minimal secretion signal is located within the last -60 C-terminal amino acids and is both necessary and sufficient to direct secretion. (Kenny et al., "Analysis of the haemolysin transport process through the secretion from E. coli of PCM, CAT or beta-galactosidase fused to the C-terminal signal domain," Mol. Microbiol. 5:2557-2568, 1991, incorporated herein by reference). Proteins targeted to the translocator carry an uncleaved, poorly conserved secretion signal at the extreme C-terminus (absolutely required for secretion) that binds to the nucleotide binding domain of ABC-ATPase (E. coli HlyB) in a reaction reversible by ATP and mimics initial movement of HlyA into the translocation channel and rapid transport of HlyA to the extracellular medium (Holland B. et al., "Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review)," Mol. Membr. Biol. 22:29-39, 2005;

incorporated herein by reference). [0076] T1SS from Pseudomonas fluorescens is also known and has been used to secrete recombinant proteins. Park et al identified a 105 amino acid polypeptide as the minimal region for recognition and transport by the lipase ABC transporter (Park et al., "Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluoresce™ for secretion and fluorescence of green fluorescent protein," Microb Cell Fact. 11 :60, 2012; incorporated herein by reference). A fusion of a target protein to this minimal region allowed secretion of a recombinant protein. The versatility of T1SS for protein secretion is seen in its wide array of transport substrates, which vary from small proteins like the hemophore HasA (19 kDa) to huge surface layer proteins up to 900 kDa in size

(Linhartova et al., "RTX proteins: a highly diverse family secreted by a common mechanism, FEMS Microbiol. Rev." 34:1076-1112, 2010; Satchell, "Structure and function of MARTX toxins and other large repetitive RTX proteins," Ann Rev Microbiol. 65:71 -90, 2011 ; all publications incorporated herein by reference). Other proteins secreted by T1SS include, for example, adenylate cyclases, lipases, and proteases.

[0077] In some embodiments, another Tl SS system that can be used in the invention is

RaxABC from Xanthomonas oryzae pv. oryzae. Phylogenetic analysis identifies RaxB as an ABC transporter (da Silva et al., "Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21 -mediated innate immune response," Mol Plant Microbe Interact. 17:593-601, 2004; incorporated herein by reference), equivalent to HlyB from E. coli. The RaxABC transport system is used to secrete AvrXa21 molecules (small sulfated polypeptides), metalloproteases, adhesion factors and glycanases (Delepelaire, supra; Reddy et al., "ToIC is required for pathogenicity of Xylella fastidioa in Vitis vinifera grape-vines," Mol Plant Microbe Interact. 20:403-410, 2007; all of which are incorporated herein by reference). These T1SS sequences and the others described above are used to promote secretion of target proteins from the magneto-endosymbionts of the invention.

[0078] Autotransporter systems, a subset of T5SS, provide potentially the simplest mechanism for extracellular secretion of recombinant proteins. In some embodiments, an autotransporter comprises an N-terminal Sec-dependent signal sequence, a passenger domain, and a C-terminal beta-motif. In this system, translocation is a two-step process. The target protein is transported into the periplasm using .Sec-dependent transport whereupon the beta- motif forms a transmembrane pore through which the passenger domain is secreted out of the periplasm (Dautin et al., "Protein secretion in gram-negative bacteria via the autotransporter pathway," Ann Rev Microbiol. 61:89-112, 2007; Thanassi et al., "Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (Review)," Mol. Membr. Biol. 22:63-72, 2005; all publications incorporated herein by reference). The beta-motif can be cleaved, allowing translocation of the target protein out of the cell (Leyton et al., "From self sufficiency to dependence:

mechanism and factors important for autotransporter biogenesis," Nat Rev Microbiol. 10:213- 225, 2012; incorporated herein by reference).

[0079] For transport in the T5SS system, the target protein is fused with the N-terminal signal sequence, the C-terminal signal, and the beta-domain that mediate translocation of a recombinant protein through the inner and outer membranes, respectively. This chimeric gene has the N-terminal signal sequence fused in frame to the N-terminal end of the target gene, and a second, in frame fusion to DNA encoding the beta-domain sequence at the C-terminal end of the target gene. In some embodiments, the passenger domain may be replaced in the fusion protein. For example, Jong et al. defined passenger domains of E. coli autotransporter hemoglobin-binding protease (Hbp) that could be replaced in a fusion protein to facilitate secretion, along with an intact beta-domain (Jong et al., "A structurally informed

autotransporter platform for efficient heterologous protein secretion and display," Microb Cell Fact. 1 1 :85, 2012; incorporated herein by reference). As such, the Hbp (NCBI

Accession no. 088093, 1377 amino acids, as shown below) passenger domains that could be replaced by heterologous proteins are: (1) 53-308;(2) 533-608; (3) 657-697; (4) 735-766; (5) 898-922 amino acids.

MNRIYSLRYSAVARGFIAVSEFARKCVHKSVRRLCFPVLLLIPVLFSAG

SLAGTVNNELGYQLFRDFAENKGMFRPGATNIAIYNKQGEFVGTLDK

AAMPDFSAVDSEIGVATLINPQYIASVKHNGGYTNVSFGDGENRYNIV

DRNNAPSLDFHAPRLDKLVTEVAPTAVTAQGAVAGAYLDKERYPVFY

RLGSGTQYIKDSNGQLTKMGGAYSWLTGGTVGSLSSYQNGEMISTSS

GLVFDYKLNGAMPIYGEAGDSGSPLFAFDTVQNKWVLVGVLTAGNG

AGGRGNNWAVIPLDFIGQKFNEDNDAPVTFRTSEGGALEWSFNSSTG

AGALTQGTTTYAMHGQQGNDLNAGKNLIFQGQNGQINLKDSVSQGA

GSLTFRDNYTVTTSNGSTWTGAGIVVDNGVSVNWQVNGVKGDNLHK

IGEGTLTVQGTGINEGGLKVGDGKVVLNQQADNKGQVQAFSSVNIAS

GRPTVVLTDERQVNPDTVSWGYRGGTLDVNGNSLTFHQLKAADYGA

VLANNVDKRATITLDYALRADKVALNGWSESGKGTAGNLYKYNNPY

TNTTDYFILKQSTYGYFPTDQSSNATWEFVGHSQGDAQKLVADRFNT

AGYLFHGQLKGNLNVDNRLPEGVTGALVMDGAADISGTFTQENGRLT

LQGHPVIHAYNTQSVADKLAASGDHSVLTQPTSFSQEDWENRSFTFDR

LSLKNTDFGLGRNATLNTTIQADNSSVTLGDSRVFIDKNDGQGTAFTL

EEGTSVATKDADKSVFNGTVNLDNQSVLNINDIFNGGIQANNSTVNISS

DSAVLGNSTLTSTALNLNKGANALASQSFVSDGPVNISDATLSLNSRP

DEVSHTLLPVYDYAGSWNLKGDDARLNVGPYSMLSGNTNVQDKGTV TLGGEGELSPDLTLQNQMLYSLFNGYRNIWSGSLNAPDATVSMTDTQ

WSMNGNSTAGNMKLNRTIVGmGGTSPFTTLTTDNLDAVQSAFVMRT

DLNKADKLVrNKSATGHDNSIWVNFLKKPSNKDTLDIPLVSAPEATAD

NLFRASTRVVGFSDVTPILSVRKEDGKKEWYLDGYQVARNDGQGKA

AATFMHISYNNFITEVNNLNKRMGDLRDINGEAGTWYRLLNGSGSAD

GGFTDHYTLLQMGADRKHELGSMDLFTGVMATYTDTDASADLYSGK

TKSWGGGFYASGLFRSGAYFDVIAKYIHNENKYDLNFAGAGKQNFRS

HSLYAGAEVGYRYHLTDTTFVEPQAELVWGRLQGQTFNWNDSGMD

VSMRRNSVNPLVGRTGVVSGKTFSGKDWSLTARAGLHYEFDLTDSA

DVHLKDAAGEHQINGRKDSRMLYGVGLNARFGDNTRLGLEVERSAF

GKYNTDDAINANIRYSF

[0080] In some embodiments, many E. coli autotransporters can also be used. For example, the YfaL autotransporter (NCBI accession no. P45508) can be used to secrete proteins ranging from 25.3 to 143 kDa from E. coli (Ko et al., "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia coli with a novel ligation- independent cloning vector based on the autotransporter YfaL," Appl Environ Microbiol.

78:3051-3058, 2012; incorporated herein by reference).

MRIIFLRKEYLSLLPSMIASLFSANGVAAVTDSCQGYDVKASCQASRQ

SLSGITQDWSIADGQWLVFSDMTNNASGGAVFLQQGAEFSLLPENETG

MTLFANNTVTGEYNNGGAIFAKENSTLNLTDVIFSGNVAGGYGGAIYS

SGTNDTGAVDLRVTNAMFRNNIANDGKGGAIYTINNDVYLSDVIFDN

NQAYTSTSYSDGDGGAIDVTDNNSDSKHPSGYTIVNNTAFTNNTAEG

YGGAIYTNSVTAPYLIDISVDDSYSQNGGVLVDENNSAAGYGDGPSSA

AGGFMYLGLSEVTFDIADGKTLVIGNTENDGAVDSIAGTGLITKTGSG

DLVLNADNNDFTGEMQIENGEVTLGRSNSLMNVGDTHCQDDPQDCY

GLTIGSIDQYQNQAELNVGSTQQTFVHALTGFQNGTLNIDAGGNVTVN

QGSFAGIIEGAGQLTIAQNGSYVLAGAQSMALTGDIVVDDGAVLSLEG

DAADLTALQDDPQSIVLNGGVLDLSDFSTWQSGTSYNDGLEVSGSSGT

VIGSQDVVDLAGGDNLHIGGDGKDGVYVVVDASDGQVSLANNNSYL

GTTQIASGTLMVSDNSQLGDTHYNRQVIFTDKQQESVMEITSDVDTRS

DAAGHGRDIEMRADGEVAVDAGVDTQWGALMADSSGQHQDEGSTL

TKTGAGTLELTASGTTQSAVRVEEGTLKGDVADILPYASSLWVGDGA

TFVTGADQDIQSIDAISSGTIDISDGTVLRLTGQDTSVALNASLFNGDGT

LVNATDGVTLTGELNTNLETDSLTYLSNVTVNGNLTNTSGAVSLQNG

VAGDTLTVNGDYTGGGTLLLDSELNGDDSVSDQLVMNGNTAGNTTV

VVNSITGIGEPTSTGIKVVDFAADPTQFQNNAQFSLAGSGYVNMGAYD

YTLVEDNNDWYLRSQEVTPPSPPDPDPTPDPDPTPDPDPTPDPEPTPAY

QPVLNAKVGGYLNNLRAANQAFMMERRDHAGGDGQTLNLRVIGGD

YHYTAAGQLAQHEDTSTVQLSGDLFSGRWGTDGEWMLGIVGGYSDN

QGDSRSNMTGTRADNQNHGYAVGLTSSWFQHGNQKQGAWLDSWLQ

YAWFSNDVSEQEDGTDHYHSSGIIASLEAGYQWLPGRGWIEPQAQVI

YQGVQQDDFTAANRARVSQSQGDDIQTRLGLHSEWRTAVHVIPTLDL

NYYHDPHSTEIEEDGSTISDDAVKQRGEIKVGVTGNISQRVSLRGSVA

WQKGSDDFAQTAGFLSMTVKW [0081] In some embodiments, a protease (e.g., a tobacco etch virus protease) is used to cleave the C-terminus of the fusion proteins to remove the beta-domain and autotransporter, resulting in secretion from the cell. For example, the E. coli serine protease Pet can be used to cleave fusion proteins and provide for complete secretion of a range of proteins varying in sizes and structures, and including multi-component proteins (Sevastsyanovich et al., "A generalized module for the selective extracellular accumulation of recombinant proteins," Microb. Cell. Fact. 11 :69, 2012; incorporated herein by reference). Pet is one of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) that releases passenger domain from the beta-domain.

[0082] An application of an autotransporter for consolidated bioprocessing uses an E. coli autotransporter Antigen 43 (Ag43) engineered to secrete a target protein. This autotransporter system is unique in that the passenger domain Ag43alpha is self-cleaved yet the secreted domain is non-covalently attached to the beta-domain, forming an integral outer-membrane protein. In order to secrete the recombinant protein, the segment of Ag43alpha containing the cleavage mechanism was fused to a target sequence. When engineered into an E. coli strain the self-cleaving autotransporter secreted target protein out of the cell (Wargacki et al., "An engineered microbial platform for direct biofuel production from brown macroalgae," Science 335:308-313, 2012; incorporated herein by reference). These autotransporter sequences are used to promote secretion of target proteins from the magneto-endosymbionts of the invention.

[0083] A very large number of proteins are secreted via the T5SS, even more than the T2SS (Jacob-Dubuisson et al., "Protein secretion through autotransporter and two-partner pathway," Biochim Biophy Acta 1694:235-257, 2004; Dautin et al., "Protein secretion in the gram-negative bacteria via autotransporter pathway," Ann Rev Microbiol. 61 :89-l 12, 2007; all publications incorporated herein by reference). Most of the T5SS secreted proteins characterized to date are virulence factors. Proteins secreted via the T5SS include adhesions, such as AIDA-I and Ag43 of E. coli, Hia of Haemophilus influenzae, YadA of Yersinia enteroliticola and Prn of Bordetella pertussis; toxins, such as VacA of Helicobacter pylori; proteases, such as IgA proteases of Neisseria gonorrheae and Neisseria meningitides, SepA of Shigella flexneri and PrtS of Serratia marcescens; and S-layer proteins, such as rOmpB of Rickettsia sp. and Hsr of Helicobacter pylori. T5bSS (TPS) secreted proteins include adhesions, such as HecA/HecB of the plant pathogen Dickeya dadantii (Erwinia

chrysanthemii), and cytolysins, such as ShIA/ShIB of Serratia marcescens, HpmA/HpmB of Proteus mirabilis and EthA/EthB of Edwardsiellla tarda. The T5SS sequences described above are used to promote secretion of target proteins from the magneto-endosymbionts of the invention.

[0084] The type IV secretion system (T4SS) is a versatile, multi-component secretion system used by both gram-negative and gram-positive bacteria to secrete proteins, DNA, and protein- DNA complexes from a wide range of targeted eukaryotic and bacterial cells (Fronzes et al., "The Structural Biology of Type IV Secretion Systems," Nat Rev Microbiol. 7(10):703-14, 2009; Backert et al., Type IV Secretion Systems and Their Effectors in Bacterial

Pathogenesis, Curr Opin Microbiol. 9(2):207-17, 2006; all publications incorporated herein by reference) and can be divided into three groups (Fronzes et al., The Structural Biology of Type IV Secretion Systems, Nat Rev Microbiol. 7(10):703-14, 2009; incorporated herein by reference). Group 1 T4SSs mediate the conjugative transfer of plasmid DNA or transposons into a wide range of bacterial species. For example, E. coli and Agrobacterium tumifaciens can deliver DNA substrates into fungal, plant, or human cells (Grohmann et al., "Conjugative Plasmid Transfer in Gram-Positive Bacteria," Microbiol Mol Biol Rev. 67(2):277-301, 2003; Lawley et al., "F Factor Conjugation Is a True Type IV Secretion System," FEMS Microbiol Lett. 224(1): 1-15, 2003; Trieu-Cuot et al., "In Vivo Transfer of Genetic Information between Gram-Positive and Gram-Negative Bacteria," EMBOJ. 4(13A):3583-7, 1985; all of which are incorporated herein by reference). T4SSs in group 2, such as those found in Helicobacter pylori and Neisseria gonorrhea, mediate the uptake and release of DNA into the extracellular environment (Smeets et al., "Natural Transformation in Helicobacter Pylori: DNA Transport in an Unexpected Way," Trends Microbiol 10(4): 159-62, 2002; Hamilton et al., "Natural Transformation of Neisseria Gonorrhoeae: From DNA Donation to Homologous

Recombination," Mol Microbiol. 59(2):376-85, 2006; all of which are incorporated herein by reference). Group 3 T4SSs deliver effector molecules into eukaryotic cells during infection. H. pylori, Brucella suis and Legionella pneumophila are examples of bacteria that use their T4SSs to inject virulence proteins into mammalian host cells (Backert et al., "Type IV Secretion Systems and Their Effectors in Bacterial Pathogenesis," Curr Opin Microbiol. 9(2):207-17, 2006; Corbel, "Brucellosis: An Overview," Emerg Infect Dis. 3(2):213-2I, 1997; Ninio et al., "Effector Proteins Translocated by Legionella Pneumophila: Strength in Numbers," Trends Microbiol. 15(8):372-80, 2007; all of which are incorporated herein by reference). A. tumeficians uses its group 3 T4SS to deliver oncogenic DNA and proteins into plant cells (Franzes et al., "The Structural Biology of Type IV Secretion Systems," Nat Rev Microbiol. 7(10):703-14, 2009; incorporated herein by reference).

[0085] Genes encoding components of the T4SS are usually arranged in a single or multiple operons. H. pylori is an example of a bacterium that encodes multiple T4SSs. H pylori has an effector protein delivery system encoded by the cag pathogenicity island and a DNA release and uptake system encoded by the comB gene cluster (Backert et al., "Type IV Secretion Systems and Their Effectors in Bacterial Pathogenesis," Curr Opin Microbiol. 9(2):207-17, 2006; Smeets et al., "Natural Transformation in Helicobacter Pylori: DNA Transport in an Unexpected Way," Trends Microbiol. 10(4): 159-62, 2002; all of which are incorporated herein by reference).

[0086] Depending on the structural components that compose a T4SS, the systems can be broadly classified as either type IVA or type IVB systems (Voth et al., "Bacterial Type IV Secretion Systems: Versatile Virulence Machines," Future Microbiol. 7(2):241-257, 201 1 ; incorporated herein by reference). A. tumifaciens T4SS and those that resemble it fall into the type IVA secretion systems. The prototypic T-DNA or VirB secretion system of A.

tumifaciens is the most well characterized T4SS. The A. tumifaciens T4SS transporter complex and others similar to it typically consist of 1 1 VirB proteins (encoded by the virBl- virBll genes) and the coupling protein VirD4, an NTPase (Tegtmeyer et al., "Role of the Cag-Pathogenicity Island Encoded Type IV Secretion System in Helicobacter Pylori Pathogenesis," FEBSJ. 278(8): 1 190-202, 201 1; incorporated herein by reference).

Agrobacterial VirB proteins are grouped into three categories: core components, pilus- associated components and energetic components. T4SSs that fall under the type IV B classification were demonstrated in Legionella pneumophilia to consist of twenty-two structural proteins and 5 chaperone proteins (Vogel et al., "Conjugative Transfer by the Virulence System of Legionella Pneumophila," Science 279(5352):873-6, 1998; Segal et al., "Host Cell Killing and Bacterial Conjugation Require Overlapping Sets of Genes within a 22- Kb Region of the Legionella Pneumophila Genome," Proc Natl Acad Sci USA 95(4): 1669- 74, 1998; all of which are hereby incorporated by reference).

[0087] Type IVA secretion systems and Type IVB secretion systems recognize different but overlapping translocation signals. A recent study showed that two Brucella effectors can be translocated by L. pneumophila demonstrating that a type IVB secretion system can recognize translocation signals from type IVA secretion system effectors (de Jong et al., "Identification of Vcea and Vcec, Two Members of the Vjbr Regulon That Are Translocated into Macrophages by the Brucella Type IV Secretion System," Mol Microbiol. 70(6): 1378- 96, 2008; incorporated herein by reference).

[0088] The following are examples of T4SS translocation signals: The A. tumifaciens translocation signal resides in a hydrophilic C-terminal region with a consensus R-X(7)-R-X- R-X-R-X-X(n) motif (Vergunst et al., "Positive Charge Is an Important Feature of the C- Terminal Transport Signal of the Virb/D4-Translocated Proteins of Agrobacterium," Proc Natl Acad Sci USA 102(3): 832-7, 2005; incorporated herein by reference). Bartonella has a BID domain and a short positively charged tail sequence that together form a bipartite C- terminal translocation signal (Schulein et al., "A Bipartite Signal Mediates the Transfer of Type IV Secretion Substrates of Bartonella Henselae into Human Cells," Proc Natl Acad Sci USA 102(3):856-61, 2005; incorporated herein by reference). In Helicobacter pylori, there is evidence that both the N- and C-terminal ends of the CagA protein have translocation signals. Hohlefeld, et al., observed that residues 6-26 of CagA are important for translocation (Hohlfeld et al., "A C-Terminal Translocation Signal Is Necessary, but Not Sufficient for Type IV Secretion of the Helicobacter Pylori Caga Protein," Mol Microbiol. 59(5): 1624-37, 2006; incorporated herein by reference). Hohlfeld et al., also show that CagA translocation depends on the presence of its 20 C-terminal amino acids. These T4SS sequences and others described above are used to promote secretion of target proteins from magneto- endosymbionts of the invention.

[0089] Type 1, 4, and 5 secretion system genes have also been identified in the MTB

Magnetospirillum sp., strain AMB-1 genome by sequence alignments. AMB-1 contains 83 genes that are involved in cell motility and secretion (Matsunga et al., "Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. AMB- 1," DNA Res. 12:157-166, 2005; incorporated herein by reference). There are at least seven genes that encode for RTX proteins in MTB. Several putative Tl SS substrates (NCBI

Accession Nos.: YP_420631.1, YP_420638.1, YP_420640.1, YP_421364.1, YP_422662.1, YP_422785.1, and YP_423419.1) have been identified in MTB (M magneticum AMB-1) (Linhartova et al., "RTX proteins: a highly diverse family secreted by a common

mechanism," FEMS Microbiol Rev. 34:1076-11 12, 2010; incorporated herein by reference). These substrates have been annotated as large exoprotein, type 5 secretory pathway, repeats- in-toxin (RTX) toxins and related Ca²⁺ binding protein, adhesion AidA, and hypothetical protein amb3422 with putative functions such as aminomethyltransferase, adhesion, and cadherin are consistent with type 1 secretion substrates in other bacteria. Moreover, genes responsible for constituting the T1SS complex (such as those described by NCBI Accession Nos. YP_422838, YP_421739, homolog of E. coli HlyB; E. coli HlyD membrane fusion protein homologs in MTB gi:83311477, 83312258, 83312575, 83313156 with locus tag amb2378; amb3159; amb3476; amb4057; E. coli TolC outer membrane protein homologs in MTB gi:8331 1344; 83312160; 83312256 with locus tag amb2245; amb3061; amb3157) are present in the MTB genome.

[0090] MTB proteins that are either secreted or are part of the cells T1SS secretion machinery have been identified in the sequenced genomes of certain MTB, and these are used to secrete target proteins from the magneto-endosymbionts of the invention.

[0091] As described in the foregoing, bacteria use diverse machinery to secrete proteins as a means for interacting with their environment, which in the case of endosymbionts includes the environment of the host cell. The invention modifies the morphology or the physiology of the host cell (as well as organism) via protein(s), nucleic acid(s), and/or other factor(s) secreted from the artificial endosymbiont through a secretion system.

Modified Magneto-Endosymbionts

[0092] In some embodiments, MTB are modified to achieve a higher level of recombinant protein secretion. Methods for genetically modifying magneto-endosymbionts are well known in the art. Typically, the magneto-endosymbiont is genetically modified to improve secretion of target molecules from the magneto-endosymbiont. Modifications may also involve increasing production of proteins or RNA by changing promoter or ribosome binding sequences, deletion or silencing of certain genes in the magneto-endosymbiont, or by other means well-known in the art.

[0093] In some embodiments, the flagellar proteins of a magneto-endosymbiont are modified so that the flagellar proteins are no longer expressed. Flagellar proteins have high homology to bacterial secretion systems suggesting a common evolutionary ancestor. In some embodiments, the flagellar proteins of a magneto-endosymbiont are modified to create a secretion system for target proteins. As described above, in some embodiments, the magneto- endosymbiont is modified so that it can no longer synthesize an essential molecule that is preferably provided by the eukaryotic host cell. In some embodiments, the magneto- endosymbiont is genetically modified so that its cell cycle is coordinated with the cell cycle of the eukaryotic host cell to maintain copy number of the magneto-endosymbiont at a sufficient level to impart the phenotype to daughter host cells. [0094] In some embodiments, genes or portions thereof of magneto-endosymbiont, such as MTN or other MTB strains, are modified. In some embodiments, the genes encoding the magnetosome are modified, for example, such as to control the size of magnetosome for magnetic hyperthermia applications. In AMB-1, magnetosome formation involves at least 100 genes, which are organized in the magnetosome island (MAI). The overexpression or deletion of the genes mamP, R, S, T, ambl006, or mmsF from the MAI results in different magnetite crystal sizes, morphology and/or chain lengths and organizations (Murat et al., "Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle," Proc. Nat'lAcad. Sci. USA 107:5593-5598, 2010; Murat et al., "The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1," Mol Microbiol. 85:684-699, 2012; all of which are incorporated herein by reference). Magnetosomes in wild-type AMB-1 range from 30 to 40 nm in diameter, and occur in single chains. Expression of magnetosome genes can be varied to change magnetosome size, shape and content. A magnetosome size range of 35-120 nm can be obtained by genetic engineering of MAI loci (Baumgartner et al., "Magnetite biomineralization in bacteria," Prog Mol Subcell Biol. 52:3-27, 2011 ; Jogler et al., "Genomics, genetics, and cell biology of magnetosome formation," Ann. Rev. Microbiol. 63:501-521, 2009; Lower et al., "The bacterial magnetosome: a unique prokaryotic organelle," J. Mol. Microbiol. Biotechnol. 23:63-80, 2013; Liu et al., "Synthesis of magnetosome chain-like structures," Nanotechnology 19:475-603, 2008; all publications incorporated herein by reference). The amount of iron present in AMB-1 can also be varied through manipulation of AMB-1 genes.

[0095] In some embodiments, various engineering approaches are used to modify magneto- endosymbionts, including: 1) engineering into the magneto-endosymbiont dedicated secretion systems that naturally exist in other bacteria; 2) engineering cell envelope mutations into the magneto-endosymbiont so that it alters the outer membrane or peptidoglycan layer permeability {e.g., Shin et al., "Extracellular recombinant protein production from and Escherichia coli lpp deletion mutant," Biotechnol Bioeng. 101 :1288-1296, 2008; incorporated herein by reference); and 3) co-expression of a lysis-promoting protein that removes the outer membrane (Ni et al., "Extracellular recombinant protein production from and Escherichia coli," Biotechnol. Lett. 31:1661-1670, 2009; incorporated herein by reference).

[0096] In some embodiments, the E. coli a-haemolysin transporter genes HlyB and HlyD are recombinantly expressed in the magneto-endosymbiont. Target proteins are then engineered by fusing them with a T1SS substrate secretion signal, which is located in C-terminal of HlyA to target them to the a-haemolysin transporter system. In some embodiments, the HlyA secretion signal comprises the sequence:

GNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDIYR YLSGYGHHIIDDDGGKDDKLSLADIDFRDVAFRREGNDLIMYKAEGN VLSIGHKNGITFRNWFEKESGDISNHQIEQIFDKDGRVITPDSLKKALEY QQSNNKASYVYGNDALAYGSQDNLNPLINEISKIISAAGNFDVKEERA AASLLQLSGNASDFSYGRNSITLTASA.

[0097] In some embodiments, the T1 SS from Pseudomonas fluorescens is used to transport recombinant proteins. The T1SS genes such as TliA for the lipase ABC transporter are engineered into the magneto-endosymbiont. The target protein is fused N-terminally to a 104 residue, minimal region comprising the sequence:

GSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGY QPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGG LWSEGVLIS

[0098] This fusion of target protein and minimal region allows secretion of recombinant protein. It is interesting to note that there are several proteins exhibiting substantial sequence homology to P. fluorescens lipase TliA such as those described by NCBI Accession Nos. YP_420640, YP_422785, YP_421364, YP_420631, and others that are present in the MTB genome. Most of this homology is located in the C-terminal domain of the proteins, which contains the transport signal. The MTB lipase ABC transport system is used to transport recombinant proteins by fusing target protein to a C-terminal signal of MTB lipase. In some embodiments, the C-terminal signal sequence comprises the sequence:

GAIGGAGAIPGITLVGNAGNDDIIGTNGNDLLLGGKGGATYRFSGGGC GSGGGWSIVQSDTNDVISAGAGDDVIYGDARLVNGNIQITGSGNDVLD GGSGNDQIHGGAGNDTIIGGTGDDVMFGDQGNDTFLFDFGFGHDVVD GGRGSNWTDTLDLTHDNQISSVNIEGVSGWAVSVDAQGHHVAQATN GAHDANGTIVVTNHDGSQDTIEFHNVEKVVW.

[0099] In some embodiments, the RaxA, RaxB and RaxC genes of Xanthomonas oryzae pv. oryzae are recombinantly expressed in the magneto-endosymbiont. The target protein is fused to RaxST, which is the recognition sequence for the RaxABC secretory system. In some embodiments, the RaxST recognition sequence comprises the sequence:

MLMGMSEEHPSNVQIDDAQRVRLLRAVFDAYYQNRQELGTVFDTNR AWCSRLTGLARLFPRSRMICCVRDVGWIVDSFERLAQSQPLRLSALFG YDPEDSVSMHADLLTAPRGVVGYALDGLRQAFYGDHADRLLLLRYD TLAQRPAQAMEQVYAFLQLPAFAHDYAGVQAEAERFDAALQMPGLH RVRRGVHYVPRRSVLPPALFDQLQELAFWESAPSHGALLV.

[0100] In some embodiments, the target protein is fused at the N-terminal end to a Sec- dependent signal sequence, and the C-terminal end of the target protein is fused to a β-motif. Translocation of such target protein fusions is a two-step process. The target protein is transported into the periplasm using Sec-dependent transport whereupon the β-motif forms a transmembrane pore in the outer-membrane through which the target protein is secreted from the periplasmic space. The β-motif is cleaved, allowing translocation of the target protein.

[0101] In some embodiments, the target protein is fused to an autotransporter (e.g., target protein replaces the passenger domain region, such as amino acids 29 to 685 of the YfaL autotransporter (Ko et al., "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia colt with a novel ligation-independent cloning vector based on the autotransporter YfaL," Appl Environ Microbiol. 78:3051-3058, 2012;

incorporated herein by reference). In some embodiments, the YfaL autotransporter sequence comprises the sequence:

MRIIFLRKEYLSLLPSMIASLFSANGVAAVTDSCQGYDVKASCQASRQ

SLSGITQDWSIADGQWLVFSDMTNNASGGAVFLQQGAEFSLLPENETG

MTLFANNTVTGEYNNGGAIFAKENSTLNLTDVIFSGNVAGGYGGAIYS

SGTODTGAVDLRVTNAMFRNNIANDGKGGAIYTINNDVYLSDVIFDN

NQAYTSTSYSDGDGGAIDVTDNNSDSKHPSGYTIVNNTAFTNNTAEG

YGGAIYTNSVTAPYLIDISVDDSYSQNGGVLVDENNSAAGYGDGPSSA

AGGFMYLGLSEVTFDIADGKTLVIGNTENDGAVDSIAGTGLITKTGSG

DLVLNADNNDFTGEMQIENGEVTLGRSNSLMNVGDTHCQDDPQDCY

GLTIGSIDQYQNQAELNVGSTQQTFVHALTGFQNGTLNIDAGGNVTVN

QGSFAGIIEGAGQLTIAQNGSYVLAGAQSMALTGDIVVDDGAVLSLEG

DAADLTALQDDPQSIVLNGGVLDLSDFSTWQSGTSYNDGLEVSGSSGT

VIGSQDVVDLAGGDNLHIGGDGKDGVYVVVDASDGQVSLANNNSYL

GTTQIASGTLMVSDNSQLGDTHYNRQVIFTDKQQESVMEITSDVDTRS

DAAGHGRDIEMRADGEVAVDAGVDTQWGALMADSSGQHQDEGSTL

TKTGAGTLELTASGTTQSAVRVEEGTLKGDVADILPYASSLWVGDGA

TFVTGADQDIQSIDAISSGTIDISDGTVLRLTGQDTSVALNASLFNGDGT

LVNATDGVTLTGELNTmETDSLTYLSNVTVNGNLTNTSGAVSLQNG

VAGDTLTVNGDYTGGGTLLLDSELNGDDSVSDQLVMNGNTAGNTTV

VWSITGIGEPTSTGIKVVDFAADPTQFQNNAQFSLAGSGYVNMGAYD

YTLVEDNNDWYLRSQEVTPPSPPDPDPTPDPDPTPDPDPTPDPEPTPAY

QPVLNAKVGGYLNNLRAANQAFMMERRDHAGGDGQTLNLRVIGGD

YHYTAAGQLAQHEDTSTVQLSGDLFSGRWGTDGEWMLGIVGGYSDN

QGDSRSNMTGTRADNQNHGYAVGLTSSWFQHGNQKQGAWLDSWLQ

YAWFSNDVSEQEDGTDHYHSSGIIASLEAGYQWLPGRGWIEPQAQVI

YQGVQQDDFTAANRARVSQSQGDDIQTRLGLHSEWRTAVHVIPTLDL

NYYHDPHSTEIEEDGSTISDDAVKQRGEIKVGVTGNISQRVSLRGSVA

WQKGSDDFAQTAGFLSMTVKW [0102] In some embodiments, a protease is included to cleave the β-motif from the target protein, such as for example, a tobacco etch virus protease, E. coli serine protease Pet, or a serine protease autotransporter of the Enterobacteriaceae (SPATEs) that releases passenger domains from the β-domain, without requiring exogenous protease. In some embodiments, an E. coli autotransporter Antigen 43 (Ag43) is used with the target protein. In some

embodiments, the Antigen 43 autotransporter sequence comprises the sequence:

MKRHLNTCYRLVWNHMTGAFVVASELARARGKRGGVAVALSLAAV

TSLPVLAADIVVHPGETVNGGTLANHDNQIVFGTTNGMTISTGLEYGP

DNEANTGGQWVQDGGTANKTTVTSGGLQRVNPGGSVSDTVISAGGG

QSLQGRAVNTTLNGGEQWMHEGAIATGTVINDKGWQVVKPGTVATD

TVVNTGAEGGPDAENGDTGQFVRGDAVRTTINKNGRQIVRAEGTANT

TVVYAGGDQTVHGHALDTTLNGGYQYVHNGGTASDTVVNSDGWQI

VKNGGVAGNTTVNQKGRLQVDAGGTATNVTLKQGGALVTSTAATV

TGINRLGAFSVVEGKADNVVLENGGRLDVLTGHTATNTRVDDGGTLD

VRNGGTATTVSMGNGGVLLADSGAAVSGTRSDGKAFSIGGGQADAL

MLEKGSSFTLNAGDTATDTTVNGGLFTARGGTLAGTTTLNNGAILTLS

GKTVNNDTLTIREGDALLQGGSLTGNGSVEKSGSGTLTVSNTTLTQKA

VNLNEGTLTLNDSTVTTDVIAQRGTALKLTGSTVLNGAIDPTNVTLAS

GATWNIPDNATVQSVVDDLSHAGQIHFTSTRTGKFVPATLKVKNLNG

QNGTISLRVRPDMAQNNADRLVIDGGRATGKTILNLVNAGNSASGLA

TSGKGIQVVEAINGATTEEGAFVQGNRLQAGAFNYSLNRDSDESWYL

RSENAYRAEVPLYASMLTQAMDYDRIVAGSRSHQTGVNGENNSVRLS

IQGGHLGHDNNGGIARGATPESSGSYGFVRLEGDLMRTEVAGMSVTA

GVYGAAGHSSVDVKDDDGSRAGTVRDDAGSLGGYLNLVHTSSGLW

ADIVAQGTRHSMKASSDNNDFRARGWGWLGSLETGLPFSITDNLMLE

PQLQYTWQGLSLDDGKDNAGYVKFGHGSAQHVRAGFRLGSHNDMT

FGEGTSSRAPLRDSAKHSVSELPVNWWVQPSVIRTFSSRGDMRVGTST

AGSGMTFSPSQNGTSLDLQAGLEARVRENITLGVQAGYAHSVSGSSAE

GYNGQATLNVTF

[0103] In some embodiments, the translocase of the outer mitochondrial membrane (TOM complex) is engineered into the magneto-endosymbiont. The TOM complex includes the receptors Tom20, Tom22, Tom70 and the channel-forming protein Tom40, and several other small subunits (reviewed in Hoogenraad et al., Biochim BiophyActa 1592:97-105, 2002; Neupert et al., Ann. Rev. Biochem. 76:723-749, 2007; and Chacinska et al., Cell 138:628-644, 2009; all publications incorporated herein by reference). Tom20 recognizes the substrate and transfers to centrally located Tom22, thereby the substrate is inserted into the Tom40 channel. Upon substrate import, TOM forms a complex with the translocase of the inner membrane (TIM complex) (Chacinska et al., EMBO J 22:5370-5381, 2003; incorporated herein by reference). The TIM complex consists of four integral membrane proteins, Tim23, Tim 17, Tim50, and Tim21. Tim23 forms the protein-conducting channel of the translocase and is tightly associated with Tom 17, whereas, Tim50 acts as regulator for the Tim23 channel and Tim21 transiently interacts with the TOM complex via Tom22 (Milisav et al., "Modular structure of the Tim23 preprotein translocase of mitochondria," J Biol Chem. 276:25856- 25861, 2001 ; incorporated herein by reference).

Intracellular Targeting of Secreted Factors

[0104] In some embodiments, the artificial endosymbionts, such as magneto-endosymbionts, secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s), where the polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) that are targeted or delivered to a specific cellular compartment, organelle or other locale of the host cell. In some embodiments, the secreted factor, particularly polypeptide(s) and nucleic acid(s) contain a region, also referred to as a targeting sequence, which targets or directs the polypeptide or nucleic acid to a specific cellular compartment or organelle of the host cell. Various intracellular targets, include by way of example and not limitation, Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane (see, e.g., U.S. Patent No. 6,455,247, incorporated herein by reference). In some

embodiments, the secrete factor is directed to the cellular cytoplasm.

[0105] In some embodiments, the secreted factors contain a nuclear localization sequence for delivery to the nucleus of the host cell(s). A nuclear localization signal (NLS) is a targeting peptide that directs proteins to the nucleus and is often a unit consisting of short basic, positively-charged amino acids. The NLS normally is located anywhere on the peptide chain. Numerous NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (PKKKLRKV; Kalderon et al., Cell 39:499-509, 1984); the human retinoic acid receptor β-nuclear localization signal

(ARRRRP); NFKB p50 (EEVQRKRQKL; Ghosh et al., Cell 62: 1019, 1990); NFKB p65 (EEKRKRTYE; Nolan et al., Cell 64:961, 1991). In some embodiments, the NLS comprises double basic NLS's, as exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasms (AVKRPAATKKAGQAKKKKLD; Dingwall et al., Cell 30:449-458, 1982; and Dingwall et al., J Cell Biol. 107:641-849, 1988).

[0106] In some embodiments, the targeting sequence is a nucleolar localization signal

(abbreviated NoLS or NOS) that target biomolecules for delivery to the nucleolus of the host cell(s). Exemplary nucleolar targeting sequences include, among others: SQDSKKKKKKKEKKKHKKHKKHKKHKKH,

SWTVQESKKKKRKKKKKGNKSASSE,

HRKSKKEKKKKKKRKHKKEKKKKDKEHRRP,

KKHSHRQNKKKQLRKQLKKPEWQVERE;

GRSTVSVSKKEKNRKRRNRKKKKKPQRVRGVSSE;

KAVLLKTKKKGQKKSGRPKKQRKQK;

AKSIIKKKKHFKKKRIKTTQKTKKQRK,

QAQAAKEKKKRRRRKKKAEENAEGG,

RRHRQKLEKDKRRKKRKEKEERTKGKKKSKK, and

QPKEQGQGDLKKKKKKKKGKLPKNYDPK.

[0107] In some embodiments, the targeting sequence is a lysosomal targeting sequence, including, for example, a lysosomal degradation sequence such as Lamp-2 (KFERQ; Dice, Ann. N. Y. Acad Sci. 674:58, 1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell Mol Biol Res. 41 :405, 1995) or Lamp-2 (LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF; Konecki et la., Biochem Biophys Res Comm. 205:1-5, 1994).

[0108] In some embodiments, the secreted factors contain a mammalian mitochondrial localization signal for delivery of the secreted factor to the mitochondria of the host cell(s). Generally, the mitochondrial targeting sequence is about 10-70 amino acid long peptide that directs a newly synthesized proteins to the mitochondria. It can be found at the N-terminus and can consist of an alternating pattern of hydrophobic and positively charged amino acids to form what is called an amphipathic helix. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the

mitochondria, such as the mitochondrial matrix. Like signal peptides, mitochondrial targeting signals are cleaved once targeting is complete. Exemplary mitochondrial localization sequence, include, among others, mitochondrial matrix sequences {e.g., yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz, Eur J Biochem. 165:1- 6, 1987); mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL; Schatz, supra); mitochondrial intermembrane space sequences (yeast cytochrome c 1 ;

MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADS LTAEAMTA; Schatz, supra) and mitochondrial outer membrane sequences (yeast 70 kD outer membrane protein; MKSFITRNKTAILATVMTGTAIGAYYYYNQLQQQQQRGKK; Schatz, supra). [0109] In some embodiments, the secreted factors contain a Golgi/endoplasmic reticulum localization signal to target or deliver the secreted factor to the Golgi/endoplasmic reticulum of the host cell(s). In some embodiments, the Golgi/endoplasmic reticulum targeting sequence comprises an amino acid ER retention sequence, such as that found in calreticulin (KDEL; Pelham, Royal Society London Transactions B; 1-10, 1992) or adenovirus E3/19K protein (LYLSRRSFIDEKKMP; Jackson et al., EMBOJ. 9:3153, 1990).

[0110] In some embodiments, the secreted factors contain a localization signal to target or deliver the factor to a peroxisome of the host cell(s). At least two types of targeting sequences have been identified for targeting to peroxisome, also referred to as peroxisomal targeting signals (PTS). In some embodiments, the peroxisomal targeting sequence is PTS1, which is typically comprised of three amino acids located on the C-terminus. An exemplary PTS1 comprises the sequence SKL. In some embodiments, the peroxisomal targeting sequence is PTS2, having in some embodiments, a general sequence (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A) (see, e.g., Rachubinski et al., Cell 83: 525-528, 1995). An exemplary PTS2 comprises RQQVLLGHL or RLQVVLGHL. Other variations of peroxisomal targeting sequences are described in Lazarow, P.B., Biochim Biophy Acta 1763(12): 1599— 1604, 2006; incorporated herein by reference.

[0111] In some embodiments, the secreted factors contain signals to deliver the secreted factor to the plasma membrane of the host cell(s). There are a large number of known secretory signal sequences which are placed 5' to a polypeptide, and are cleaved from the polypeptide region to target the polypeptide to the secretory pathway. Secretory signal sequences and their transferability to unrelated proteins are well known (see, e.g., Silhavy, et al., Microbiol Rev. 49, 398-418, 1985; incorporated herein by reference). In some

embodiments, a secreted extracellular polypeptide is useful for binding to the surface of, or affecting the physiology of, a target cell that is other than the host cell. In some embodiments, the cell surface membrane localization signal comprises a signal peptide, which is generally a short (about 5-30 amino acids long) peptide present at the N-terminus of the majority of synthesized proteins that are destined towards the secretory pathway. Proteins that contain such signals are destined for either extra-cellular secretion, the plasma membrane, the lumen or membrane of either the (ER), Golgi or endosomes. Generally, the core of the signal peptide contains a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. In addition, many signal peptides begin with a short positively charged stretch of amino acids, which may help to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. Suitable signal peptides are well known, including sequences from IL-2 (MYRMQLLSCIALSLALVTNS; Villinger et al., J Immunol 155:3946, 1995), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al., Nucleic Acids Res. 7:30, 1979); preproinsulin

(MAL WMRLLPLL ALL AL WGPDP AAAF VN ; Bell et al., Nature 284:26, 1980); influenza HA protein (MKAKLL VLLY AF V AGDQI ; Sekiwawa et al., Proc Natl Acad Sci USA 80:3563, 1983), and cytokine IL-4 MGLTSQLLPPLFFLLAC AGNF VHG .

[0112] In some embodiments, the secreted factors contain membrane anchoring domains along with signals sequences to target the secreted factor to the plasma membrane of the host cell(s) for directed expression on the cell surface of the host cell(s). In some embodiments, the targeting sequence is a membrane anchoring signal sequence. For extracellular presentation, a membrane anchoring region is provided at the carboxyl terminus of the polypeptide. The transmembrane proteins are inserted into the membrane such that the regions encoded 5' of the transmembrane domain are extracellular and the sequences 3' become intracellular. In some embodiments, the transmembrane domains are placed 5' of the polypeptide, thus serving to anchor it as an intracellular domain, which may be desirable in some embodiments. Exemplary membrane-anchoring sequences include, but are not limited to, those derived from CD8, ICAM-2, IL-8R, CD4 and LFA-1. Useful sequences include sequences from: (1) class I integral membrane proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal sequence, 241-265 are the transmembrane residues; see

Hatakeyama et al., Science 244:551, 1989; and von Heijne et al, Eur J Biochem. 174:671, 1988) and insulin receptor beta chain (residues 1-27 are the signal, 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic domain; Ebina et al., Cell 40:747, 1985); (2) class 1 1 integral membrane proteins such as neutral endopeptidase (residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmic domain; see Malfroy et al., Biochem Biophy Res Commun. 144:59, 1987); (3) type III proteins such as human cytochrome P450 NF25 (Hatakeyama, supra); and (4) type IV proteins such as human P-glycoprotein

(Hatakeyama, supra). Particularly preferred are CD8 and ICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at the extreme 5' end of the transcript. These comprise the amino acids 1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al., Proc Natl Acad Sci USA 82:5126, 1985) and 1-21 in the case of ICAM-2 (MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature 339:61, 1989). These leader sequences deliver the construct to the membrane while the hydrophobic transmembrane domains, placed 3' of the random candidate region, serve to anchor the construct in the membrane. Transmembrane domains are encompassed by amino acids 145-195 from CD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra) and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR; Staunton, supra).

[0113] In some embodiments, the membrane anchoring sequence comprises a GPI anchor sequence, which results in a covalent bond formation between the molecule and a glycosyl- phosphatidylinositol moiety, thus anchoring the protein to the lipid bilayer. An exemplary GPI anchor sequence is contained in the sequence

PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (see, e.g., Homans et al., Nature 333(6170):269-72, 1988; and Moran et al., J Biol Chem. 266:1250, 1991).

[0114] In some embodiments, the membrane anchoring domain comprises a myristylation sequence. For example, myristylation of c-src recruits the protein to the plasma membrane. The first 14 amino acids of the protein are solely responsible for this function:

MGSSKSKPKDPSQR (Cross et al., Mol Cell Biol. 4(9): 1834, 1984; Spencer et al., Science 262:1019-1024, 1993; both publications hereby incorporated by reference). Other modifications such as palmitoylation can be used to anchor polypeptides in the plasma membrane; for example, palmitoylation sequences from the G protein-coupled receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL; Stoffel et al., J Biol Chem. 269:27791, 1994); from rhodopsin (KQFRNCMLTSLCCGK PLGD; Barnstable et al., J Mol Neurosci. 5(3):207, 1994); and the p21 H-ras 1 protein

(LNPPDESGPGCMSCKCVLS; Capon et al., Nature 302:33, 1983).

[0115] It is to be understood that while the expression of secreted polypeptides targeted to specific cellular locations is under control of the regulatable control region, in some embodiments of the present disclosure, a method for introducing a phenotype into a host cell can comprise expressing a polypeptide, nucleic acid or other factor in an artificial endosymbiont in a eukaryotic host cell, where the polypeptide, nucleic acid or other factor comprises a intracellular localization signal, e.g., targeting sequence. The polypeptide, nucleic acid or other factor expressed in the artificial endosymbiont is secreted into the host cell, and induces a phenotype in the host cell by localization to specific intracellular target(s), e.g., an organelle or cellular membrane. In some embodiments, the expression can be constitutive, without the use of a regulatable control region. Thus, for each and every embodiment herein using localization signals to induce a phenotype in the host cell, it is contemplated that expression in the artificial endosymbiont can be constitutive or regulatable.

Nucleic Acids and Control Regions

[0116] The nucleic acids of the invention include those that encode at least in part the individual peptides, polypeptides and proteins secreted in the method of the disclosure. The peptides, polypeptide and proteins can be natural, synthetic or a combination thereof. The nucleic acids of the invention also include the nucleic acids that are secreted from the magneto-endosymbiont into the host cell. The nucleic acids of the invention may be RNA, mRNA, microRNA, siRNA, shRNA, DNA or cDNA.

[0117] The nucleic acids of the invention also include expression constructs, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA. Expression constructs can contain a nucleic acid sequence that enables the construct to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. In eukaryotic host cells, e.g., mammalian cells, the expression construct can be integrated into the host cell chromosome and then replicate with the host chromosome.

Similarly, constructs can be integrated into the chromosome of prokaryotic cells.

[0118] Expression constructs also generally contain a selection gene, also termed a selectable marker. Selectable markers are well-known in the art for prokaryotic and eukaryotic cells, including host cells of the invention. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the constructs containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well-known in the art. In some embodiments, the selectable marker is the target protein or encoded by the nucleic acid secreted by the magneto-endosymbiont into the host cell.

[0119] In some embodiments, the expression construct for producing a heterologous polypeptide contain an inducible control region that is recognized by the host RNA polymerase and is operably linked to the nucleic acid encoding the protein or polypeptide, or expression of the nucleic acid that is to be secreted into the host cell. Inducible or constitutive promoters (or control regions) with suitable enhancers, introns, and other regulatory sequences are well-known in the art. In particular, the control regions comprise regulatable control regions, where expression from the control region can be induced or inhibited.

[0120] Many inducible control regions are well-known in the art and can be used in the expression constructs of the invention. In some embodiments, the inducible control regions that may be used include heat shock control regions that are induced to express gene products when a cell is undergoing a heat shock response. Such heat shock control regions include, for example, the magneto-endosymbiont homologues of the mammalian Hsp60 or Hsp70 proteins, that is, the chaperones GroE or DnaK in E. coli (see also, e.g., U.S. Patent No.

7,285,542, incorporated herein by reference). The expression constructs of the invention may also utilize temperature-sensing RNA structures from the 5' untranslated regions of thermally regulated mRNAs. These RNA structures respond to temperature changes, and can be used in tandem with heat shock control regions to thermally regulate expression in the magneto- endosymbiont (Klinkert et al., "Microbial thermosensors," Cell. Mol. Life Sci. 66:2661-2676, 2009; incorporated herein by reference). In some embodiments, expression constructs with thermally regulated control regions and RNA hairpins are used for spatiotemporal control of gene expression from magneto-endosymbionts inside host cells. Gene expression based on heat shock control regions can be regulated by methods that can raise the temperature sufficiently to induce expression from the heat shock control region. These include, among others, alternating magnetic fields, ultrasound, thermal (infrared) radiation, laser illumination (e.g., 532 nm irradiation; see, e.g., Ramos et al., BMC Dev Biol. 6:55-70, 2006), radio- frequency, and microwave radiation. In some embodiments, the heat shock control region can be natural or synthetic, including hybrid heat shock control regions.

[0121] In some embodiments, the regulatable control region can be controlled with a chemical agent. Numerous control regions regulatable with a chemical agent can be used in the present invention. Exemplary control regions and corresponding chemical agents that can be used, include, by way of example and not limitation, lac promoter (e.g., IPTG), trp promoter (e.g., indoleacrylic acid), P. putida cmt promoter (e.g., cumate; see aczmarczyk et al., Appl Environ Microbiol. 79(21):6795-802, 2013), tet promoter -tTA/TetR (e.g., doxycycline), rapamycin inducible promoters (e.g., rapamycin and analogs; see Wang et al., Gene Ther. 13: 187-190, 2006), and ecdysone regulatable promoters (e.g., ecdysone; see, e.g., U.S. Patent No. 7,091,038).

[0122] In some embodiments, the regulatable control region is a control region that can be controlled using light, also referred to as light-switchable expression systems. Such systems include, among others, those based on photoreceptor phytochrome, flavin chromophore, and photolyase like crytochromes (Shimizu-Sato et al., Nature Biotech. 20:1041-1044, 2002; U.S. Patent No. 6,858,429; U.S. patent publication 20130345294; all publications incorporated herein by reference).

[0123] It is to be understood that in some embodiments, regulated expression from a control region generally requires presence of one or more accessory molecules, such as a repressor protein, that controls expression from the control region. For heterologous control regions, it is contemplated that the bacterial host cell of the invention expresses these accessory proteins necessary for controlling expression. Such accessory proteins can be encoded on

extrachromosomal nucleic acids (e.g., plasm ids) or integrated into the genome of the host cell. Methods, genes and vectors for expressing such accessory molecules in a host cell are well known and well within the skill of those in the art. In some embodiments, it may be necessary to de-repress expression as a way of controlling expression.

[0124] In some embodiments, it may be desirable to modify the polypeptides of the invention. One of skill will recognize many ways of generating alterations in a given nucleic acid construct encoding a polypeptide. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see, e.g., Giliman and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328: 731-734, 1987; each publication incorporated herein by reference).

[0125] In some embodiments, the recombinant nucleic acids encoding the polypeptides of the invention are modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism. [0126] The polynucleotides of the invention also include polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides of the invention. Polynucleotides according to the invention can have at least about 80%, more typically have at least about 90%, and even more typically have at least about 95%, sequence identity to a polynucleotide of the invention. The invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above. The polynucleotides of the invention also encompass those nucleic acids which will hybridize under stringent conditions to a polynucleotide of the invention. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.

[0127] Nucleic acids which encode protein analogs in accordance with this invention (i.e., wherein one or more amino acids are designed to differ from the wild type polypeptide) may be produced using site directed mutagenesis or PCR amplification in which the primer(s) have the desired point mutations. For a detailed description of suitable mutagenesis techniques, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and/or Ausubel et al., editors, Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y. (1994), which are hereby incorporated by reference in its entirety for all purposes. Chemical synthesis using methods described by Engels et al., Angew Che Intl Ed. 28:716-734, 1989; which is hereby incorporated by reference in its entirety for all purposes, may also be used to prepare such nucleic acids.

[0128] "Recombinant variant" refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, such as enzymatic or binding activities, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology. [0129] Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0130] "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

[0131] Alternatively, where alteration of function is desired, insertions, deletions or non- conservative alterations can be engineered to produce altered polypeptides or chimeric polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.

[0132] Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, or degradation/turnover rate.

[0133] In a preferred method, polynucleotides encoding the novel nucleic acids are changed via site-directed mutagenesis. This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183, 1983. A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller et al., Nucleic Acids Res. 10:6487-6500, 1982.

[0134] PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the target at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasm id and this gives the desired amino acid variant.

[0135] A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315, 1985; which is hereby incorporated by reference in its entirety for all purposes; and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al., supra.

Host Cells

[0136] In another aspect, the present invention provides a eukaryotic host cell containing a artificial endosymbiont, particularly a magneto-endosymbiont, wherein the artificial endosymbiont imparts a phenotype to the host cell through secretion of proteins, nucleic acids, and/or other factors from the artificial endosymbiont into the host cell. In some embodiments, the artificial endosymbiont is heritable.

[0137] In some embodiments, the host cells of the invention are animal cells. In some embodiments, the host cells are mammalian, such as mouse, rat, rabbit, hamster, human, porcine, bovine, or canine. Mice routinely function as a model for other mammals, most particularly for humans (see, e.g., Hanna et al., "Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin," Science 318:1 20-1923, 2007;

Holtzman et al., "Expression of human apolipoprotein E reduces amyloid-β deposition in a mouse model of Alzheimer's disease," J Clin Invest. 103(6):R15-R21, 1999; Warren et al., "Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis," J. Clin. Invest. 95:1789-1797, 1995; all publications incorporated herein by reference). Exemplary animal cells include, among others, fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, and hematopoietic cells.

[0138] In some embodiments, the host cell is a cancer cell, including human cancer cells. In some embodiments, the cancer cells are cancer cell lines, many of which are well known to those of ordinary skill in the art, including common epithelial tumor cell lines such as Coco- 2, MDA-MB231 and MCF7; and non-epithelial tumor cell lines, such as HT-1080 and HL60, and the NCI60-cell line panel (see, e.g., Shoemaker, "The NCI60 human tumor cell line anticancer drug screen," Nature Reviews Cancer 6:813-823, 2006; incorporated herein by reference). Additionally, those of ordinary skill in the art are familiar with obtaining cancer cells from primary tumors. Cancer cells also include, for example, solid tumor cell types, hematopoietic cancer cells, carcinomas, sarcomas, leukemias, lymphomas, gliomas, as well as specific tissue related cancers such as prostate cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, and liver cancer.

[0139] In some embodiments, the host cells are stem cells. Those of ordinary skill in the art are familiar with a variety of stem cell types, including for example, embryonic stem cells, inducible pluripotent stem cells, hematopoietic stem cells, neural stem cells, epidermal neural crest stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, olfactory adult stem cells, testicular cells, and progenitor cells (e.g., neural, angioblast, osteoblast, chondroblast, pancreatic, epidermal, etc.). Stem cells of the invention may be pluripotent, oligopotent, or unipotent.

[0140] In some embodiments, the host cell is a cell of the circulatory system of a mammal, including humans. For example, red blood cells, platelets, plasma cells, T-cells, natural killer cells, or the like, and precursor cells of the same. As a group, these cells are defined to be circulating host cells of the invention. The present invention may be used with any of these circulating cells. In some embodiments, the host cell is a T-cell. In some embodiments, the host cell is a B-cell. In some embodiments, the host cell is a neutrophil. In some

embodiments, the host cell is a megakaryocyte.

[0141] In some embodiments, the host cell is a cell that is resident in the tissues and organs of a living animal. [0142] In some embodiments, the host cell is a fungal cell, including, but not limited to, the genera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula,

Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

[0143] In some embodiments, at least one gene from the host cell is genetically altered, as described herein. For example, in some embodiments, mutual nutritional dependence, sometimes referred to as biotrophy, may be established between the artificial endosymbiont and the host cell by genetic modification of the host cell, using appropriate molecular biology techniques specific to the target host cell type known to those of ordinary skill in the art, creating host cell dependence on the artificial endosymbiont for some essential

macromolecule, and thus establishing the environmental pressures for biotrophy. In some embodiments, nutritional dependence for a artificial endosymbiont on the host cell may be established by genetically altering the host cell to eliminate the ability of the host cell to synthesize various metabolites, cofactors, vitamins, nucleotides, or other essential molecules. In such embodiments, the essential molecule may be provided by the artificial endosymbiont. In some embodiments, the host cell gene encoding the enzyme serine

hydroxymethyltransferase, which converts serine into glycine at the terminus of the 3- phosphoglycerate biosynthetic pathway for amino acid production, may be modified.

Methods of Introducing Magneto-Endosymbionts into Host Cells

[0144] In the embodiments herein, the single-celled organisms of the invention can be introduced into host cells by a number of methods known to those of skill in the art including, but not limited to, microinjection, natural phagocytosis, induced phagocytosis,

macropinocytosis, other cellular uptake processes, liposome fusion, erythrocyte ghost fusion, electroporation, receptor mediated methods, and the like (see, e.g., Microinjection and Organelle Transplantation Techniques, Celis et al., Eds., Academic Press, New York, 1986, and references cited therein, which are hereby incorporated by reference in its entirety).

[0145] In some embodiments, a single-celled organism is introduced to the host cell by microinjection into the cytoplasm of the host cell. A variety of microinjection techniques are known to those skilled in the art. Microinjection is the most efficient transfer technique available (essentially 100%) and has no cell type restrictions (Microinjection and Organelle Transplantation Techniques, 1986; Xi et al., "Characterization of Wolbachia transfection efficiency by using microinjection of embryonic cytoplasm and embryo homogenate," Appl Environ Microbiol. 71(6):3199-3204, 2005; Goetz et al., "Microinjection and growth of bacteria in the cytosol of mammalian host cells," Proc Natl Acad ScL USA 98: 12221-12226, 2001; all publications incorporated herein by reference).

[0146] Naturally phagocytotic cells have been show to take up bacteria, including MTB (Burdette et a!., "Vibrio VopQ induces PI3-kinase independent autophagy and antagonizes phagocytosis," Molecular microbiology 73:639, 2009; Wiedemann, et al., "Yersinia enterocolitica invasin triggers phagocytosis via βΐ integrins, CDC42Hs and WASp in macrophages," Cellular Microbiology 3:693, 2001 ; Hackam et al., "Rho is required for the initiation of calcium signaling and phagocytosis by Fey receptors in macrophages," J Exp Med. l86(6):955-966, 1997; Matsunaga et al., "Phagocytosis of bacterial magnetite by leucocytes," Applied Microbiology and Biotechnology 31(4):40l-405, 1989; all publications incorporated herein by reference).

[0147] Studies have shown that non-phagocytotic cell types can be induced to endocytose bacteria when co-cultured with various factors, such as media, chemical factors, and biologic factors, for example, baculovirus, protein factors, genetic knock-ins, etc. (see, e.g., Salminen et al., "Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes," J Virol. 79(5):2720-2728, 200S;

Modalsli et al., "Microinjection of HEp-2 cells with coxsackie Bl virus RNA enhances invasiveness of Shigella flexneri only after prestimulation with UV-inactivated virus," APMES 101:602-606, 1993; Hayward et al., "Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella," EMBO J. 18:4926-4934, L 999; Yoshida et al.,

"Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Racl activity and efficient bacterial internalization," EMBO J. 21.2923-2935, 2002; Bigildeev et al., J. Exp Hematol., 39: 187, 2011; and Finlay et al., "Common themes in microbial pathogenicity revisited," Microbiol. andMoL Biol Rev. 61:136-169, 1997; all publications incorporated herein by reference).

[0148] The related process, macropinocytosis or "cell drinking," is a method numerous bacteria and viruses employ for intracellular entry (Zhang, In: Molecular Imaging and Contrast Agent Database (MICAD) (database online); Bethesda (MD): National Library of Medicine (US), NCBI; 2004-2011, 2004; incorporated herein by reference in its entirety for all purposes). Various protocols exist which can be employed to induce cells to take up bacteria. Several agents, such as nucleic acids, proteins, drugs and organelles have been encapsulated in liposomes and delivered to cells (see, e.g., Ben-Haim et al., "Cell-specific integration of artificial organelles based on functionalized polymer vesicles," Nam Lett. 8(5): 1368-1373, 2008; Lian et al, "Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles," Experimental Cell Research 313(l):53-64, 2007; Heng et al., "Immunoliposome-mediated delivery of neomycin phosphotransferase for the lineage-specific selection of

differentiated/committed stem cell progenies: Potential advantages over transfection with marker genes, fluorescence-activated and magnetic affinity cell-sorting," Med Hypotheses 65(2):334-336, 2005; Potrykus, Ciba Found Symp. 154:198, 1990; each publication incorporated herein by reference). This method is inexpensive, relatively simple and scalable. Additionally, liposome uptake can be enhanced by manipulation of incubation conditions, variation of liposome charge, receptor mediation, and magnetic enhancement (see, e.g., Pan et al., Int. J. Pharm. 358:263, 2008; Sarbolouki et al., "Storage stability of stabilized MLV and REV liposomes containing sodium methotrexate (aqueous & lyophilized)," JPharm Sci Techno. 52(10):23-27, 1998; Elorza et al., "Comparison of particle size and encapsulation parameters of three liposomal preparations, J Microencapsul. 10(2):237-248, 1993;

Mykhaylyk et al., "Liposomal Magnetofection," Methods Mol Bio. 605:487-525, 2010; each publication incorporated herein by reference).

[0149] Erythrocyte-mediated transfer is similar to liposome fusion and has been shown to have high efficiency and efficacy across all cell types tested (Microinjection and Organelle Transplantation Techniques, Celis et al. Eds., Academic Press: New York (1986);

incorporated herein by reference). Typically erythrocytes are loaded by osmotic shock methods or electroporation methods (Schoen et al., "Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles," Gene Therapy 6:823-832, 1999; Li et al., "Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation," BiophyJ. 71:479-486, 1996;

Carruthers et al., "A rapid method of reconstituting human erythrocyte sugar transport proteins," Biochemistry 23:2712-2718, 1984; each publication incorporated herein by reference). Alternatively, erythrocytes may be loaded indirectly by loading hematopoietic progenitors with single-celled organisms and inducing them to differentiate and expand into erythrocytes containing single-celled organisms.

[0150] Electroporation is a commonly used, inexpensive method to deliver factors to cells. (Potrykus, "Gene transfer methods for plants and cell cultures," Ciba Found Symp 154: 198- 208, 1990; Wolbank et al., "Labeling of human adipose-derived stem cells for non-invasive in vivo cell tracking," Cell Tissue Bank 8: 163-177, 2007; each publication incorporated herein by reference).

[0151] In some embodiments, a host cell that naturally endocytoses bacteria (e.g., Chinese hamster ovary (CHO)) is used. In some embodiments, the modified single-celled bacteria are added to the CHO culture directly. CHO cells can be cultured by standard procedures, for example, in Ham's F-12 media with 10% fetal calf serum media prior to infection with the MTB. Post infection, the media is augmented with additional iron (40 to 80 μΜ) as either ferric malate or FeCl₃. Numerous other cell types that internalize bacteria by endocytosis, or more specifically phagocytosis; endosymbionts or parasites, have their own methods for cellular entry, and these natural processes can be exploited for internalization of the magneto- endosymbionts resulting in the generation of so-called symbiosomes. In some embodiments, symbiosomes from one cell can be transplanted to another cell type (e.g., one incapable of endocytosis of magneto-endosymbionts) using microinjection, organelle transplantation, and chimera techniques. These host cells are cultured in typical media and techniques for the specific cell type.

[0152] In some embodiments, a single-celled organism is introduced to the host cell by a liposome mediated process. Mitochondria and chloroplasts, which are larger than MTB, have been efficiently introduced into eukaryotic cells when encapsulated into liposomes (see, e.g., Bonnett, H. T. Planta 131 :229, 1976; Giles et al., "Liposome-mediated uptake of chloroplasts by plant protoplasts," In Vitro Cellular & Developmental Biology - Plant 16(7) : 581 -584, 1976; each publication incorporated herein by reference). Numerous liposome fusion protocols and agents are available and can be used by the skilled artisan without undue experimentation (see, e.g., Ben-Haim et al., "Cell-specific integration of artificial organelles based on functionalized polymer vesicles," Nano Lett. 8(5): 1368-1373, 2008; Lian et al., "Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles," Exp Cell Res. 313(l):53-64, 2007; Heng et al., "Immunoliposome-mediated delivery of neomycin phosphotransferase for the lineage-specific selection of differentiated committed stem cell progenies: Potential advantages over transfection with marker genes, fluorescence-activated and magnetic affinity cell-sorting," Med Hypotheses 65(2):334-336, 2005; Potrykus, Ciba Found Symp. 1(54):198, 1990; each publication incorporated herein by reference).

[0153] In some embodiments, a single-celled organism is introduced to the host cell by an infectious process much like naturally occurring intracellular pathogens. In some embodiments, a single-celled organism is introduced to the host cell by a mechanism related to Listeria infection. In some embodiments, a single-celled organism is introduced to the host cell by a mechanism related to Salmonella infection. In some embodiments, a single-celled organism is introduced to the host cell by a mechanism related to Rickettsia infection. In some embodiments, a single-celled organism is introduced to the host cell by a mechanism related to Chlymidia infection.

Use of Host Cells with Artificial endosymbionts

[0154] In some embodiments, the artificial endosymbionts of the invention introduce into host cells nucleic acids, peptides/polypeptides, and/or other factors. These polypeptides, nucleic acids, or other factors can alter gene transcription or translation, post translational modifications, host cell differentiation, remodeling, proliferation, sensitivity, cell surface proteins or response to external and/or internal stimuli, metabolic, anabolic or other biochemical processes. The artificial endosymbiont can control host cells through expression, availability, and delivery of certain transcription factors, growth factors, cell surface markers or other recombinant proteins. The artificial endosymbiont may also introduce a desirable phenotype to the host cell through the polypeptides, nucleic acids or other factors that are secreted into the host cell from the artificial endosymbiont. As described herein, in some embodiments, the artificial endosymbiont comprises a magneto-endosymbiont, e.g., a magnetotactic bacterium.

[0155] The proteins, nucleic acids, or other factors secreted from an artificial endosymbiont into the host cell can be used for cell viability, proliferation, differentiation, de- differentiation, growth, detoxification, cell labeling, treating a pathology or deficiency, creating energy, inducing cell death, inducing angiogenesis, neurogenesis, osteogenesis, or wound healing, modifying cell signaling, modifying gene expression, neutralizing intracellular proteins or nucleic acids, or modifying cell function by providing: nutrients, growth factors, proteins, minerals, nucleic acids, therapeutic agents, small molecules, ions, chemokines, polysaccharides, lipids, metals, cofactors, or hormones. The artificial endosymbiont and host cells may also be used to manufacture bioremediation agents, enzymes, neurotransmitters, polypeptides, carbohydrates, pesticides, fertilizers, etc.

[0156] In some embodiments, the artificial endosymbiont can secrete a protein or other factor that provide a beacon for the host cell from a reporter such as a fluorescent protein (e.g., GFP, RFP, YFP, CFP), and/or luciferase. In some embodiments, the artificial endosymbiont can secrete a protein or other factor that provide a cell surface marker for the host cell from a protein such as a nerve growth factor, immune cell markers, adhesion molecules or other proteins present on the cell surface. In some embodiments, the artificial endosymbiont can secrete an amino acid into the host cell, including, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations of these amino acids.

[0157] In some embodiments, the artificial endosymbiont can secrete into a host cell a nucleic acid, RNA, mRNA, hnRNA, shRNA, siRNA, microRNA, antisense RNA or DNA, or DNA. Genetic material exchange between an artificial endosymbiont and a host cell can be used for gene therapy, to silence a gene, to transcribe a gene, to replace a gene, to modify expression of a gene, to modify a gene, to introduce a gene or nucleic acid fragment, to bind nucleic acids, to interact with nucleic acids, or the like.

[0158] In some embodiments, the artificial endosymbiont may introduce into the host cell signal pathway molecules such as, for example, receptors, ligands (hormones,

neurotransmitters, etc.), ion channels, kinases, phosphatases, DNA binding proteins

(transcription factors), Yamanaka factors (Oct4, Oct3, Sox2, Klf4, c-Myc, NANOG, and Lin28) for reprogramming mature cells into iPS cells.

[0159] In some embodiments, an artificial endosymbiont and its host cell are used to deliver therapeutic proteins to locations in an organism's body. For example, immune cells that migrate to sites of autoimmune disease, such as T cells that migrate to the pancreas in diabetics, or to myelin in multiple sclerosis, could be engineered with artificial

endosymbionts to express immunomodulatory proteins, IL-12p40 or other immune proteins. Alternatively, host cells with artificial endosymbionts could be implanted into sites in the organism. For example, cells that produce insulin from an artificial endosymbiont could be transplanted under the kidney capsule, or other tissue site, to make an artificial pancreas to treat diabetes. The artificial endosymbiont expression of insulin can be controlled using magnetic hyperthermia, and/or other means, providing genetic control in a location that is difficult to access repeatedly.

[0160] In some embodiments, an artificial endosymbiont can secrete transcription factors used to reprogram a cell like Oct4. In some embodiments, an artificial endosymbiont can secrete an enzyme to replace deficient enzymes in a lysosomal storage disease or ALDH2. In some embodiments, a magneto-endosymbiont can secrete a moiety to label a cell such as: Calcein, superparamagnetic iron oxide, gadolinium containing reagents, fluorescent proteins, luminescent proteins, magnetic reporters, other reporter proteins (e.g., Gilad et al., "MRI Reporter Genes," JNucl Med. 49: 1905-1908, 2008; incorporated herein by reference in its entirety). In some embodiments, a magneto-endosymbiont can secrete proteins related to energy generation or exchange such as hydrogenase, nitrogenase, laccase.

Methods of Controlling Gene Expression in Artificial Endosymbionts while in Host Cells

[0161] In some embodiments of the present invention, gene expression in the artificial endosymbiont in a eukaryotic host cell, particularly a magneto-endosymbiont in a eukaryotic host cell can be controlled using various methods. Generally, the method of controlling gene expression is selected based on the type of control region employed to regulate expression of the nucleic acid or polypeptide of interest (e.g., a gene encoding a polypeptide) in the bacterial cell.

[0162] In some embodiments, the control region comprises a heat shock control region, which as described above, can be controlled by hyperthermia and can be induced by treatments with various external signals, such as magnetic fields, ultrasound, laser illumination, radio-frequency, microwave, and thermal (infrared) radiation.

[0163] In some embodiments, where the artificial endosymbiont is a magneto-endosymbiont, an alternating magnetic field of sufficient strength, duration and frequency is used to activate expression from the heat shock control region. In some embodiments, magneto- endosymbionts selectively absorb energy from alternating magnetic fields relative to surrounding tissues, which is subsequently dissipated as heat, a process called magnetic hyperthermia. In some embodiments, the alternating magnetic field can be applied at a low frequency. In some embodiments, the, alternating magnetic field comprises magnetic fields at frequencies in the range of about 100kHz to about 500kHz. Generally, magnetic fields in these frequency ranges are weakly absorbed by biological tissues and thus highly penetrating. It allows essentially infinite depth of action, but limiting absorption to those cells engineered with magneto-endosymbionts.

[0164] In some embodiments, selective hyperthermia is used to control expression from a bacterial heat shock control region that expresses a gene of interest. The extent of heating from magnetic hyperthermia can be modulated for gene expression, or alternatively, increased to ablate the magneto-endosymbionts, or magneto-endosymbiont and its host cell, or the magneto-endosymbiont, its host cell, and surrounding tissue, at any location in the body.

[0165] In some embodiments, the magneto-endosymbiont are directed to specific target tissues, and the entire body treated with alternating magnetic fields such that only the tissues containing the magneto-endosymbiont are heated. In some embodiments, this local hyperthermia is used to control gene expression, protein folding and function, or other naturally occurring or engineered thermally sensitive cellular process (including controlled release of any molecule) such that function can be specifically manipulated noninvasively.

[0166] Without being bound by theory, models for temperature rise induced by magnetic hyperthermia are well-known in the art (see, e.g., Andra et al., "Synthesis of supermagnetic MgFe₂O₄ nanoparticles by coprecipitation," J Magnetism Magnetic Mat. 194: 1-7, 1999; incorporated herein by reference). For example, the equation:

calculates the temperature increase in a subcellular compartment containing nanogram amounts of magnetosomal iron. In this equation, m is mass of iron, SLP is specific loss power, K is thermal conductivity of the surrounding tissue, and D is the sub-cellular compartment diameter. This equation calculates approximately 5°C increase in temperature at the magneto-endosymbiont site, assuming an iron loading level of 2 ng Fe per mammalian cell, an SLP of 1500 W/g, a thermal conductivity of 0.64 W/m (muscle-like tissue) and a sub-cellular compartment diameter of 0.25μm.

[0167] In some embodiments, MRI is used for real time temperature mapping. In some embodiments, proton resonance frequency shift (PRFS) is used for the real time temperature mapping (see, e.g., Hofstetter et al., "Fat-referenced MR thermometry in the breast and prostate using IDEAL," Magnet Reson Imag. 36:722-732, 2012; incorporated herein by reference). In some embodiments, the amount of temperature rise and the area affected are controlled by varying the amount of iron in the magnetosomes, the frequency of the alternating magnetic field, and/or the time of exposure to the alternating magnetic field. In some embodiments, exposure of magneto-endosymbionts to higher amplitude alternating magnetic fields for short periods of time raises the temperature in smaller areas, e.g., just the magneto-endosymbiont, whereas lower amplitude magnetic fields used for longer periods of time raise the temperature in a larger area, e.g., the host cell and/or surrounding tissues. [0168] In some embodiments, the magneto-endosymbiont delivers, controls, permits visualization, and elimination of modulators that control cellular fates and function in vivo. In some embodiments, different genes or groups of genes are expressed at different times through differential response to temperature or other factors (physical or biochemical). For example, different RNA hairpins can be designed to inhibit expression up to different temperature ranges, and when heat shock is induced, the different RNAs will produce gene products at different temperatures. In some embodiments, the temperature is controlled by magnetic hyperthermia so that desired genes are expressed at desired times. In some embodiments, the frequency and intensity of alternating magnetic fields are modulated to achieve specific temperatures. Lower levels will target control of gene expression and hence the developmental fates and functions of the host cells. At higher levels, heating can be used to ablate the magneto-endosymbionts or host cells without adversely affecting the surrounding tissue, unless ablation of the surrounding tissue is desired.

[0169] In some embodiments, other treatments can be used to regulate expression from heat shock control regions in an artificial endosymbiont. In some embodiments, ultrasound is used to induce expression of genes under the control of heat shock control regions (see, e.g., Sontag et al., Ultrasound in Medicine and Biology 35(6):1032-1041, 2009; Eker et al., Radiology 258(2):496-504, 2011 ; all publications incorporated herein by reference). In some embodiments, selective hyperthermia can be induced by focused ultrasound. Ultrasound is demonstrated to cause hyperthermia in treated cells and tissues. In some embodiments, ultrasound, particularly focused ultrasound, of sufficient frequency, amplitude and duration is used to induce expression from the heat shock control regions. In some embodiments, the ultrasound is used at a frequency from 10 to about 20 MHz, particularly at about 10 MHz. The duration of ultrasound treatment can vary from 1 min to about 30 min or more, particularly, about 5 min to about 20 min. In some embodiments, the ultrasound treatment can be continuous or pulsed. In some embodiments, the focused ultrasound treatment can be based on temperature imaging with MRI, which allows delivery of ultrasound to specified temperatures. In particular, an MRI feedback system is used to control the ultrasound treatment to a specific temperature or temperature range.

[0170] In some embodiments, radio-frequency (RF) radiation is used to induce expression from heat shock control regions (see, e.g., U.S. Patent Publication No. 20080140063). The theoretical basis for RF radiation based hyperthermia is that RF radiation absorbed by matter causes molecules to vibrate, which in turn causes heating. More specifically, RF waves interact with matter by causing molecules to oscillate with the electric field. Generally, the interaction is highly effective for molecules that are polar, i.e., having their own internal electric field, such as water. Radio frequency waves have low tissue specific absorption, which can provide for whole body radiation, in some instances. In some embodiments, RF radiation of sufficient frequency, amplitude, and duration is used to induce expression of genes under the control of heat shock control regions. The RF spectrum is generally between 3 kHz and 300 GHz, but for hyperthermia it generally refers to frequencies below the microwave range. Microwaves occupy the general EM frequency spectrum between 300 MHz and 300 GHz. In some embodiments, the RF frequency used for inducing hyperthermia is between 10 to 20 MHz. Common RF frequencies used include 13.56 and 27.12 MHz, which have been used in diathermy applications. The treatment with RF frequencies can be continuous or pulsed to produce the desired hyperthermia and subsequent activation of expression from the heat shock control regions.

[0171] In some embodiments, electromagnetic radiation in the microwave range is used to regulate expression from the heat shock control regions. In some embodiments, microwave radiation of sufficient frequency, amplitude, and duration is used to induce expression of genes under the control of heat shock control regions. In some embodiments, microwave in the range of about 400 to about 3000 MHz is used. Commonly used microwave frequencies in hyperthermia include 433, 915, and 2450 MHz. In some embodiments, the microwaves can be coupled into tissues by waveguides, dipoles, microstrips, or other radiating devices. In some embodiments, a probe that generates microwave radiation in the desired frequency is used to direct and focus energy into tissues by direct radiation from the probe. In some embodiments, an array of microwave generating probes can be used to increase the volume of cells treated. Similar to use in ultrasound applications to measure and control temperature, MRI thermography can be adapted to monitor and control microwave based hyperthermia (Wlodarczyk et al., JMagn Reson Imaging 8:165, 1998; incorporated herein by reference).

[0172] In some embodiments, laser illumination of sufficient frequency, amplitude and duration is used to induce hyperthermia and activate expression from heat shock control regions. Laser mediated induction of heat shock genes are described in, for example, Ramos, supra; Halfon et al., Proc Natl Acad Sci USA 94:6255-6260, 1997; Due et al., Mol Vis.

18:2380-7, 2012; Rylander et al., Lasers in Surgery and Medicine 39:731-746, 2007; and Halloran et al., Development 127, 1953-1960, 2000; all publications incorporated herein by reference). Laser illumination in the range of about 400 nm to about 1000 nm wavelength {e.g., 440, 532, and 810 nm) have been used successfully to induce hyperthermia in cells and subsequent expression from heat shock promoters. In some embodiments, cells can be heat shocked with nanosecond bursts of laser, e.g., 2 200 ns, at a frequency of 1-10 Hz, for a duration of 0.2 to 5 min. In some embodiments, the laser can be used for in vivo situations by use of a catheter or endoscope to direct the illumination to a specific target tissue or cells.

[0173] In some embodiments, the heat shock control region is regulated using thermal radiation. The thermal radiation can be from a heated element or probe, infrared light source (e.g., infrared laser), or any other known sources of infrared radiation. In some embodiments, the thermal radiation can be directed to a specific target cell or tissue. In some embodiments, the thermal probe, heating element, or infrared laser is applied to the target cell or tissue in sufficient frequency, amplitude and duration to induce expression from the heat shock control region. In some embodiments, the thermal radiation is delivered by an infrared laser (Deguchi et al., Dev Growth Differ. 51 :769-775, 2009; Kamei et al., Nat Methods 6:79-81, 2009; all publications incorporated herein by reference).

[0174] In some embodiments, where control regions inducible by chemical agents are used to regulate gene expression in the artificial endosymbiont, the gene expression can be regulated by contacting the cell or tissue, or administering to the organism a specific chemical agent that induces expression. As discussed above, the control regions (e.g., promoters) regulatable with chemical agents include, among others, lac promoter (inducible with IPTG), trp promoter (inducible with indoleacrylic acid), P. putida cmt promoter (inducible with cumate), tet promoter -tTA/TetR (inducible with tetracycline or analog doxycycline), rapamycin inducible promoters (inducible with rapamycin and rapamycin analogs), and ecdysone regulatable promoters (inducible with ecdysone ore related steroids). The chemical agent can be provided to the cells, tissues or animals at a sufficient dose, as a single bolus or by repeated administration, to induce expression from the specific control region. In some embodiments, chemical agents that have minimal or no adverse effects on the eukaryotic cell or animal is used to control gene expression in the bacterial cell (see, e.g., Freundlieb et al., J Gene Med. 1 :4 -12, 1999; U.S. Patent No. 7,807,417).

[0175] In some embodiments, where control regions regulatable with light are used to control gene expression, the gene expression is regulated by illumination with light of sufficient duration, amplitude (intensity) and frequency, depending on the type of light switchable control region used, for example phytochrome, flavin chromophore (e.g., phototropin), or crytochromes (e.g., aureochrome). The light sources include, but are not limited to LED, incandescent, fluorescent, and laser light sources. Exemplary illumination intensities can vary from 0-0.8 W/m², and illumination can vary from 1 s (illumination) every 30 s, 1 s every 60 s, or 1 s every 120 s.

[0176] As discussed in the present disclosure, over the past decades advances in genetics, synthetic biology and microbiology have created a large number of engineered microbes for various bio-industrial, biopharmaceutical, and other commercial applications. A limitation of these systems is that the engineered microbe needs to support the genetics and metabolism for both life (i.e., housekeeping) and the engineered pathways. Artificial endosymbionts provide an alternative strategy because the housekeeping functions can be provided by the host, which allows more resources to be dedicated to the engineered functionality in the artificial endosymbiont. For example, in a medical setting, drug eluting artificial endosymbionts could treat cellular functions underlying numerous diseases; returning insulin production to pancreatic cells, restoring hormone or metabolic deficiencies, etc. In an industrial setting, artificial endosymbionts could increase the yields of numerous biosynthesized materials, biofuels, etc.

[0177] In some embodiments, the artificial endosymbiont, particularly a magneto

endosymbiont, introduces the Yamanaka factors, sox2, klf-4, c-myc, and oct-3/4, to reprogram differentiated cells into induced pluripotent stem (iPS) cells. In some

embodiments, a fibroblast host cell contains a artificial endosymbiont that expresses Hnf4a and Foxal, 2, and 3 to direct differentiation of the fibroblast into a hepatocyte. In some embodiments, a fibroblast host cell contains an artificial endosymbiont that expresses myoD to convert the fibroblast into a myocyte. Other transcription factors for differentiation of cells include those for cardiomyocytes (Gata4, Mefic, Tbx5, and others), and neuronal progenitors (TRA2B, Ascll, SHCBP1). Host cells with artificial endosymbiont expressing these factors can be tested in 1) treatment of cardiac infarcts with cardiomyocytes, 2) use of neuronal progenitors to treat stroke, and 3) implantation of myocytes into skeletal muscle.

[0178] In some embodiments, the artificial endosymbionts can provide carbon, energy (like the endosymbiotically derived mitochondria, hydrogensomes, plastids, mitosomes and mitochondrion-derived organelles) or other metabolites that could be very useful

commercially. For instance, enabling animal cells to derive carbon and/or ATP though the Calvin cycle and photosynthesis could be used in lab grown food stocks. Attempts to transfer chloroplast from plant cells to animal cells have been reported previously (Bonnett et al., "On the mechanism of the uptake of Vaucheria; chloroplasts by carrot protoplasts treated with polyethylene glycol," Planta 13: 229-233, 1976; Giles et al., "Liposome-mediated uptake of chloroplasts by plant protoplasts," In Vitro Cellular & Developmental Biology - Plant 16:581-584, 1980; publications incorporated herein by reference). ATP production using an electron donor other than oxygen could be used to enable various host cells to inhabit new niches, potentially even extraterrestrial. Microorganisms have many ways to produce ATP: phototrophy, chemotrophy, photolithotrophy (examples: cyanobacteria, Chromatiaceae, Chlorobiceae), photoorganotrophy (example Rhodospirillaceae), chemolithotrophy

(examples: hydrogen-oxidizing bacteria, thiobacilli, nitrosomonas, nitrobacter, methanogens, acetogens) and chemoorganotrophy (examples: pseudomonads, bacillus, sulfate reducers, Clostridia, lactic acid bacteria). Other such uses will be apparent in light of the guidance in the present disclosure.

[0179] In some embodiments, the artificial endosymbionts are used to affect epigenetic changes in the host cell. These epigenetic changes can affect various biological processes, such as cellular differentiation, response to environmental agents, genomic imprinting, tumorigenesis, etc. In some embodiments, the epigenetic changes can be effected by expressing in a regulated manner genes and corresponding gene products in the magneto- endosymbiont to induce epigenetic changes in a host cell. In some embodiments, the host cell, for example a stem cell or contains a magneto-endosymbiont that expresses a DNA methylase (e.g., CpG methylase), RNA methylase, DNA demethylase, RNA demethylase, histone acetylase, histone deacetylase, histone methylase, and the like. In some embodiments, the gene is induced to express or expression repressed during a specific stage in development of an organism containing the host cell.

[0180] The invention will be better understood from the experimental details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative, and are not intended to be limiting of the present invention.

EXAMPLES

Example 1. Translocation of Proteins into Host Cells Using a Type I Secretion

System from an Magneto-Endosymbiont

[0181] The type I secretion systems (T1SS) in Gram-negative bacteria can be used to export a variety of proteins of various sizes and diverse functions (their cognate substrates). The

MTB genome encodes T1SS genes (Matsunga et al., "Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. AMB-1," DNA Res.

12:157-166, 2005; incorporated herein by reference in its entirety). [0182] Target protein, green fluorescent protein (GFP), or red fluorescent protein (RFP), is N-terminally fused to C-terminal 200 amino acids of protein describe by NCBI Accession Nos. YP_420640 (RTX toxins and related Ca²⁺ binding protein); YP 423419 (RTX toxins and related Ca²⁺ binding protein); YP_422785 (amb3422); or other MTB T1 SS substrates. Alternatively, the proteins in MTB, such as those described by NCBI Accession Nos.

YP_420502; YP_420631.1 ; YP_420638.1 ; YP_420640.1 ; YP_421364.1 ; YP_422662.1 ; YP_422785.1; and YP 423419.1; may be used for the fusion by taking their C-terminus (the 200 C-terminal amino acids, which contains the secretion signal) to target the recombinant fusion protein to the secretion system. The DNA encoding the fusion of GFP or RFP with 200 C-terminal amino acids of YP_420640 (RTX toxins and related Ca²⁺ binding protein), YP_423419 (RTX toxins and related Ca²⁺ binding protein), YP_422785 (amb3422), or other MTB T1SS substrates is cloned into pBBR-MSC (Kovach et al., "pBBRlMCS: a broad-host- range cloning vector," Biotechniques 16:800-2, 1994; incorporated herein by reference). Translocation of the fusion protein out of the magneto-endosymbiont is detected by fluorescence, or immunofluorescence, and/or immunoblotting.

[0183] The hemolysin (Hly) secretion system of E. coli is one of the best studied type I secretion systems (T1SS). Secretion of the hemolysin A toxin (HlyA) is catalyzed by a membrane protein complex (Bakkes et al., "The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system," J Biol Chem. 285(52): 40573-40580, 2010; incorporated herein by reference) that consists of HlyB, an inner membrane ATP binding cassette transporter (Davidson et al., "ATP-binding cassette transporters in bacteria," Ann. Rev. Biochem. 73:241-268, 2004; incorporated herein by reference); TolC, the outer membrane protein (Koronakis et al., "Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export," Nature 405(6789):914-919, 2000;

incorporated herein by reference); and HlyD, the membrane fusion protein that is anchored to the inner membrane (Johnson et al., "Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps," JAfo/ 5/^'o/. 287(3):695-715, 1999; incorporated by reference). Export of HlyA requires ATP hydrolysis by HlyB (Thanabalu et al., "Substrate-induced assembly of a contiguous channel for protein export from E. coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore," EMBO J. 17(22):6487-6496, 1998; incorporated herein by reference). The last 218 C-terminal amino acids of HlyA have been shown to direct the secretion of a large variety of polypeptides through the TISS (Kenny et al., "Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta- galactosidase fused to the Hly C-terminal signal domain," Mol. Microbiol. 5:2557-2568, 1991; Mackman et al., "Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin," EMBOJ. 6:2835-2841, 1987; Holland et al., "The mechanism of secretion of hemolysin and other polypeptides from gram-negative bacteria," J Bioenerg Biomembr. 22(3):473-491, 1990; all publications incorporated herein by reference).

[0184] In order to translocate a protein, such as GFP or RFP, out of the bacterial cell into a target host cell via the T1SS, the protein, GFP, for example (GenBank: ABG78037.1) is fused to the last 218 C-terminal amino acids of HlyA (HlyAN-term 218, UniProtKB/Swiss- Prot: P09983.1, bolded-underlined sequence):

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY KGNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGN DIYRYLSGYGHHIIDDDGGKDDKLSLADIDFRDVAFRREGNDLIMY KAEGNVLSIGHKNGITFKNWFEKESGDISNHOIEOIFDKDGRVITP DSLKKALEYOOSNNKASYVYGNDALAYGSOGNLNPLINEISKnSA

AGNFDVKEERAAASLLOLSGNASDFSYGRNSITLTASA

For, example, the GFP-HlyAC-term 218 fusion is secreted out of the bacterial cell into the host cell via the HlyB-HlyD-TolC complex. The GFP-HlyAC-term 218 fusion as well as the

HlyB-HlyD-TolC complex is engineered into the pBBRlMCS-2 plasmid and expressed under the control of the tac promoter. Translocation of the GFP-HlyAC-term 218 fusion into target host cells is monitored by fluorescence microscopy.

[0185] Translocation/Secretion assays to demonstrate that proteins are secreted from MTB can use the following procedure. Overnight cultures of MTB strains harboring the appropriate recombinant plasmids are diluted (1 : 10) into fresh MG media supplemented with antibiotics (for culture conditions, see Greene, et al., "Analysis of the CtrA pathway in

Magnetospirillum reveals an ancestral role in motility in alphaproteobacteria," J itoc/.

194:2973-2986, 2012; incorporated by reference). Cells carrying the defined plasmid combinations are grown to an optical density of 400 nm (OD400) of 0.2 before IPTG or arabinose is added to induce the expression of target protein fusions. Proteins in the supernatants are precipitated with 10-20% trichloroacetic acid for 30 min at 4°C. The precipitated proteins are collected by centrifugation and washed in 80% acetone. Cell pellets are washed once in 20 mM Tris (pH 8.0), 1 mM EDTA. Cell pellets and precipitates are resuspended in IX sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and amounts equivalent to 0.2 OD unit are analyzed by SDS-PAGE and immunoblotting. Proteins are stained with Coomassie brilliant blue and/or probed by immunoblotting using specific antibodies. MTB isolates that secrete GFP are introduced to the mammalian cell line MDA-MB231 , using the magnet assisted entry method. Cells with magneto-endosymbionts are obtained, and translocated protein is detected by fluorescence, or immunofluorescence or immunoblotting.

Example 2: Translocation of a Protein into a Host Cell Using a Type IV Secretion

System from a Magneto-Endosymbiont

[0186] CagA is secreted through a T4SS system by engineering a CagA protein by replacing the last 20 amino acids with 24 amino acids (residues 684-709) from the C-terminal end of the RSFIOIO MobA protein. Translocation of CagA-MobA into the host cell is monitored by observation of the hummingbird phenotype caused by CagA in the host cell. The

hummingbird phenotype is "characterized by spreading and elongated growth of the cell, the presence of lamellipodia (thin actin sheets present at the edge of the cell), and filapodia (finger-like protrusions containing a tight bundle of actin filaments; see Segal, et al. "Altered States: Involvement of Phosphorylated Caga in the Induction of Host Cellular Growth Changes by Helicobacter Pylori," Proc Nail Acad Sci USA 96(25): 14559-64, 1999;

incorporated herein by reference).

[0187] The hummingbird phenotype resembles the morphological changes induced by hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF) in epithelial cells (Sugiyama, T., "Development of gastric cancer associated with Helicobacter pylori infection," Cancer Chemother Pharmacol. 54(Suppl. 1):S12-S20, 2004; incorporated herein by reference). Hepatocyte growth factor, also known as scatter factor, evokes a unique morphogenic activity, e.g., induces kidney, or mammary gland-derived epithelial cells, to form branching ducts in three-dimensional collagen gels (Ohmichi, H., et al., "Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development," Development 125: 131 -1324, 1998; incorporated herein by reference).

[0188] Other proteins, including GFP, are fused to the C-terminal residues of MobA (see sequence below) in order to be translocated from Helicobacter pylori with a T4SS into a MDM-MB231 host cell. In the case of GFP, translocation of this protein into the MDM- MB231 host cells are monitored by looking for the presence of GFP fluorescence outside of the magneto-endosymbiont.

[0189] GFP amino acid sequence (non-underlined sequence; GenBank: ABG78037.1) fused to MobA residues 684-709 (bolded-underlined sequence; GenBank: AAA26445.1):

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY KLARAELARAPAPRORGMDRGGPDFSM

[0190] Alternatively, other proteins, including GFP, is fused to the C-terminal 50aa residues of VirD5 in order to be translocated from A. tumifaciens with a T4SS into a MDM-MB231 host cell. In the case of GFP, translocation of this protein into the MDM-MB231 host cell is monitored by looking for the presence of GFP fluorescence.

[0191] GFP amino acid sequence (non-underlined sequence; GenBank: ABG78037.1) fused to VirD5 residues 787-836 (bolded-underlined sequence; NCBI Reference Sequence:

YP_001967549.1):

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY KISALDRTARLISTSPSKARSKAETEKATOELDDRRVYDPRDRAOD KAFKR

Example 3: Translocation of a Nucleic Acid into a Host Cell Using a Type IV

Secretion System from an Magneto-Endosymbiont

[0192] The type 4 secretion system (T4SS) is used to transfer plasmid DNA from a magneto- endosymbiont into a mammalian host cell. Sequence analysis of the MTB genome reveals that components of the T4SS system are present in MTB.

[0193] Plasmid pBBR-MSC is engineered to include (oriT+trwABC) for transfer of the plasmid by a T4SS system. The plasmid is also engineered to contain an expression cassette encoding the target protein under the control of the HCMV IE1 promoter-enhancer-first intron. The target protein is a selectable marker such as, DHFR or glutamate synthetase, or a reporter such as GFP, or a transcription factor such as cMyc.

[0194] A mammalian selectable marker is used as the target gene. These include puromycin N-acetyl-transferase gene for puromycin resistance; blasticidin S deaminase for balstadin S resistance; and aminoglycoside 3 '-phosphotransferase for G418 resistance. If MDA-MB231 is used as a host cell line, 2 ug/ml puromycin, 5 ug/ml blastacidin S, or 1 mg/ml G418 is used for selection.

[0195] Plasmid DNA harboring a DNA fragment of interest along with an antibiotic resistance cassette (the choice of cassette will vary depending on the conditions needed) can be introduced into MTB via conjugation with a mating strain of E. coli that is auxotrophic to diaminopimelic acid (DAP). Successful transfer of the plasmid DNA to MTB will result in growth of MTB on MG agar plates in the presence of antibiotic. The E. coli mating strain auxotrophic to (DAP) will ensure that any growth seen on the MG agar plates + antibiotic will be strictly MTB as no E. coli colonies should survive on plates not containing DAP. MTB colonies harboring the plasmid DNA will be cultured in liquid MG growth medium to be used in subsequent experiments where MTB will be introduced into mammalian cells. MTB with the plasmid are introduced into mammalian cells, such as the human breast cancer cell line MDA-MB231. The plasmid will then integrate into the host cell chromosome via non-homologous recombination. Transfer of plasmid nucleic acid from MTB, the magneto- endosymbiont, to the host cell (MDA-MB231) is detected by selection for host cells that grow in the presence of 2 ug/ml puromycin, 5 ug/ml blastacidin S, or 1 mg/ml G418. Selected cells are grown and then suitable assays are performed to detect chromosomal integration of the plasmid into the host cell genome, such as for example, in situ hybridizations, or Southern hybridization.

Example 4: Magnetic Hyperthermia Control of Magneto-Endosymbionts in Mice

[0196] Mammary carcinoma host cells with magneto-endosymbionts are injected into FVB mice subcutaneously in the flank. Mice are imaged with 7T MRI using a cell tracking pulse sequence to confirm cell cluster location. Surface skin temperature is mapped using a thermal imaging camera, for example a FLIR Systems Infrared Camera, temperature is also mapped internally using MR thermography methods. Peak and average values of temperature as a function of AMF application time, for various field amplitudes and frequencies, are recorded. Engraftment and viability are assessed using luciferase/GFP reporters expressed from the magneto-endosymbionts and/or the host cells. The magneto-endosymbionts are also followed using multiplexed BLI through expression of the bacterial Lux operon that encodes luciferase and the biosynthetic enzymes for the substrate, (see Contag et al., "Photonic detection of bacterial pathogens in living hosts," Mol Microbiol. 18:593-603, 1995; incorporated herein by reference) and survival of the labeled cells through expression of Luc (Contag et al., "Bioluminescent indicators in living animals," Nat Med. 4:245-247, 1998; Contag et al., "In vivo patterns of heme oxygenase-1 transcription," JPerinatol. 21(suppl):Sl 19-S124, 2001; Contag et al., "Advances in in vivo bioluminescence imaging of gene expression," Ann Rev Biomed Eng. 4:235-260, 2002; all publications incorporated herein by reference).

[0197] Mammary carcinoma host cells containing magneto-endosymbionts are embedded in matrigel and transplanted subcutaneously into the flanks of mice. AMF is applied at selected frequencies and amplitude, and for a time period to induce gene expression by raising the temperature. The effectiveness of gene expression is assessed using optical reporter genes. Temperatures are mapped on the skin surface by a thermal imaging camera, and throughout the 3D volume of the mice by MR thermography.

[0198] Mice are transplanted with host cells containing magneto-endosymbionts, as described above. AMF is applied at the desired frequency and amplitude, and for a time period to ablate the host cells containing the magneto-endosymbionts. The ablation of host cells is assessed by histological analysis of postmortem tissue. Temperatures are mapped on the skin surface of the mice by thermal imaging camera, and throughout the 3D volume of the mouse by MR thermography.

Example 5: Tet Activator Expression from Magneto-Endosymbionts

[0199] Magneto-endosymbiont are engineered to express a secreted tet activator (tTA) with a host cell nuclear translocation signal into the cytoplasm of host cells. The mammalian host cell is engineered to contain a tet-regulated control region that expresses a GFP-luciferase reporter gene. The tet-reporter construct is derived from a human ubiquitin C control region (huUbiqC) with seven binding sites for the tTA (tet-70) upstream of GFP fused to click beetle luciferase (CBL). In the presence of the small molecule doxycycline, the tTA will bind to the tTA binding sites and activate expression of the GFP-luciferase reporter.

[0200] In a second embodiment, the tet activator is placed under the control of a heat shock control region such as the Hsp control region. This engineered magneto-endosymbiont is placed inside a host cell and the host cell is subjected to AMF at selected frequencies and amplitude and time period, for example about 100kHz frequency, lOkA/m amplitude, with times ranging from a 2 min to more than 30 min, to induce gene expression by raising the temperature. Localization of the tet activator to the nucleus of the host cell is identified by GFP-luciferase expression induced by the tet activator.

Example 6: Tissue Regeneration using Host Cells with Magneto-Endosymbionts [0201] Magneto-endosymbiont are engineered to express the transcription factors, Hnf4a and Foxal, -2 and -3. These magneto-endosymbionts are placed into mammary carcinoma host cells. Gene expression and translocation to the host nucleus by these transcription factors is observed using adult hepatic markers (e.g., ALB, CX32, CYP1A1, CYP1A2, CYP2B6 and CYP3A4), and liver progenitor markers (e.g., DKK1, DPP4, DSG2, CX43 and K19), capacity to form colonies in vitro, and cellular function (Buyl et al., "Characterization of hepatic markers in human Wharton's Jelly-derived mesenchymal stem cells," Toxicol in Vitro 28:113-1 19, 2013; incorporated herein by reference).

[0202] These hepatocytes derived from fibroblasts are transplanted into livers of normal mice to assess longevity and control of gene expression. These hepatocytes are also transplanted into a murine model of hereditary tyrosinemia type 1 (HTl) (see Vogel et al., "Chronic liver disease in murine hereditary tyrosinemia type I induces resistance to cell death," Hepatology 39:433-443, 2004; incorporated herein by reference). In this model, healthy

fumarylacetoacetate hydrolase deficient mice (Fah-/-) are protected from liver injury by the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-l ,3-cyclohexanedione (NTBC), and the tyrosine metabolite homogentisic acid (HGA) causes rapid hepatocyte death. The Fah-/- mice are available through Jackson Laboratories (stock number 018129), and fine-tuning of the model is possible through the use of the small molecules, NTBC and HGA.

[0203] In some embodiments, magneto-endosymbiont-tagged hepatocytes expressing luciferase are used in a murine model of hereditary tyrosinemia type 1 (HTl). After treatment with homogentisic acid (HGA) to cause rapid hepatocyte death (Vogel et al., Chronic liver disease in murine hereditary tyrosinemia type I induces resistance to cell death, Hepatology 39:433-443, 2004, hereby incorporated by reference), engineered hepatocytes with magneto- endosymbionts are introduced into the mice, and BLI is used to localize the transplanted cells, MRI is used to assess tissue volume and to localize the magneto-endosymbionts, and liver enzyme assays are used as an indicator of restored liver function.

Example 7: Using Magneto-Endosymbionts to Make Induced Pluripotent Stem

Cells

[0204] Magneto-endosymbiont are engineered to express the Yamanaka factors (sox2, klf-4, c-myc, and oct-3/4). The expression constructs for the Yamanaka factors use heat shock inducible control regions, such as bacterial heat shock proteins, or DNAK.

[0205] These engineered magneto-endosymbiont are placed into blood or skin host cells, and the host cells with the magneto-endosymbiont are treated with AMF at selected frequencies and amplitude, and for a time period to induce gene expression by magnetic hyperthermia. Induced pluripotent stem cells are identified in AMF treated cell cultures by their

morphology and cell surface marker profile.

[0206] Magneto-endosymbiont are engineered to be infectious to target cells and can be introduced systemically and target specific cells, including stem cells, in the body.

Example 8: Imaging of Host Cells with AMB-1 in a Mouse

[0207] AMB-1 expressing luciferase ("ME") was introduced into 231 BR cells. These 231 BR cells containing ME were administered by intracranial injection into mice. Mice received 10,000, 1,000, or 100 231 BR cells containing MEs. The mice receiving 10,000, 1,000, or 100 231 BR cells containing MEs were imaged my MRI and a bSSFP sequence was used to acquire images with 150um isotropic resolution, TR TE 10, 8 phase cycles, 1 average, FA 15. The mice receiving single 231 BR cells containing MEs fixed, and a GRE MRI sequence was used with resolution of 80x80x100 urn, slice thickness of 100 Mm, TE 17, TR 35, FA 12, BW 3, NEX 4.

[0208] MRI images of 231 BR cells containing ME that were injected into mice are shown in FIG. 1 A-D. FIG. 1A shows an image from a mouse that received 104 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME. FIG. IB shows an image from a mouse that received 103 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME. FIG. 1C shows an image from a mouse that received 102 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME. FIG. IC demonstrates that as few as 100 231 BR cells with ME can be visualized in vivo in a mouse with MRI.

[0209] 231 BR cells containing ME were administered by ultrasound-guided intracardiac injection into nude mice. These injections resulted in a countable number of sparsely distributed single 231BR cells throughout the brain, with a subset of these proliferating to form macroscopic brain metastases. FIG. ID shows an MRI image with detection of single cells of 231 BR with ME in mouse brain. The arrow indicates the location of single cells of 231 BR with ME.

[0210] Based on this work, we identified an optimal pulse sequence for in vivo single cell ME imaging as a 3D gradient echo sequence with 3D voxel size 80x80xl00um, echo time 17ms, repetition time 35ms, flip angle 12ms, receive bandwidth 3kHz, number of averages 4, and a scan time of ~lh. Example 9: Inducing Magnetic Hyperthermia with SPIOs or Magnelles

[0211] A graph showing induction of hyperthermia in a sample containing magnetite nanoparticles of 25 nm is shown in FIG. 2. The magnetite nanoparticles (25 nm) were purchased from Azano Scientific. FIG. 2 shows an induction of about a 6 oC increase in temperature by placing a sample containing 100 μΐ of magnetite nanoparticles in a

Magnetherm System (Nanotherics) and exposing the sample to an RF field of 25 mT at a frequency of 1 1 1 kHz for 800 seconds

[0212] Magnelles (AMB-1) were placed into 0.5 mL of media to give a concentration of 4 X 1011 Magnelles per mL. These Magnelles were subjected to an RF field of 25 mT at a frequency of 1 11 kHz. The RF field was generated by Magnatherm system from

Nanotherics. After 30 minutes, the temperature rose by 8 oC, and this temperature increase was 4 oC greater than that of a control (media without Magnelles) placed in the 25 mT RF field.

Example 10: Expression of Luciferase in AMB- 1

[0213] LuxCDABE was expressed in AMB-1 and detectable light output was obtained using an extrachromosomal, medium copy, broad-host-range cloning plasmid pBBRlMCS-2. FIG. 3 shows the relative light units (RLU) of lux+AMB-1 in increasing concentration from left to right as follows: 3.91E+05, 7.81E+05, 1.56E+06, 3.13E+06, 6.25E+06, 1.25E+07, 2.50E+07, 5.00E+07, and 1.00E+08 AMB-1 per well. Triplicates at each concentration were assessed across three rows shown. The image was taken using an I VIS Lumina system. FIG. 3 shows that AMB-1 expressed luciferase and the recombinant luciferase produced detectable light.

[0214] All publications, patents and patent applications discussed and cited herein are incorporated herein by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0215] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the scope of the invention(s) of the present disclosure.

Claims

We claim:

1. A method for introducing a phenotype into a host cell, comprising:

subjecting a eukaryotic host cell comprising a artificial endosymbiont, wherein the artificial endosymbiont comprises a regulatable control region operably linked to a nucleic acid encoding a secreted polypeptide, to a treatment that induces expression of the polypeptide, wherein the polypeptide is secreted from the artificial endosymbiont into the host cell to produce the phenotype.

2. The method of claim 1, wherein the artificial endosymbiont comprises a magneto-endosymbiont.

3. The method of claim 2, wherein the magneto-endosymbiont comprises a magnetotactic bacterium.

4. The method of claim 1, wherein the artificial endosymbiont is derived from a nonmagnetic prokaryote.

5. The method of claim 1, wherein the non-magnetic prokaryote is a bacterium selected from E. coli, Salmonella and Listeria.

6. The method of claim 1, wherein the regulatable control region comprises a control region inducible with an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, or infrared radiation, and the treatment is with an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, or infrared radiation, respectively.

7. The method of claim 1, wherein the regulatable control region comprises a heat shock control region, and the treatment results in hyperthermia that induces expression from the heat shock control region.

8. The method of claim 7, wherein the treatment inducing hyperthermia is selected from an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, and infrared radiation.

9. The method of claim 8, wherein the ultrasound radiation is focused ultrasound.

10. The method of claim 1, wherein the regulatable control region comprises a control region inducible with a chemical agent, and the treatment comprises a chemical agent that induces expression from the control region.

1 1. The method of claim 1, further comprising a step of culturing the eukaryotic host cell comprising the artificial endosymbiont.

12. The method of claim 1, wherein the encoded polypeptide comprises a selectable marker.

13. The method of claim 1, wherein the encoded polypeptide comprises a reporter protein.

14. The method of claim 14, wherein the reporter protein is a fluorescent protein.

15. The method of claim 1, wherein the encoded polypeptide comprises a Yamanaka factor.

16. The method of claim 15, wherein the Yamanaka factor is selected from Oct 4, Oct 3, Sox 2, Klf 4, c-Myc, NANOG, Lin 28 protein, and combinations thereof.

17. The method of claim 1, wherein the eukaryotic host cell comprises a pluripotent cell.

18. The method of claim 17, wherein the encoded polypeptide induces the pluripotent cell to differentiate.

19. The method of claim 18, wherein the encoded polypeptide comprises a polypeptide selected from Hnf4a, Foxal, Foxa2 protein, Foxa3 protein, and combinations thereof.

20. The method of any one of claims 1 to 19, wherein the eukaryotic host cell is present in an intact animal.

21. The method of claim 20, wherein the eukaryotic host cell is present in an intact organ or tissue of a living animal or human.

22. A method for introducing a phenotype into a host cell present in an organ or tissue of a living animal,

(a) administering into an animal an artificial endosymbiont which enters into specific cell types of the animal, wherein the artificial endosymbiont comprises a regulatable control region operably linked to a heterologous nucleic acid encoding a secreted polypeptide; and (b) subjecting the animal to a treatment that induces expression of the polypeptide, wherein the polypeptide is secreted from the artificial endosymbiont into the host cell to produce the phenotype.

23. The method of claim 22, wherein the artificial endosymbiont comprises a magneto-endosymbiont.

24. The method of claim 22, wherein the magneto-endosymbiont comprises a magnetotactic bacterium.

25. The method of claim 22, wherein the artificial endosymbiont is derived from a nonmagnetic prokaryote.

26. The method of claim 25, wherein the artificial endosymbiont is derived from a nonmagnetic bacterium selected from E. coli, Salmonella, and Listeria.

27. The method of claim 22, wherein the regulatable control region comprises a control region inducible with an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, or infrared radiation, and the treatment is with an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, or infrared radiation, respectively.

28. The method of claim 22, wherein the regulatable control region comprises a heat shock control region, and the treatment results in hyperthermia that induces expression from the heat shock control region.

29. The method of claim 28, wherein the treatment inducing hyperthermia is selected from an alternating magnetic field, ultrasound radiation, laser illumination, radio frequency radiation, microwave radiation, and infrared radiation.

30. The method of claim 29, wherein the ultrasound radiation is focused ultrasound.

31. The method of claim 22, wherein the regulatable control region comprises a control region inducible with a chemical agent, and the treatment is with a chemical agent that induces expression from the control region.

32. The method of claim 22, wherein the encoded polypeptide comprises a selectable marker.

33. The method of claim 22, wherein the encoded polypeptide comprises a reporter protein.

34. The method of claim 33, wherein the reporter protein is a fluorescent protein.

35. The method of claim 22, wherein the encoded polypeptide comprises a Yamanaka factor.

36. The method of claim 35, wherein the Yamanaka factor is selected from Oct 4, Oct 3, Sox 2, Klf 4, c-Myc, NANOG, Lin 28 protein, and combinations thereof.

37. The method of claim 22, wherein the eukaryotic host cell comprises a pluripotent cell.

38. The method of claim 37, wherein the encoded polypeptide induces the pluripotent cell to differentiate.

39. The method of claim 38, wherein the encoded polypeptide comprises a polypeptide selected from Hnf4a, Foxal, Foxa2 protein, Foxa3 protein, and combinations thereof.