OA20578A - Probiotic delivery of guided antimicrobial peptides. - Google Patents

Abstract

The present disclosure pertains to a treatment strategy to combat select bacteria in the gut, such as H. pylori. The strategy uses a probiotic-based system to express and deliver a guided antimicrobial peptide to the gut. The guided antimicrobial peptide is expressed from a hybrid gene that codes for an antimicrobial peptide fused to a guide peptide, the latter binding to a protein of the target bacterium. This technology can eliminate the target bacterium selectively and specifically from the gut microbiota. The specificity of the targeting, being at the strain, species or genus level, depends on the sequence of the guide peptide used to provide the targeting. The treatment can be administered orally, such as by using an ingestible probiotic.

Description

PROBIOTIC DELIVERY OE GUIDED ANTIMICROBIAL PEPTIDES

BACKGROUND

[0001] The present disclosure relates to a means of eliminating a spécifie gut bacterial species, such as Hélicobacter pylori, without altering the microbiome.

[0002] The microbiota of the gut affects human health in many ways. The gut microbiome contains 100+ trillion bacteria and is largely involved in mediating the host's immune response while also performing other essential functions including the extraction of nutrients and energy from food. The bacterial makeup of the gut prédisposés humans to health issues ranging from obesity to cancer to psychoiogical disorders. Disruption to the microbiome (dysbiosis) results în an imbalance in the types and number of bacteria thaï comprise a person’s normal, protective microflora. There are a number of factors that lead to dysbiosis including ingestion of pathogenic bacteria and antibiotic-mediated or immunosuppressive mediated déplétion of the microbiome. Dysbiosis has been linked to numerous human diseases including both intestinal as well as extra-intestinal disorders. The literature indicates dysbiosis in the pathogenesis of IBS, inflammatory bowel disease, and colorectal cancer as well as allergies, cardiovascular disease, and mental illness. Additionally, gut microbiota hâve been implicated as precursor for autoimmune diseases given that severity and/or incidence of disease has been shown to be reduced in germ-free animal models.

[0003] In other cases, changes to gut bacteria resuit from ingestion of a dangerous pathogen that can produce an intestinal disease. There are few, if any, reported means to effectively knock out a spécifie bacterial species which is causing problcms in the gut, either as an active pathogen or as a player in the microbiome that prédisposés humans to various disorders.

[0004] Hélicobacter pylori is a gut bacterium that is the primary cause of peptîc ulcers and gastric cancer. Gastric cancer causes the third most fatalities worldwide among cancers and is especially common in the Far East (Bahkti étal., 2020). Only 1 in 5 patients survive gastric cancer 5 years after diagnosis. H. pylori is recognized by the International Agency for Research on Cancer as a Group 1 carcinogen. Il is estimated that 4.4 billion people are infected with H. pylori, with developing countries having the highest infection rates (70% prevalence in Africa) (Hooi et al., 2017). In lhe United States, H. pylori occurs twice as frequently in the ηοπ-white population as in the white population (Everhart et al.,

2000) and is associated with lower socio-economic status worldwide.

[0005] No commercial vaccine exists against H. pylori. Though some progress has been seen in lowered H. pylori prevalence in some countries using antibiotic treatment, large increases in antibiotic résistance rates are now being seen in H. pylori isolâtes. The prevalence of clarithromycin-resistance in H. pylori rose from 11% to 60% in just 4 years (2005-2009) in Korea, with similar increases recorded in China and Japan (Thung et al., 2016). Though the standard treatment is in fact a triple antibiotic therapy, antibiotic résistance rates continue to rise. Thus, it is difficult to see a path forward with H. pylori treatment via antibiotics. Other bacteria offer similar challenges.

SUMMARY

[0006] The présent disclosure pcrtaîns to a treatment strategy to combat select bacteria in the gut, such as H. pylori. The strategy uses a probiotic-based System for the expression and delivery of a guided antimicrobial peptide to the gut. The guided antimicrobial peptide is expressed from a hybrid gene in the probiotic bacterium's DNA, and can be the sequence coding for an antimicrobial peptide fused to the sequence coding for a guide peptide, with the latter peptide responsible for binding to a protein of the target bacterium. The fusing can occur with or without a linker sequence, that is, independent of the presence of a linker sequence. This technology can eliminate the target bacterium selectively and specifically from the gut microbiota. The specificity of the targeting, being al the strain, species or genus level, dépends on the guide protein used to provide the targeting. The treatment can be admînistered orally, such as by using an ingestible probiotic.

[0007] Preferred embodiments described herein relate to a method for the control of a target bacterium such as H. pylori which does not in volve antibiotics. For delivery of the active protein, this method uses engîneered probiotic bacteria. Preferred embodiments utilize lactic acid bacteria, including Lactococcus and Lactobacillus species, such as Lactococcus lactis and Lactobacillus acidophilus, which are food grade bacterium thaï are safe for human consumptîon or hâve been granted GRAS status (Generally Regarded As Safe) by the FDA and are in widespread commercial use for processing dairy food products. Probiotics constitute a well-established technology which is inexpensive, highly scalable, and very successful commercially. These commercial traits make this technology especialiy amenable to large-scale application, particularly in developing countries.

[0008] The probiotic bacterium can be formulated as a recognizable food product that is commonly found in the probiotics market, such as dried yoghurt peilets, which can be stored without réfrigération for months. In this format, the product may be taken by travelers to foreign countries or by long-term expatriâtes or soldiers with food, perhaps twice per week, as a preventative (“prophylactic”) to disease. The treatment could also serve as a therapy, being eaten after the patient is sick.

[0009] The présent technology is important and advantageous because it utîlizes guided antimicrobial peptides that eliminate only the target bacterium while leaving ail the other members of the microbîal community undistuibed. The use of probiotic bacteria that are ingested and remain active in the digestive system in order to secrete the guided recombinant antimicrobial peptide directly în the gut of the patient is also significantly different from previous technologies.

BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1 shows the pE-SUMOstar vector carrying AMP for expression in E. coli BL21 cells. SUMO protease site is between SUMO and A12C-AMP.

[0011] FIG. 2 shows expression of SUMO/AMP in E. coli and cleavage of AMP free of SUMO fusion partner.

[0012] FIG. 3 shows log values for minimum inhibitory concentrations (MIC) in μΜ for non-targeted and targeled analogues of eurocin and plectasin against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aurais and Staphylococcus epidermidis.

[0013] FIG. 4 shows the cell-kinetic profile for B. subtilis, S. epidermidis, S. aureus and E. faecalis (clockwise), created by plotting log CFU/ml of the bacteria grown in the presence of each peptide.

[0014] FIG. 5 shows biofilm inhibition activity evaluated by plotting the absorbance of crystal violet (540 nm) against the concentration of 4 AMPs on the 4 bacteria - B. subtilis, S. epidermidis, S. aureus and E. faecalis.

[0015] FIG. 6 shows resuits of a PCR analysis of slomach reverse gavage extracts demonstrating the presence of Lactococcus lactis harboring the cmpty vector, the vector with antimicrobial peptide, and the the vector with antiinicrobial peptide with the guide peptide from multimerin in the stomachs of mice three days after ingestion.

[0016] FIG. 7 shows a vector for transformation aï Lactococcus lactis in accordance with preferred embodiments described herein.

[0017] FIG. 8 shows the viability of E. coli in the presence of different antibiotic dilutions and supernatants of broth cultures of Lactococcus lactis secreting antimicrobial peptide with or without a guide peptide.

[0018] FIG. 9 shows an exemplary vector for Lactococcus lactis sécrétion of AMPs and gAMPs.

[0019] FIG. 10 shows resuits of qPCR on VacA gene, showing élimination of H. pylori by co-culturing in vitro with L. lactis expressing gAMPs or AMPs.

[0020] FIG. 11 shows growth of Lactobacillus plantarum after 24 hours coculturing with L. lactis expressing empty vector (pTKR), AMPs (alyteserin, laterosporulin, or CRAMP), or gAMPs (MMl-alyteserin, MM 1-laterosporulin, or MM 1-CRAMP).

[0021] FIG. 12 shows growth of Esherichia coli after 24 hours co-culturing with L. lactis expressing empty vector (pTKR), AMPs (alyteserin, laterosporulin, or CRAMP), or gAMPs (MM1-alyteserin, MMl-laterosporulin, or MM1-CRAMP).

[0022] FIG. 13 shows a standard curve for CFU/μΙ of H. pylori culture with qPCR Ct values.

[0023] FIG. 14 shows a therapeutic test, with the CFU/μΙ of H. pylori vs days after inoculation, in mice treated with Lactococcus lactis probiotic secreting AMPs or gAMPs on Day 5 after inoculation with H. pylori.

[0024] FIG. 15 shows a prophylactic test, with the CFU/μΙ of H. pylori in mouse stomach fluid for control mice (Null) and mice inoculated with empty vector (pTKR) or Lactococcus lactis probiotic secreting AMPs or gAMPs (where MM1 = Multimerin 1 guide peptide), before inoculation with H. pylori on Day 4.

[0025] FIG. 16 shows the différences in taxonomie diversity for mouse stomach bacterial populations with four different treatments without the presence of H pylori: Antibiotic treatment, L. lactis probiotic with empty vector, buffer mock inoculation, probiotic expressing AMP, probiotic expressing gAMP.

[0026] FIG. 17 shows différences in relative abundance of four bacterial indicator species under different treatments; Staphylococcus and Acinetobacter are associated with dysbiosis while Lactobacillus and Muribacter are associated with microbiota health; Day 0 is before any treatment; Day 5 is after 5 days of H. pylori infection; Days 8 and 10 are 3 and 5 days, respectively, after various therapeutic treatments (probiotics with either empty vector or expressing AMP or gAMP).

[0027] FIG. 18 shows taxonomie différences (distance) in sequencing data for bacterial species found in mouse stomach in four treatment groups, Empty (probiotic carrying only an empty vector), Null (mock inoculation with buffer), Targeted (probiotic expressing gAMP), and Non-targeted (probiotic expressing AMP), compared to Empty.

[0028] FIG. 19 shows taxonomie différences (distance) in sequencing data for bacterial species found in mouse stomach in four treatment groups, Empty (probiotic carrying only an empty vector), Null (mock inoculation with buffer), Targeted (probiotic expressing gAMP), and Non-targeted (probiotic expressing AMP), compared to Null.

[0029] FIG. 20 shows cumulative taxonomie différences (Shannon entropy) accruing over five days in sequencing data for bacterial species found in mouse stomach after four different treatments: Empty (probiotic carrying only an empty vector), Null (mock inoculation with buffer), Targeted (probiotic expressing gAMP), and Non-targeted (probiotic expressing AMP).

DETAILED DESCRIPTION O F PREFERRED EMBODIMENTS

[0030] The présent disclosure relates to a means for targeting and eliminating a target bacterium using a probiotic that expresses and sécrétés a protein that kîlls the disruptive bacterium without harming other bacteria.

[0031] In preferred embodiments, the présent technology pertaîns to a probiotic bacterium that has been transformed to include a DNA construct for a guidcd antimicrobial peptide. In preferred embodiments, the probiotic bacterium is a bacterium that is safe for human consumption, such as Lactococcus lactis. The sequence coding for the guided antimicrobial peptide includes the sequence coding for a targeting (guide) peptide fused to the sequence coding for an antimicrobial peptide and expressed by the probiotic bacterium as a hybrid protein. The guide peptide îs spécifie for the target bacterium and limits the action of the antimicrobial peptide to that particular bacterium.

[0032] Accordingly, preferred embodiments described herein relate to a probiotic for the prévention or treatment of a condition caused by a target bacterium living in the gastrointestinal tract of a subject, comprising a probiotic bacterium. The probiotic bacterium îs preferably a lactîc acid bacterium, such as a Lactococcus bacterium, and preferably Lactococcus lactis. The probiotic bacterium has been transformed to comprise a DNA construct expressing a guided antimicrobial peptide, wherein the sequence coding for the guided antimicrobial peptide comprises the sequence coding for an antimicrobial peptide fused to the sequence coding for a guide peptide that binds to a protein of the target bacterium. The protein of the target bacterium may be a virulence factor. In preferred embodiments, the target bacterium is//. pylori and the virulence factor îs VacA. The guide peptide may be multimerin-1. The guided antimicrobial peptide kills the target bacterium in the gastrointestinal tract of the subject. The guided antimicrobial peptide also minimally disrupts other bacteria found in the gastrointestinal tract of the subject when compared to unguidcd antimicrobial peptides, antibiotics, or other broad spectrum treatments.

[0033] As used herein, “minimally disrupts” means the guided antimicrobial peptide does not cause a disruption that would cause a health effect, as opposed to a technical change în bacterial abondance. “Minimally disrupts” also means the guided antimicrobial peptide does not significantly disrupt other ηθη-target bacteria, where the disruption would cause a health effect.

[0034] Preferred embodiments relate to a probiolic system which delivers antimicrobial peptides (AMPs) to the gut. Antimicrobial peptides are natural products produced by plants, animais and fungi to protect against bacterial infection (Ngyuen et al., 2011). However, an AMP by itself has broad spectrum activity, similar to an antibiotic. The broad activity of antibiotics has been well-documented to lead to microbiota dysbiosis. Many publications hâve demonstrated connections between antibiotic-induced dysbiosis p and rheumatoid arthritis, inflammalory bowel disease, diabètes, obesity and other disorders (for a review, see Keeney et al., 2014). This is one of the conséquences of the overuse of antibiotics and nonselective AMPs share the same weakness. Exemplary AMPs used în preferred embodiments described herein include laterosporulin, alyteserin, and cathelin5 related anti-microbial peptide (CRAMP).

[0035] To solve this problem of dysbîosis, the preferred embodiments described herein include a guide peptide fused to an AMP, produced from a corresponding guideAMP hybrid gene of the probiotic baclerium. This enables the resulting gui de d AMP (gAMP) to bind specifically to the targeted bacterium such as H. pylori, leaving the 10 commensal bacteria of the gut largely undisturbed. In this way, a probiotic expressing gAMP will multiply in the stomach and sélectively kill the target pathogen, H. pylori without the health issues associated with antibiotics and other broad-spectrum treatments. Other targeted bacterium can be treated similarly, and H. pylori is used herein as an example.

[0036] In preferred embodiments, the specificity of the guide peptide described 15 herein is based on the natural specificity of a bacterial virulence factor and the host receptor to which it binds. VacA is a virulence factor protein produced by ail isolâtes of H. pylori (Fitchen étal., 2005). It is secreled but also adhères to the surface of H. pylori cells (Fitchen et al., 2005). VacA natural 1 y binds to the human receptor protein, multimerin-l. Preferred embodiments described herein utilize the VacA-binding sequence (aa 321-340) of the multimerin-l protein (Satoh et al., 2013) to serve as the guide peptide for the gAMPs. In this way, these gAMPs will be localîzed to the surface of the H. pylori via binding to VacA and the AMP portion can then act to destabilize the bacterial membrane and specifically kill the H. pylori cell.

[0037] The probiotic gAMPs described in preferred embodiments are distinct from 25 similar technologies. They possess a seiectivity not found în antibiotics and unguided AMPs. The use of probiotics makes it possible to produce probiotic gAMPs much more cheaply than gAMP proteins purified from a heterologous expression System or synthesized chemically. This combination of seiectivity and low-cost scalability is essential for any replacement for cheap and abundant antibiotics to be successful commercially and therefore 30 reach the intended patients.

P [0038] Preferred embodiments disclosed herein relate to an edible Lactococcus lactis probiotic bacterium, wherein lhe probiotic baclerium has been transformed to comprise a DNA construct expressing a guided antimicrobial peptide, wherein the sequence coding for the guided antimicrobial peptide comprises the sequence coding for an 5 antimicrobial peptide fused to the sequence coding for a guide peptide that binds to the VacA peptide of H. pylori, produced from the corresponding hybrid gene of the L. lactis bacterium, wherein the antimicrobial peptide is laterosporulin, aiyteserin, or cathelin-related anli-microbial peptide, and wherein the guided antimicrobial peptide kills H. pylori in the gastrointestinal tract of the patient without causing a significant disruptive effect on other 10 bacterial species. In other words, lhe probiotic bacterium expressing the guided antimicrobial peptide will not disrupt the taxonomie balance of the slomach microbiota and will not cause long-term damage.

[0039] Additional preferred embodiments relate to a method for treating a disease or condition associated with H. pylori by administering an edible probiotic to a subject, 15 where the edible probiotic is ingested and remains active in the subject’s gut long enough to secte te a guided antimicrobial peptide that kills H. pylori.

[0040] In another aspect of the présent invention there is provided a probiotic composition including a therapeutically effective amount of a transformed probiotic lactis bacterium expressing a guided antimicrobial peptide and an acceptable excipient, adjuvant, 20 carrier, buffer or stabiliser. A “therapeutically effective amount” is to be underslood as an amount of an exemplary probiotic that is sufficient to show inhibitory effects on H. pylori. The actual amount, rate and time-course of administration will dépend on the nature and severity of the condition or disease being treated. Prescription of treatmenl is within the responsibilîty of general practitioners and other medical doctors. The acceptable excipient, 25 adjuvant, carrier, buffer or stabiliser should be non-toxic and should not interfère with the efficacy of the secreted antimicrobial protein. The précisé nature of the carrier or other material will dépend on lhe route of administration, which is preferably oral.

[0041] The L. lactis bacteria useful în the disclosed probiotic composition may be provided as a live culture, as a dormant material or a combination thereof. Those skilled in 30 the art will appreciate thaï lhe L. lactis bacteria may be rendered dormant by, for example, a lyophilization process, as îs well known to those skilled in the art.

[0042] An example of an appropriate lyophilization process may begîn with a media carrying appropriate L. lactis bacteria to which an appropriate protectant may be added l'or cell protection prior to lyophilization. Examples of appropriate protectants include, but are not limited to, distilled water, polyethylene glycol, sucrose, trehalose, skim milk, xylose, 5 hemicellulose, pectin, amylose, amylopectin, xylan, arabinogalactan, starch (e.g., potato starch or rice starch) and polyvinylpyrrolidone. Gasses useful for the lyophilization process include but are not limited to nitrogen and carbon dioxide.

[0043] In one aspect, the L. lactis bacteria in the disclosed probiotic composition may be provided as a dispersion in a solution or media. In another aspect, the L.

lactis bacteria in the disclosed probiotic may be provided as a semi-solid or cake. In another aspect, the L. lactis bacteria in the disclosed probiotic may be provided in powdered form.

[0044] Quantities of appropriate/.. lactis bacteria may be generated using a fermentation process. For example, a stérile, anaérobie fermentor may be charged with media, such as glucose, polysaccharides, oligosaccharides, mono- and disaccharides, yeast 15 extract, protein/nîtrogen sources, macronutrients and trace nutrients (vitamins and minerais), and cultures of the desired L. lactis bacteria may be added to the media. During fermentation, concentration (colony forming units per gram), purity, safety and lack of contaminants may be monitored to ensure a quality end resuit. After fermentation, the L. lactis bacteria cells may be separated from the media using various well known techniques, 20 such as filtering, centrifuging and the like. The separated cells may be dried by, for example, lyophilization, spray drying, heat drying or combinations thereof, with protective solutions/media added as needed.

[0045] The probiotic compositions may be prepared in various forms, such as capsules, suppositories, tablets, food/drink and the like. The probiolic compositions may 25 include various pharmaceuticaliy acceptable excipients, such as microcrystalline cellulose, mannitol, glucose, defatted milk powder, polyvinylpyrrolidone, starch and combinations thereof.

[0046] The probiotic composition may be prepared as a capsule. The capsule (i.e., the carrier) may be a hoilow, generally cylindrical capsule formed from various substances, 30 such as gelatin, cellulose, carbohydrate or the like. The capsule may receive the probiotic bacteria therein. Optionally, and in addition to the appropriate probiotic bacteria, the capsule may include but is not limited to coloring, flavoring, rice or other starch, glycerin, caramel color and/or titanium dioxide.

[0047] The probiotic composition may be prepared as a suppository. The suppository may include but is not limited to the appropriate probiotic bacteria and one or more carriers, such as polyethylene glycol, acacia, acetylated monoglycerîdes, carnuba wax, cellulose acetate phthalale, corn starch, dibutyl phthalate, docusate sodium, gelatin, glycerin, iron oxides, kaolin, lactose, magnésium stéarate, methyl paraben, pharmaceutical glaze, povidone, propyl paraben, sodium benzoate, sorbitan monoleate, sucrose talc, titanium dioxide, white wax and coloring agents.

[0048] The probiotic composition may be prepared as a tablet. The tablet may include the appropriate probiotic bacteria and one or more tableting agents (i.e., carriers), such as dibasic calcium phosphate, stearic acid, croscarmellose, silica, cellulose and cellulose coating. The tablets may be formed using a direct compression process, though those skilled in the art will appreciate that various techniques may be used to form the tablets. A capsule may also be used to contaîn the composition.

[0049] The probiotic composition may be formed as food or drink or, alternativeiy, as an additive to food or drink, wherein an appropriate quantily of probiotic bacteria is added to the food or drink to render the food or drink the carrier.

[0050] The concentration of probiotic bacteria in the probiotic composition may vary depending upon the desired resuit, the type of bacteria used, the form and method of administration, among other things. For example, a probiotic composition may be prepared having a count of probiotic bacteria in the préparation of no less than about lxl0⁶colony forming units (CFUs) per gram, based upon the total weight of the préparation.

[0051] When lactic acid bacteria are used as gut expression vehicles, various dairy products, such as youghurt, youghurt pellets, or other milk products may be used as the physîcal carrier for oral administration, with or without the above mentioned adjuvants or carriers.

[0052] In another aspect, there is provided the use in the manufacture of a médicament of a therapeutically effective amount of a probiotic as defined above for administration to a subject.

[0053] The term “therapeutically effective amount” means a nontoxic but sufficient amount of the probiotic to provide the desired therapeutic effecl. The amount that is “effective” will vary from subject to subject, dependîng on the âge and general condition of the individual, the particular concentration and composition being administered, and the like. Thus, it is not always possible to specîfy an exact effective amount. However, an appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine expérimentation. Furthermore, the effective amount is the concentration that is within a range sufficient to permit ready application of the formulation so as to deiiver an amount of the drug that is within a therapeutically effective range.

[0054] The probiotic in its final form is expected to hâve a very low production cost and be highly scalable. In addition, it should hâve a long shelf life and not require réfrigération. A physicîan's prescription may not be requîred. Thus, the market is expected to be unusually wide. The probiotic is expected to provide sophistîcated control at a very low price.

[0055] The probiotic compositions described herein can be used to prevent or treat H. pylori infections, or diseases or disorders caused by H. pylori, in humans and animais. The probiotic compositions may be administered as a prophylactic, prior to an exposure or challenge with H. pylori. The probiotic compositions may be administered therapeutically, after an infection with H. pylori has occurred. The probiotic compositions may be încorporated into animal feed or animal drinking water.

EXAMPLE 1

[0056] Engineered proteins that specifically kill certain pathogenic bacteria without harming unrelated commensal bacteria hâve been developed. The specificity of killing is due to a targeling (guide) peptide attached to an antimicrobial peptide as expressed from a hybrid gene. In the present example the skin pathogen, Staphylococcus aureits, was targeted using purified guided antimicrobial protein produced from an E. coli expression system. However, the targeling system can be modified to specifically kill any bacterium.

[0057] In this exampie, two commonly used antimicrobial peptides (AMPs), plectasin and eurocin, were genetically fused to the targeting peptide A12C, which selectîvely binds to Staphylococcus species. Il should be noted that A12C peptide was developed using a generic bîopanning technique; in theory, any bacterium can be targeted using this method for producîng guide proteins. A12C was developed by another laboratory to serve as a guide protein for vesicles, which also illustrâtes that peptides developed for other purposes can be repurposed to serve as guide proteins for antimicrobial peptides. The targeting peptide did not decrease activity against the largeted Staphylococcus aureus and Staphylococcus epidermidis, but drastically decreased activity against the non-targeted species, Enterococcus faecalis and Bacillus subtilis. This effect was equaily évident across two different AMPs, two different species of Staphylococcus, two different négative control bacleria, and against bîofilm and planktonic forms of the bacteria.

[0058] Methods:

[0059] Reagents. The pE-SUMOstar vector (LifeSensors) was grown in 10-β and BL21 E. coli (New En gland Biolabs) and AMP was released from expressed fusion/AMP using Ulpl protease produced in house. The AMPs plectasin (GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY (SEQ ID NO:1); MW 4408) and eurocin (GFGCPGDAYQCSEHCRALGGGRTGGYCAGPWYLGHPTCTCSF (SEQ ID NO:2); MW 4345) were expressed from pE58 SUMOstar as were A12C-plectasin (MW 6137) and A12C-eurocin (MW 6074), both of which had the A12C targeting peptide (underlined) plus a short linker (GVHMVAGPGREPTGGGHM) (SEQ ID NO:3) genetically fused to the Nterminus of the respective AMP sequences. As a control, plectasin and eurocin were also conjugated with the AgrDl bacterial pheromone sequence (YSTCYFIM)(SEQ ID NO:4) (Mao et al. 2013) at the N- terminus. Synthetic A12C peptide (Biosynthesis) was used as a “target peptide only” control. FIG. 1 shows the pE-SUMOstar vector carrying AMP for expression în£. coli BL21 cells. SUMO protease site is between SUMO and A12C-AMP.

[0060] Expression, Purification and Analysis of Fusion Proteins. The DNA sequences for the AMPs were synthesized (Integrated DNA Technologies) and ligated into the pE66 SUMOstar vector and cloned into E. coli 10-beta cells. Plasmid from these were used to transform E. coli BL21 cells for protein expression. Transformed cultures were grown out and induced with IPTG according to standard procedures. The resulting bacterial pellets were resuspended in PBS/25 mM imidazole/0.1 mg/ml lysozyme and frozen overnight. The cells were then thawed, sonicated, and ultracentrifuged at 80,000 x g for 1 h at 4°C and the 6his/SUMO/AMP fusion protein in the supernatant was purified by nickel column chromatography. The AMP was separated from SUMO by proteolysis using Ulpl (1U per 100 ug fusion protein) at 4°C overnight and the cleavage was evaluated by SDSPAGE. Yields were calculated from the SDS-PAGE data, using NIH ImageJ to measure band density and the marker lane bands for mass reference. Mass spectrometry was used to ensure the proper cleavage of the AMP from the SUMO carrier protein. In-gel tryptic digest (Thermo Fisher) was performed on the AMP excised from the SDS-PAGE gel. The digest was examined by LC-ESI-MS (Synapt G2-S, Waters) at the Baylor University Mass Spectrometry Center. The analysis of the MS data was done by MassLynx (v4.1) The spcctra of each protein, both non-targeted and targeted, were peak centered and MaxEntS processed and then matched against hypolhetical peaks from peptides generated by simulated trypsin digestion of the respective proteins.

[0061] Hemolytic Activity Assay. Guided AMPs, non-guided AMPs and synthetic A12C peptide were assessed for human hemolytic activity via exposure to washed human érythrocytes. Whole blood cells were collected a healthy volunteer using standard procedures (Evans et al. 2013) and cells were diluted in phosphate buffered saline to 5x108 cells/ml. To initiale hemolysis, 190 μΐ of the cells was added to 20 μΐ of a 2-fold serially diluted peptide/ test reagent in phosphate buffered saline. Wells without peptide were used as négative Controls, while wells containing 1% 85 Triton X-100 were used as positive Controls.

[0062] In Vitro Bactericidal Activity Assay. The Ulp-1 protease-cleaved proteins were tested for antimicrobial assays against four strains of bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalîs and Bacillus subtilis. These four species were selected because they are gram positive and the AMPs plectasin and eurocin are specifically active against gram positive bacteria (Mygind et al. 2005, Oeemig et al. 2012). The component Controls were free SUMO protein and synthetically produced A12C peptide. Vancomycin was used as the positive control. The standard protocol for a microtiter plate assay with serial dilution was used în which serial 2-fold dilutions of test peptide were made across a 96-well plate containing unîform bacterial inoculum across the peptide dilutions.

After bacterial growth in the presence of peptide, cell viability was assayed with resazurin.

Expenments with ail peptides against ail bacterial species were performed with >5 repheates each.

[0063| In Vitro cell kinetics study. Ulp-1 protease-cleaved peptides were assayed to détermine their dynamic action against the bacteria in a growing culture. The bacteria were grown at 37°C with shaking and diluted to ~lx!08 CFU/mL To these cultures were added plectasin or eurocin, at 3x the respective minimum inhibitory concentrations, or the A12C-targeted versions at these same respective concentrations. The vancomycin control concentration was the mean of the molar concentration of plectasin and eurocin used. Growth was then monitored from 2-10 h after addition of the peptides, diluting 10 pl of culture in medium and plating onto Mueller-Hinton agar plates. The number of colonies was recorded the next day.

[0064] In Vitro biofilm inhibition assay. In addition to planktonic cultures, biofilm cultures were used to assay inhibition by the peptides, using standard procedures (O’Toole 2011). Briefly, overnight cultures were diluted 1:100 and added to serially diluted peptides. Biofilms were aliowed to grow for 24-36 h of unshaken culture. The liquid was removed and the biofilms were washed, dried and fixed with methanol and then slained with Crystal Violet, which was later dissolved with 30% acetic acid and the resulting solution measured for absorbance at 540 nm to quantify the amount of biofilm formed. Ail assays were run in triplicate or greater.

[0065] Results:

[0066] Protein Expression and Purification. AMP/SUMO fusion proteins, with or without the A12C targeting domain, were highly expressed in E. coli BL21 cells. These were successfully cleaved with SUMO protease (Ulp-1) into their component AMP and SUMO carrier protein and were clearly visualized with SDS-PAGE as 4-6 kDa free AMP and -17 kDa SUMO/AMP fusion proteins. FIG. 2 shows expression of SUMO/AMP in E. coli and cleavage of AMP free of SUMO fusion partner, where Lane 1: free SUMO control and Lanes 2-9: Intact fusion proteins (even lanes) and cleaved products (odd lanes) in the following order: SUMO/plectasin, SUMO/A12C-plectasin, SUMO/eurocin, SUMO/A12Ceurocin. Arrows: free AMP The average yields (n>=3) of the proteins plectasin, A12Cplectasîn, eurocin and A12C-eurocin were 15-26 mg (3-4 pmoles) per L of culture. For peptide confirmation, peptides were extracted from the SDS-PAGE gel bands, digested by trypsin and analyzed by mass spectrometry. Peptide identifies were confirmed using the MassLynx (v4.1) application (Waters).

[0067] Hemolytic Activity Assay. In concordance with previously pubtished individual studies on plectasin and eurocin (Mygind et al. 2005, Oeemig et al. 2012, Yacoby et al. 2006), both guidcd and un-guided fusion peptides, along with the free A12C peptide control, displayed no hemolytic effect on human érythrocytes in comparison to a 20% Triton-X positive control (data not shown).

[0068] In Vitro Bactericidal Activity Assay. Differential toxicity against off target bacteria was observed with the A12C targeting peptide added to the AMPs. A12C-AMPs retained their toxicity against both of the targeted staphylococci bacterial species but showed a dramatic decrease in toxicity against the off target bacterial species relative to unmodified AMPs. FIG. 3 shows log values for minimum inhibitory concentrations (MIC) în μΜ for non-targeted and targeted analogues of eurocin and plectasin against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. The boxed régions represent 50% of the values while the bars represent 95%. Unmodified plectasin and eurocin had the expected mean MIC values of 3-6 μΜ, which are typical values for AMPs with sequential tri-disulfide bonds produced in E. coli expression Systems (Li et al. 2010, Parachin et al. 2012, Li et al. 2017). In contrast, the addition of the A12C guide peptide rendered these AMPs essentially noninhibitory to the off target bacteria, with MIC values >70 μΜ. In ail cases, the MIC values for A12C/AMP versus AMP were significantly different for both of the off target bacteria, E. faecalis and B. subtilis (p<0.001; ANOVA 2139 tailed test). Négative Controls (SUMO alone and A12C alone) showed no antimicrobiai activity (data not shown) and these were run for ail experiments.

[0069] In Vitro cell kinetics study. Growth kinetics over an 8 to 10 hour period more conclusively demonstrated the loss of antimicrobiai activity of the A12C/AMP against the off target bacterial species. For these bacteria, A12C/AMP treatment resulted in bacterial growth that lagged only slightiy behind buffer control treated cultures. FIG. 4 shows the cell-kinetic profile for B. subtilis, S. epidermidis, S. aureus and E. faecalis (clockwîse), created by plotting log CFU/ml of the bacteria grown in the presence of each peptide for 810 hours collected in 2-3 hour intervals. Unmodified AMPs were bactericidal similar to the vancomycin control. In contrast, ali peptides - both guided and unguided - demonstrated a strong bactericidal effect against the target bacteria S. epidermidis and 5. aureus, similar to the vancomycin positive control. The relatively flatter growth curve for the B. subtilis control cultures reflects its growth kinetics, which is far slower than that of other bacteria.

[00701 In Vitro biofilm inhibition assay. Growing bacterial cultures with the peptides demonstrated the preferential inhibition of bacterial biofilm of lhe Staphylococciis strains by the targeted AMPs over the non-Staphylococcus bacteria. FIG. 5 shows biofilm inhibition activity evaluated by plotting the absorbance of crystal violet (540 nm) against the concentration of 4 AMPs on the 4 bacteria - B. subtilis, S. epidermidis, S. aureus and E.

faecalis (clockwise). (* = p<0.1, ** = p<0.05, n>=3). The absorption reading (hence, the quantity of biofilm formed) decreased with the increase in peptide concentration for ail the 4 bacteria when treated with unguided peptides but the guided peptides did not hâve similar effects on B. subtilis and E. faecalis with significant (p <0.10 or p<0.05) différence in the absorbance values between targeted and non-targeted AMPs at concentrations beyond 6.25 15 μΜ.

[0071] This example demonstrates successful targeting of the AMPs plectasin and eurocin against two staphylococcal bacteria. Importantly, this was achieved by essentially eliminating the activity against the two off target bacteria tested. This is the expected outcome for an antimicrobial therapy that préserves the commensal members of the 20 microbiome while killing the pathogenic target bacteria. This is also the outcome that was achieved against S. aureus by Mao et al. (2013) with the use of a bacterial pheromone peptide for targeting of plectasin. Other than a lower MIC for the unmodified plectasin itself, the same drastic degree of réduction in the activity against the off target bacteria, E. faecalis and B. subtilis was seen, as was reported by Mao et al. (2013). Th us, it is demonstrated that 25 a biopannîng-derived ligand works as efficiently as a pheromone-derived ligand, which is the class of targeting peptide used in ail targeted AMPs to date. It should be noted that the pheromone-derived ligand was more spécifie than A12C, wîth activity against 5. aureus but not S. epidermis, while A12C/plectasîn was highly active against both species.

[0072] Four main sources of ligands exist for use as guide peptides for AMPs. First, 30 bacterial pheromones are species-specific peptide signais which trigger the development of compétence, virulence, or other capabilities, and pheromone peptides hâve been determîned for many pathogenic bacteria (Monnet et al. 2016). Second, biopannîng is a means of screening random lîbraries of peptides for the ability to bind to a target sequence, such as a receptor on a bacterîal cell. Usually, a bactériophage is used to display the members of the peptide library (Wu et al. 2016). Third, bactériophage receptor binding proteins can be used as a resource for the development of targeting peptides for AMPs. The receptor binding proteins of phages against many pathogenic bacteria hâve already been characterized (Dowah and Clokie 2018, Nobrega et al. 2018). In addition, screens for new phages against lesser studied bacterîal pathogens can be carried out (Weber-Dqbrowska et al. 2016). Fourth, virulence factors of the targeted bacterîal pathogen can be targeted by using targeting (guide) peptides consisting of the sequence of the host receptor that îs bound by the bacterîal virulence factor. In this way, the host receptor sequence is used as a guide peptide to direct an AMP back to the bacterîal pathogen. This is demonstrated in the experiments of this patent application.

EXAMPLE 2

[0073] An exemplary probiotic bacterium, Lactococcus lactis, has been shown to survive well in the stomach of mice. Mice were force fed the recombinant probiotic by oral gavage and recombinant bacterîal DNA was recovered from the stomachs of the mice a full 3 days after introduction. In FIG. 6, it îs seen that Lactococcus lactis harboring the pTlbinl expression vector with the open reading frames of eîther the antimicrobial peptide laterosporulin (AMP1) or with the antimicrobial peptide alyssaserin (AMP2) or with laterosporulin genetically fused to the guide peptide open reading frame derived from multimerin (targeted AMP1) were ail présent 3 days after the introduction of these bacteria to the mice by oral gavage, as evidenced by PCR (using vector-specific primers) of the stomach reverse gavage extracls. This indicates that recombinant Lactococcus lactis was thrîving in the stomachs of the mice. Force feeding (oral gavage) was used to ensure that a consistent amount of bacterium was delivered to each mouse. Reverse oral gavage was used to flush mouse stomach with buffer and collect the stomach contents for PCR analysis. In FIG. 6, the Positive Control was PCR of the pTlbinl/laterosporulin DNA and the Négative Control was PCR of no template DNA, with the same vector-specific primers used in both of these control PCRs as was used for the PCRs for the mouse extracts in the other lanes. The last lane of FIG. 6 is a marker lane with a DNA ladder. Ail positive bands comprised DNA of the expected size.

[0074] A vector has also been developed that greatly facilitâtes Lactococcus lactis engineering. To creale this vector (shown in FIG. 7), the original Lactococcus lactis vector, pTINX, was modified by the addition of and E. coli origin of réplication and a kanamycin résistance cassette, both from the SUMO-basedE. coli expression vector, pE-SUMOstar. In FIG. 7, the kanamycin résistance block represents both the kanR cassette and the E. coli origin of réplication. This bînary vector (pTlbinl) can be grown ii\E. coli to facilitate the addition of AMP or guide sequence inserts by recombinanl DNA techniques. Generous quantities of plasmîd can be produced via standard plasmid préparation techniques in order to ease the transformation of Lactococcus lactis. This iatter transformation îs diffîcult to achieve with ligation products, but is easier with DNA from plasmid préparations.

[0075] It has been demonstrated în vitro that engineered Lactococcus lactis secreting antimicrobial peptide kilis other bacteria în vitro. This is reported in FIG. 8 as the survival of E. coli in the presence of broth culture of Lactococcus lactis secreting antimicrobial peptide with or without a guide peptide. FIG. 8 shows the viability of E. coli in the presence of different antibiotic dilutions and supernatants. It should be noted that the legend is in reverse order of the lines, top to bottom, with the upper line in the graph being the buffer control and the lower line being vancomycin. To obtain the results shown in FIG. 8, cultures of Lactococcus lactis contaîning either the empty pTlbinl vector, pTlbinl harboring the antimicrobial peptide laterosporulin, or pTlbinl harboring laterosporulin genetically fused to the guide peptide from multimerin were centrifuged to remove bacterial cells and the resulting supernatants were added to separate starter cultures of E. coli to check for inhibition of E. coli growth. The starter culture used supplying ail replicates consisted of 500 μΐ of overnight culture of E. coli diluted in 50 ml of LB broth. Three replicates of each treatment were conducted and each point in the graph represents an average with correspoding error bars. To run the treatments and replicates, a 96-well microtiter plate was used. For each well, 100 μΙ of diluted Lactococcus lactis supernatant was added to 100 μΐ of E. coli starter culture. As seen in the x-axis of FIG. 8, the dilutions used ranged from no dilution (100 μ] of 100% supernatant added to the 100 μΐ of E. coli) down to 1/200 dilution of supernatant (100 μΐ of 0.5% supernatant added). Antibiotic positive Controls were diluted similarly, with the starting concentrations (undîluted) stated in the legend. The y-axis of FIG. 8 represents the inhibition of E. coli viability by these supernatant and antibîotic dilutions. E. coli viability was measured by platîng onto LB agar plates the cultures in each well after 4 hours of exposure to supernatant or antibiotic. The resulting colonies appearing on the plates were recorded, with the undiluted buffer control treatment being sel to 100% and ail other treatments being convertcd to a fraction of this value, as plotted on the y-axis.

[0076] Looking at FIG. 8, it can be seen that the buffer control did not inhibit E. coli. VÏQ'xeveï, Lactococcus lactis broth culture (with cells removed) did inhibit E. coli even with no recombinant antimicrobial peptide present (empty vector control). This is considered the baseline for examinïng the effect of the secreted recombinant proteins. The expression of laterosporulin by Lactococcus lactis resulted in a sîgnificant decrease in viability of E. coli compared to this baseline. However, there was no sîgnificant différence seen between the empty vector baseline and the multimerin-guîded (targeled) lactosporulin. This means that the guide peptide completely abolished antimicrobial activity of laterosporulin against the nontarget bacterium E. coli. This is in agreement with results shown in Example 1 with Staphylococcus. This data supports the ability of these extracts to kill différent target bacterium, such as Hélicobacter pylori.

EXAMPLE 3

[0077] In vitro control of Hélicobacter pylori by Lactococcus lactis expressîng gAMPs.

[0078] Purpose

[0079] This example demonstrates that antimicrobial peptide (AMP) fused to the multimerin-derived guide peptide spécifie for Hélicobacter pylori, expressed from a hybrid gene and secreted from the probîotic Lactococcus lactis, can specifically kill H. pylori when the probiotic is co-cultîvated with H. pylori în vitro. This was an in vitro proof of principle before conducting the in vivo studies in mice.

[0080] Experimental Design

[0081] In the co-cultures, different dilutions of L. lactis were used but each well had 10 μΐ of H. pylori culture (-3000 CFUs). The L. lactis secreted AMP, gAMP or contained an empty expression vector. Alyteserin and CRAMP were the AMPs tested. These were constructed either genetically fused to the multimerin-derived guide peptide (guide AMP or gAMP) or not (AMP). The amount of H. pylori present in the co-culture at any given time point was measured by qPCR, using primers spécifie for the VacA gene itself, which codes for the receptor protein to which the gAMP bmds. The entire expenment was run in tnplicate and the growth of H. pylori after 24 h in the presence of L. lactis expressing various AMPs or gAMPs is shown in FIG. 10.

[0082] Methods

[0083] Genetic Constructs

[0084] FIG. 9 shows the vector for Lactococcus lactis sécrétion of AMPs and gAMPs. The ORFs of the AMPs, codon-optimized for Lactococcus lactis, were cloned into the modified pTINX-kanR (pTKR) vector for L. lactis expression/secretion in between the restriction enzyme sites BamHI and Spel by replacing the spaX protein of the original plasmid. The PI promoter upstream of the BamHI cut-sîte Controls the downstream expression as a constitutive promoter which is upreguiated by low p/L The usp45 gene immedîately upstream of BamHI site codes for an endogenous signal peptide of L. lactis that allows sécrétion of the resulting fusion peptide. After ligation of the AMP/tAMP into pTKR vector, it was transformed into E. coli (10β, NEB) and plated onto kanamycin sélective plate. The pTINX plasmid (LMBP 3498) has erythromycin résistance but was modified to create pTKR as shown în FIG. 9, which also has kanamycin résistance for clonîng into electrocompetent E. coli (10β, NEB) for plasmid propagation. Extracted plasmid from the E. coli was then electroporated into electrocompetent L. lactis MG 1363 (LMBP 3019) and plated on erythromycin sélective GM17 plates (30° C, microaerobic, overnight). After screening for the presence of the AMP/gAMP ORFs with PCR, selected colonies were propagated in liquid cultures of M17 broth with glucose (0.5% w/v) in the presence of erythromycin (5 pg/ml).

[0085] AMP/gAMPs used in this experiment

[0086] The following AMPs and guided AMPs (gAMPs) were cloned into the sécrétion vector pTKR. The multimerinl (MM1) guide peptide sequence MQKMTDQVNYQAMKLTLLQK (SEQ ID NO:5) is underlined and the serine/glycine linker sequence is in boid.

AMP/ gAMP Peptide Sequence

Laterosporulin MM1-	ACQCPDAISGWTHTDYQCHGLENKMYRHVYA1CMNGTQVYCRTEWGSSC (SEQID NO:6) MQKMTDQVNYQAMKLTLLQKSGGGSACQCPDAISGWTHTDYQCHGLENK
LaterosporulIn Alyteserin MM1-	MYRHVYAICMNGTQVYCRTEWGSSC (SEQ ID NO:7) GLKDIFKAGLGSLVKGIAAHVAN (SEQID NO:8) MQKMTDQVNYQAMKLTLLQKSGGGSGLKDIFKAGLGSLVKGIAAHVAN
Alyteserin	(SEQIDNO:9)
CRAMP MM1-CRAMP	ISRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE (SEQ ID NQ:10) MQKMTDQVNYQAMKLTLLQKSGGGSISRLAGLIRKGGEKIGEKLKKIGQKIK
	NFFQKLVPQPE (SEQ ID NO:11)

Underline = Multimerinl, Bold = linker

[0087] S L. lactis/H. pylori co-culture and qPCR analysis

[0088] L. lactis AMP/gAMP clones were propagated from glycerol stocks and grown in GM 17 broth overnight with erythromycin (5 pg/ml) with no shaking. H. pylori 5 stocks were first propagated on Blood-TS agar overnight with microaerobic condition and >5% CO2 environment. Then colonies from the plate were transferred to a TS broth with newborn calf sérum (5%) and grown overnight under microaerobic condition and >5% CO2 environment. The L. lactis cultures were seriatly diluted în a 96-well culture plate with TSB broth to make up a volume of 100 pL. To each well, 10 pL of the overnight H. pylori culture 10 was added and each well volume was brought up to 200 pL with more TS broth. The plate was left to grow overnight in a microaerobic environment with >5% CO2. After 24 h, well contents from the culture plate were transferred to a 96-well PCR plate. That PCR plate was sealed and heated for 15 min at 100° C and chilled at 4° C for 5 min. Then the plate was centrifuged at 2000 g for 2 min and lhe supernatant was used as the template for qPCR. The 15 qPCR was done using primers for VacA gene to quantify H. pylori (forward: 5'ATGGAAATACAACAAACACAC-3' (SEQ ID NO:12), reverse: 5'CTGCTTGAATGCGCCAAAC-3' (SEQ ID NO: 13) and primers for acma gene for quantifyîng L. lactis. Standard curves for H. pylori and L. lactis were constructed by determining Ct values for different dilutions of the overnight cultures of the respective bacteria (1/10,1/100,1/1000,1/10000) in the qPCR plates, the CFUs for the dilutions were determined by plating on their respective agar plates.

[0089] Results

[0090] FIG. 10 shows the results of qPCR on VacA gene of H. pylori co-cultured with L. lactis expressing gAMPs or AMPs. L. lactis expressing AMPs with or without guide peptides knocked down the H. pylori culture to below the baseline of détection for this experiment (Ct value of 40). Plain AMPs are represented with open symbols while gAMPs are represented with solid gray symbols. Alyteserin was not very effective unless fused to the guide peptide. The control experiment (solid line), with L. lactis carrying the empty vector, showed that the L. lactis probiotic, by itself, had little to no influence on the growth oiH. pylori over 24 hours. Error bars represent 95% confidence limits.

[0091] Conclusions

[0092] L. lactis expressing two different AMPs was able to knock down, to baseline levels, a vigorous H. pylori culture in vitro. The multimerin guide peptide sequence was shown to not interféré with AMP toxicity in CRAMP, with targeted and untargeted CRAMP cqually toxic to H. pylori. In ail cases, the gAMP (“MM1” prefix) was more toxic (lower on y-axis) than the corresponding AMP. In the case of the alyteserin AMP/gAMP pair, the guide peptide appeared to be a requirement for high toxicity to H. pylori.

EXAMPLE 4

[0093] Effect on off-target bacteria of probiotic gAMPs,

[0094] Purpose

[0095] To détermine the effect of L. lactis probiotic expressing AMP or gAMP on off-target bacteria in vitro.

[0096] Experimental Design.

[0097] The experimental design was identical to that described above in Example 3, with the exception of the off-target bacterium replacing the targeted H. pylori. The off-target bacteria used were Lactobacillus plantarum (gram positive) and Escherichia colt (grain négative).

[0098] Methods

[0099] Ail methods are described above in Exampie 3.

[0100] Results

[0101] FIG. 11 shows growth of Lactobacillus plantarum after 24 hours coculturing wîthE. lactis expressing empty vector (pTKR), AMPs (alyteserin, laterosporulin, or CRAMP), or gAMPs (MM 1-alyteserin, MMl-laterosporulin, or MM1-CRAMP). Compared to the probiotic only control (Lactococcus lactis with empty vector pTKR), cocultivation of Lactobacillus plantarum with probiotic expressing either AMP or gAMP led to a réduction in off-target titer with increasing amounts of probiotic deployed. However, there was significantly more négative effect on off-target growth by probiotic/AMP treatment than with probiotic/gAMP treatment for ail three AMPs tested. Specifically, at the 100,000/μΙ CFU level which was was maximally efficacious for H. pylori kill in Example 3, ail probiotic/AMP treatments led to Lactobacillus levels undetectably low by qPCR. In contrast, probiotic/gAMP levels were at 10,000 CFU/μΙ for alyteserin and laterosporulin gAMPs and 2500 for CRAMP gAMP. At lower probiotic levels, a 5 to 7-fotd differential occurred between gAMP and AMP probiotic treatment, with probiotic/gAMPs significantly less deleterious to off-target Lactobacillus than probiotic/AMPs. Error bars represent 95% confidence limits.

[0102] FIG. 12 shows growth of Escherichia coli after 24 hours co-culturing with L. lactis expressing empty vector (pTKR), AMPs (alyteserin, laterosporulin, or CRAMP), or gAMPs (MMl-alyteserin, MMl-laterosporulin, or MM1-CRAMP). As seen in FIG. 12, results were similar for off-target effects against E. coli as they were for L. plantarum described above.

[0103] Conclusions

[0104] It can be concluded from these in vitro off-target results that gAMPs are significantly less deleterious to these two off-target bacterial examples than AMPs in a probiotic delivery system. These in vitro results show at least a portion of the picture of off target effects of probiotic AMPs and gAMPs. As discussed more below, averaged across the entire mouse stomach microbiota, the probiotic gAMPs hâve no more disruptive effect than unengineered Lactococcus lactis probiotic.

EXAMPLE 5

[0105] Therapeutic control of H. pylori in mice.

[0106] Purpose

[0107] The control of H. pylori by Lactococcus lactis expressing gAMPs was tested in vivo in mouse. A therapeutic test is more stringent than a prophylactic test since the pathogen is given time to establish and replicate in lhe mouse before the probiotic is introduced. This most stringent test was chosen to evaluate the effect of different AMPs, testing three different AMPs, in guided and unmodified forms.

[0108] Experimental design

[0109] Probiotic control mice. These mice received only the probiotic, prepared as described in the previous example. Stomach samples were collected on Day 0 before inoculation with reverse-oral gavage; resuspended L. lactis were fed to the mice by oral gavage; stomach samples were taken on Days 3, 5 and 7.

[0110] Therapy treatment of H. pyton-infected mice. These mice were inoculated with H. pylori and the H. pylori was allowed to establish itself in the mouse stomach for 3 days, with daily inoculations to ensure establishment. The mice were then given L. lactis secreting AMP or gAMP to therapeutically treat the H. pylori infection. Stomach samples were collected on Day 0 before H. pylori inoculation; resuspended H. pylori were fed b y oral gavage once daily for 3 consecutive days; stomach samples were then collected on Day 5 to test for H. pylori presence and on Day 5 resuspended L. lactis were fed to the mice; subséquent stomach samples were collected on Day 8 and 10.

[0111] Untreated H. pylori infection control mice. These mice were infected with H. pylori and no prophylatîc or therapy was provided. Stomach samples were collected on Day 0 before H. pylori inoculation; resuspended H. pylori were fed by oral gavage once daily for 3 consecutive days; stomach samples were then collected on Day 5, 8 and 10 to test for H. pylori presence.

[0112] Methods

[0113] Admînistering L. lactis and H. pylori in mice by oral gavage. The L. lactis cultures were propagated overnight as described above. The overnight cultures were spun down at 4000 g for 15 min at 4o C. The pellets were resuspended in stérile PBS. H. pylori stocks were grown overnight on Blood-TS agar as described above and then scraped by a stérile loop and resuspended in stérile PBS. The either bacterîal suspension were fed to the mîce using 1.5 ga oral gavage needle not exceeding half their stomach volume (-250 pL). The colony forming units (CFUs) of the resuspension being fed were determined by diluting the resuspension 1/1000 and 1/10000 times and plating on appropriate plates. Prc- and postinoculation samples from the mouse stomach were collected by flushing their stomach with excess PBS (-300 pL) and the stomach fluid was collected by reversing the oral gavage injection until the vacuum was maintained.

[0114] Assay for H. pylori and L. lactis presence by qPCR. The stomach samples collected were heated at 100ο C for 15 min and chilled at 4o C for 5 min. The supernatants were collected and plated in a 96-well plate and qPCR was performed with primers for the VacA gene to quantify for H. pylori and primers for the acma gene to quantify for L. lactis. Standard curves for each bacterium against their CT values were constructed by including different dilutions of the overnight cultures of the respective bacteria (1/10, 1/100,1/1000, 1/10000) in the qPCR and plating those dilutions on respective plates to détermine the corresponding CFU values. Each data point represents at least 3 replicale mice.

[0115] qPCR value standardization. The standard curve for CFU/ pl of H. pylori culture with CT values îs shown în FIG. 13. This was used to generate CFU/pl data from qPCR CT values in FIG. 14.

[0116] Resuits

[0117] Before inoculation with H. pylori, at Day 0, mice had very low levels of native H. pylori with the VacA gene (8-200 CFU/pl) (Figure 14). At 5 days after inoculation with H. pylori, 2,000-12,000 CFU/ul of H. pylori was recorded, indicating strong réplication in the mouse stomach. At Day 5, mice were inoculated with probiotics, except for the Null control. H. pylori continued replicating well m the Null control mice, increasing 3-fold after Day 5 and reaching 40,000 CFU/μΙ at Day 10. After probiotic therapy treatment at Day 5, the pTKR (empty vector) probiotic control increased 2-fold to Day 10. In the mice used for pTKR treatment, the H. pylori inoculation was not as effective and thus the H. pylori titer was lower at Day 5 than the other mouse groups, even before probiotic treatment.

[0118] In contrast, ali mice given probiotics expressing AMP or gAMP experîenced a strong décliné in stomach H. pylori after probiotic therapy delivered at Day 5 (Figure 14). This décliné was between 15-fold and 320-fold depending on the AMP or gAMP treatment, which led to final H. pylori levels 100 to 1000-fold less than the Null control, which received no probiotic therapy and had continued H. pylori growth after Day 5. Furthermore, within each of the three AMP/gAMP pairs, the AMP treatment was significantly less effective at controlling H. pylori than the gAMP treatment. Specifically, at Day 10, for alyteserin and CRAMP, there was 15-fold more H. pylori with the AMP versus the gAMP, while for iaterosporulin, there was 2.5-foid more H. pylori for the AMP versus the gAMP. Error bars represent 95% confidence limits.

[0119] FIG. 14 shows the CFU/μΙ of H. pylori in mouse stomach fluid for control mice (Null) and mice inoculated with empty vector (pTKR) or Lactococcus lactis probiotic secreting AMPs or gAMPs (where MM1 = Muitimerinl guide peptide).

[0120] Conclusions

[0121] The expression of AMP or gAMP in the probiotic L. lactis led to significant réduction (15 to 320-fold) in H. pylori liters in mouse stomach previously inoculated with H. pylori. Thus, probiotic L. lactis engineered to express AMP or gAMP can be expected to serve as a strong therapeutic treatment for H. pylori. Furthermore, a significant distinction can be drawn between AMP and gAMP effector proteins, and this differentîal holds up for ail three AMPs tested. gAMPs were 2.5, 15, and 15-fold more effective at eliminating H. pylori than AMPs for Iaterosporulin, alyteserin, and CRAMP, respectively. Thus, gAMP technology is functionally superior in killing efficacy to AMP technology when delivered via probiotics for application against H. pylori.

EXAMPLE 6

[0122] Prophylactic control of H. pylori in mice.

[0123] Purpose

[0124] Mice were inoculated with the probiotic, L. lactis, secreting gAMP or AMP as a prophylactic treatment in order to prevent the establishment of H. pylori after challenge inoculation 3 days later. Though any medical application of probiotic gAMP technology would be expected to be therapeutic rather than prophylactic, and though this is a less slringent test of effectiveness than the therapeutic test, this experiment was run for completeness.

[0125] Experimental design

[0126] Probiotic control mice. These mice received only the probiotic, prepared as in the examples above. Stomach samples were collected on Day 0 before inoculation with reverse-oral gavage; resuspended L. lactis were fed to the mîcc by oral gavage; stomach samples were taken on Days 3, 5 and 7.

[0127] H. pylori challenge to probiotic prophylactic treatment of mice. These mice received a probiotic expressing AMP or gAMP and then were challenged 3 days later with H. pylori. Stomach samples were collected on Day 0 before L. lactis inoculation; resuspended L. lactis were fed by oral gavage on the same day; stomach samples were then collected on Day 3 to test for L. lactis presence and on Day 3 resuspended H. pylori were fed to the mice once daily for 3 consecutive days; subséquent stomach samples were collected on Day 8 and 10.

[0128] Untreated H. pylori infection control mice. These mice were infected with H. pylori and no prophylatic or therapy was provided. Stomach samples were collected on Day 0 before H. pylori inoculation; resuspended H. pylori were fed by oral gavage once daily for 3 consecutive days; stomach samples were then collected on Day 5, 8 and 10 to test for H. pylori presence.

[0129] Results

[0130] FIG. 15 shows the CFU/μΙ of H. pylori in mouse stomach fluid for control mice (Nul!) and mice inoculated with empty vector (pTKR) or Lactococcus lactis probiotic secreting AMPs or gAMPs (where MM1 = Multimerinl guide peptide), followed by additional feeding of H. pylori. Ail mice started with 170-300 CFU/μΙ of native H. pylori at Day 0, before L. lactis inoculation. By Day 4, native H. pylori had increased to 500-700 CFU/μΙ just before exogenous/ί pylori challenge. By Day 12, H. pylori had increased only to 1500 (gAMP) and 2000 (AMP) CFU/μΙ in the probiotic prophylactic mice treated with MMl-Alyteserin (gAMP) or Alyteserin (AMP). In contrast, H. pylori increased to 13,000 and 18,000 CFU/μΙ in mice given a prophylactic pre-treatment with empty vector (pTKR) or no prophylactic probiotic, respectively. Error bars represenl 95% confidence limits.

[0131] Conclusions

[0132] Probiotics engineered to deliver AMP or gAMP both provided strong prophylactic protection against H. pylori challenge. H. pylori increased only 2-foid in 7 days after H. pylori challenge with probiotic/AMP or probiotic/gAMP pre-treatment. In contrast, with empty vector probiotic, H. pylori increased 26-fold in 7 days. This demonstrates that prophylactic treatment is very effective against H. pylori infection.

EXAMPLE 7

[0133] Microbiome sequence analysis demonstrates only slight disruption to the mouse stomach microbiota

[0134] Purpose

[0135] The stomach microbial communities of mice from the prophylactic and therapeutic experiments were examined by next génération sequencing. The effect of these treatinents on the microbial diversity in the stomach will be determined. It was expected that, due to the sélective toxicily of gAMPs, the microbiota of the probiotic/gAMP-treated mice will be more diverse than that of the probiotic/AMP-treated mice.

[0136] Background

[0137] H. pylori has been found to cause dysbiosîs of the gut microbiota in humans (Liou et al., 2019). In humans, it has been found that gut microbial diversity decreases with increasing H. pylori infection while the éradication of H. pylori is often associated with an increase in microbial diversity (Li et al., 2017). However, antibiotic treatment, in general, is associated with a decrease both taxonomically and in terms of bacterial abundance in the gut (Lange et al., 2016). In this study, mice treated with H. pylori were given a varîety of therapeutic treatments at Day 5 and then compared. In this way, a comparison of the effecl of H. pylori infection on taxonomy versus infection treated with probiotic alone, probiotic/AMP, probiotic/gAMP, or antibiotics was possible.

[0138] Experimental Design

[0139] The experiments for the therapeutic and prophylactic studies generated mouse reverse-oral gavage sampies that were used for qPCR in Examples 5 (therapy) and 6 (prophylactic) above. These same sampies were analyzed for population shifts in the stomach microbiota using next génération sequencing. Hence, the experimental design is identical to Examples 5 and 6.

[0140] Methods

[0141] As described for Examples 5 and 6, the mouse stomach sampies collected by reverse oral gavage were heated at 100°C for 15 min and chilled ai 4°C for 5 min. The supernatants were collected and plated in 96 well plate for upslream processing for Next G en sequencing. The sampies were amplified with 16s primers and then with Illumina index primers with subséquent clean-up and purification. The sampies were pooled into a library and sequenced using Illumina MÎSeq v3 kit. The data was demultiplexed, denoised and analyzed using QIIME2.

[0142] Results

[0143] Effects of therapeutics on stomach total bacterial diversity: Raréfaction estimâtes. Raréfaction curves derived from Illumina MiSeq next génération sequencing were used to estimate total bacterial abundance. These represent the number of species (operational taxonomie units, OTUs) that were detected withing different portions of the data set. The different portions of the data set are randomly chosen subsamples. Raréfaction curves were used to détermine the minimum number of sampies that can be used while still w representing the entire range of OTUs in order to reduce computer load in calculations. For our purposes, this standard graphie reveals the species diversity from each treatment.

[0144] Strikîng différences ΐπ bacterial diversity were observed in data from Day 8 and 10 of the therapeutic study detailed in Example 5 (FIG. 16). In this study, H. pylori 5 infection h ad developed for 5 day s by Day 5. On Day 5, the therapy was administered. By Day s 8 or 10, the therapy h ad 3 or 5 day s to affect the stomach microbiota, respectively. As shown in FIG. 16, the use of the combination antibiotic tetracycline/amoxicilin resulted in the lowest species diversity. Importantly, the use of AMPs delivered by the probiotic (data from ail three AMPs represented here) led to less diversity compared to the use of probiotic 10 with the empty vector or no therapy, but more diversity compared to the use of antibiotics. The maximal diversity resulted from treatment with the probiotic expressing gAMP (data from ail three AMPs represented in FIG. 16).

[0145] The differential seen in the in vitro experiments of Example 2 in terms of off-target effects was likely seen at a broad scale in this in vivo data. With reduced off-target 15 effects, the expression of gAMP by probiotics led to a broader range of bacterial species suiviving compared to AMPs. It is likely that lower diversity seen with probiotic/empty vector or no therapy was due to their ineffectiveness in killing H. pylori, which has been shown to reduce bacterial diversity in previous studies (Lange et al., 2016). Even though AMPs and antibiotics are able to killif. pylori, their own broad scale toxicity was seen here 20 to decrease bacterial diversity.

[0146] Effects of therapeutics on indicator species

[0147] There are only a few publications identifying mouse stomach bacteria as bénéficiai to the gut microbiota. Muribacter mûris (syn. Actinobacter mûris) is a common mouse commensal bacterium and has been used as a niche replacement for the successful 25 élimination of the pathogen Haemophilus influenzae in mice resulting in lowered inflammation (Granland et al., 2020). Lactobacillus murinus, a prédominant mouse gut commensal bacterium, has been shown to reduce gut inflammation (Pan et al., 2018). Lactobacillus reuteri has been shown to stop autoimmunity in mouse gut (He et al., 2017) and has been used to protect mice against enterotoxigenic E. coli infection (Wang et al., 30 2018) and also has been shown to hâve anti-inflammatory effects in humans in many studies (Mu et al., 2018). Since ail of these species were found to predominate in our next

P génération sequencing results we used them as indicator species for a healthy gut microbiota.

[0148] In order to pick microbiota dysbiosis indicators, the two bacterial généra among the top 10 most abundant bacteria that spiked duringH. pylori infection were chosen, 5 namely, Staphylococcus and Acinetobacter.

[0149] The abundances of these bacteria, as determined from next génération sequencing data, are shown in FIG. 17 for various time points and treatments. It can be seen that the bénéficiai indicators, Lactobacittus and Muribacter, both decreased in response to H. pylori infection, but rebounded to levels greater than pre-infection levels after treatment 10 with probiotic/gAMP. This rebound effect was greater than seen with probiotic/AMP. For the dysbiosis indicators, both Staphylococcus and Acinetobacter increased greatly in abundance in response to H. pylori infection, but were greatly reduced in response to probiotic expressing either gAMP or AMP.

[0150] It is difficult to analyze the thousands of bacteria detected by next génération 15 sequencing in the mouse stomach in these therapeutic experiments. Furthermore, it is difficult to examine such single-species data given the paucity of published information concerning the benefit or détriment of single bacterial taxa on mouse stomach microbiota populational heaith. However, these four bacteria do hâve significance to mouse stomach microbiota heaith and the effect of probiotics expressing gAMPs on these four indicator 20 species supports our general hypothesis of the bénéficiai effects of probiotic/gAMP treatment. Probiotic/gAMP treatment increased the abundance of the known bénéficiai indicator species and decreased the most abundant dysbiosis indicator species following therapy of H. pylori infection.

[0151] Effects of treatments on noninfected mice over time

[0152] The effects of various therapeutic treatments over time on noninfected mice is an important question to ask. Any therapy or prophyiactic treatment should hâve as minimal négative impact on the native gut flora as possible. As a standard treatment baseline, it has been shown that antibiotics hâve a devastating effect on gut microbial diversîty (Lange et al., 2016). Both the infection by H. pylori and the therapeutic élimination 30 of H. pylori are expected to be négative and positive confounding factors, respectively, in tenns of diversity évaluation (Liou et al., 2019; Ei et al,, 2017). Thus, the proper experimental design would not include H. pylori. For this reason, the effects of the various the therapeutic treatments on uninfected mice were compared.

10153] The mouse stomach microbiota consists of thousands of species of bacteria. In order to depict changes in number in each of these species that occur before and after treatment, it is necessary to use certain statistical indices. The following indices indicate that gAMP treatment causes far less change to the stomach microbiota than AMP treatment.

[0154] In FIG. 18, ail of the bacterîal species from mouse stomach are compared between four treatment groups: Empty (probiotic carrying only an empty vector), Null (mock inoculation with buffer), Guided (probiotic expressing gAMP), and Unguided (probiotic expressing AMP). In this figure, the latter three treatments are compared to the Empty treatment. Generalîy speaking, the y-axis represents the taxonomie distance of the collection of bacterîal species in each treatment compared to the collection of bacterîal species in the Empty treatment. Specifically, the index used (y-axis) îs a plugin from QIIME called the Nonparametric Microbial Interdependence Test (NMIT) (Zhang et al,, 2017).

[0155] Imporlantly it îs seen that the gAMP treatment (“Guided”) is much more closely related to a simple probiotic treatment (“Empty”) than is the AMP treatment (“Unguided”) or mice given only a mock inoculation with buffer (“Nul]”), This means that treatment with probiotic expressing gAMP is much more like a normal probiotic treatment.

[0156] In FIG. 19, the same index is used. but with a comparison to the “Null” (mock inoculated) treatment. Again, the species assemblage found in the probiotic/gAMP (“Guided”) treatment is more closely related to the mock inoculated stomach microbial assemblage, as is the empy vector control. The Unguided (probiotic/AMP) assemblage is again more distantly related,

[0157] FIG. 20 measures the différences seen in species assemblages from the same treatment but at different time points, The index used is Shannon's entropy and it îs reported in the y-axis. A more négative value (lower on the y-axis) indicates more change in the population over the 5 days since the inoculation of the mice on Day 0. It can be seen that the probiotic AMP (Unguided) treatment led to the greatest populational change over the 5 days. In contrast, the négative Controls (Empty and Null) and the probiotic/gAMP (Guided) treatments led to only modest populational change. Error bars represent 95% confidence hmits for ail three figures.

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Claims (31)

1. A probiotic for the prévention or treatment of a condition caused by a target bacteriuin living in the gastrointestinal tract of a subject, comprising:

a probiotic bacterium, wherein the probiotic bacterium has been transformed to comprise a DNA construct expressing a guided antimîcrobial peptide, wherein the sequence coding for the guided antimîcrobial peptide comprises the sequence coding for an antimîcrobial peptide fused to the sequence coding for a guide peptide that binds to a protein of the target bacterium, wherein the guided antimîcrobial peptide kills the target bacterium in the gastrointestînal tract of the subject, and wherein the guided antimîcrobial peptide minimally disrupts other bacteria found in the gastrointestînal tract of the subject when compared to unguided antimîcrobial peptides or antibiotics.

2. The probiotic of claim 1, wherein the probiotic bacterium comprises a lactic acid bacterium.

3. The probiotic of claim 2, wherein the lactic acid bacterium comprises a Lactococcus bacterium.

4. The probiotic of claim 3, wherein the Lactococcus bacterium comprises Lactococcus lactis.

5. The probiotîc of claîm 1, wherein the protein of the target bacterium is a virulence factor.

6. The probiotîc of claim 5, wherein the virulence factor is the VacA peptide.

7. The probiotîc of claim 1, wherein the antimîcrobial peptide is laterosporulin, alyteserin, or cathelin-related anti-microbial peptide.

8. The probiotic of claim 1, wherein the target bacterium comprises H. pylori.

9. The probiotîc of claim 1, wherein the guide peptide has a sequence comprising SEQ ID

NO:5.

10. The probîotic of claim 1, wherein the antîmicrobial peptide has a sequence comprising SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NQ:10.

11. The probiotic of claim 1, wherein the guided antîmicrobial peptide has a sequence comprising SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

12. A probîotic composition for the prévention or treatment of a condition caused by a target bacterium living in the gastrointestinal tract of a subject, comprising:

the probîotic of claim 1; and an acceptable excipient or carrier.

13. The probiotic composition of claim 1, wherein the probîotic bacterium is edible, and wherein the acceptable excipient or carrier is edible.

14. Use of a probiotic bacterium thaï has been transformée! to comprise a DNA construct expressing a guided antîmicrobial peptide, wherein the sequence coding for the guided anlimicrobiai peptide comprises the sequence coding for an antîmicrobial peptide fused to the sequence coding for a guide peptide that binds to a protein of the target bacterium, in the manufacture of a médicament for preventing or treating a condition in a subject caused by a target bacterium found in the gastrointestinai tract of the subject.

15. The use of claim 14, wherein the probîotic bacterium comprises a lactic acid bacterium.

16. The use of claim 15, wherein the lactic acid bacterium comprises a Lactococcus bacterium.

17. The use of claim 16, wherein the Lactococcus bacterium comprises Lactococcus lactis.

18. The use of claim 14, wherein the protein of the target bacterium is a virulence factor.

19. The use of claim 18, wherein the virulence factor is the VacA peptide.

20. The use of claim 14, wherein the antîmicrobial peptide is laterosporulin, alyteserin, or cathelin-related anti-niicrobial peptide.

21. The use of claim 14, wherein the target bacterium comprises H. pylori.

22. The use of claim 14, wherein the guide peptide has a sequence comprising SEQ ID NO:5.

23. The use of claim 14, wherein the antimicrobial peptide has a sequence comprising SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NQ:10.

24. The use of claim 14, wherein the guided antimicrobial peptide has a sequence comprising SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

25. The use of claim 14, wherein the subject is an animal.

26. The use of claim 14, wherein the subject is a human.

27. The use of claim 14, wherein the medicamentis edible.

28. The use of claim 14, wherein the médicament îs for administered orally.

29. A probiotic for the prévention or treatment of a condition caused by Hélicobacter pylori living in the gastrointestinal tract of a subject, comprising:

a Lactococcus lactis probiotic bacterium, wherein the Lactococcus lactis probiotic bacterium has been transformed to comprise a DNA construct expressing a guided antimicrobial peptide, wherein the sequence coding for the guided antimicrobial peptide comprises the sequence coding for an antimicrobial peptide fused to the sequence coding for a guide peptide that binds to the VacA peptide of H. pylori, wherein the guided antimicrobial peptide kills H. pylori in the gastrointestinal tract of the subject, and wherein the guided antimicrobial peptide minimally disrupts other bacteria found in the gastrointestinal tract of the subject when compared to unguided antimicrobial peptides or antibiotics.

30. The probiotic of claim 29 wherein the guide peptide is derived from multimerin-1 sequence.

31. The probiotic of claim 29, wherein the antimicrobial peptide is laterosporulin, alyteserin, or cathelin-related anti-microbial peptide.