CN113045627B

CN113045627B - Antibacterial polypeptide SA-2 and preparation method and application thereof

Info

Publication number: CN113045627B
Application number: CN202110293357.3A
Authority: CN
Inventors: 周长林; 许鹏飞; 黄晓伟
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2022-07-12
Anticipated expiration: 2041-03-18
Also published as: CN113045627A

Abstract

The invention discloses an antibacterial polypeptide SA-2, a preparation method and an application thereof, wherein the amino acid sequence of the antibacterial polypeptide SA-2 is SEQ ID NO.1: YYRRLLRVLRRRW are provided. The polypeptide SA-2 prepared by the invention has novel design, easily obtained raw material source and large-scale production; the polypeptide SA-2 has good in-vivo and in-vitro antibacterial activity, has good antibacterial activity on Candida albicans and cryptococcus neoformans, can obviously reduce the load of the cryptococcus neoformans in the lung and brain tissues of a mouse, and has potential application value in the research and development of novel medicaments for treating the infection of the lung and brain tissues of the cryptococcus neoformans.

Description

Antibacterial polypeptide SA-2 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of polypeptide medicines in biochemistry, and particularly relates to an antibacterial polypeptide SA-2 and a preparation method and application thereof.

Background

With the increase of people with immune function deficiency, the incidence and mortality of invasive fungal infection are increased year by year, and the human health is seriously threatened. At present, triazole (such as fluconazole), polyene (such as amphotericin B), echinocandin (such as caspofungin) and the like are commonly used in clinic to resist invasive fungal infection, however, the drugs can not meet the clinical requirement, and the death rate of invasive fungal infection is still high. Therefore, the development of new antifungal drugs is urgently needed.

The antibacterial peptide is a novel polypeptide drug different from small molecule antibiotics and has wide sources. The unique membrane breaking mechanism makes it difficult to generate the drug resistance phenomenon. Part of the antibacterial peptide is derived from natural protein, so that the safety is good, and the antibacterial peptide possibly has the function of immune regulation and control, and therefore, the research and development prospect of the antibacterial peptide is concerned.

Human saliva contains a number of antibacterial substances including cysteine protease inhibitors. At present, the protein cysteine protease inhibitor (SA) with the molecular weight of 14KDa is separated from saliva of oral cavity of healthy people, and the protein can be combined with actinomycetes and has certain antibacterial activity on the actinomycetes, but has no effect on fungi. In order to obtain the antifungal polypeptide with high safety, the antibacterial spectrum of the polypeptide is improved, the drug resistance is not easy to generate, and the like. Arouse the wide attention of researchers at home and abroad.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides an antibacterial polypeptide SA-2, which has good in vitro and in vivo antibacterial activity and better antibacterial activity on candida albicans and cryptococcus neoformans, can also obviously reduce the carrying capacity of the cryptococcus neoformans in lung and brain tissues, and is expected to become a novel medicine for treating the infection of the cryptococcus neoformans in lung and brain tissues.

The invention also provides a preparation method and application of the antibacterial polypeptide SA-2.

The technical scheme is as follows: in order to achieve the purpose, the amino acid sequence of the antibacterial polypeptide SA-2 is SEQ ID NO.1: YYRRLLRVLRRRW are provided.

The preparation method of the antibacterial polypeptide SA-2 comprises the following steps:

according to a cysteine protease inhibitor sequence, a sequence with strong hydrophobicity and high helicity in an SA protein sequence is selected as a polypeptide template by bioinformatics, a polypeptide SA-1 with a sequence of SEQ ID NO.2: YYRRLLRVLRARE is obtained, a nonpolar amino acid alanine (A) on the SA-1 sequence is replaced by a cationic amino acid arginine (R), a negatively charged amino acid glutamic acid (E) is replaced by a polar amino acid tryptophan (W), and then the polypeptide SA-2 is synthesized by a solid phase synthesis method. The invention relates to application of the antibacterial polypeptide SA-2 in preparing a medicament for resisting pathogenic bacteria infection.

Further, the anti-pathogenic-fungus-infection medicine is a pathogenic-fungus-infection medicine.

Further, the pathogenic fungi is candida albicans or cryptococcus neoformans.

The anti-pathogenic-bacteria-infection pharmaceutical composition comprises the antibacterial polypeptide SA-2 and a pharmaceutically acceptable carrier thereof.

According to the sequence of the cysteine protease inhibitor, the sequence with strong hydrophobicity and high helicity in the SA protein sequence is selected as a polypeptide template by utilizing the related technologies of bioinformatics. A polypeptide (SA-1) of sequence SEQ ID No.2: YYRRLLRVLRARE was obtained, which sequence contained 13 amino acids with a positive charge of 5. In order to further improve the positive charge and the hydrophobicity of the polypeptide, the nonpolar amino acid alanine (A) in the sequence is replaced by cationic amino acid arginine (R), the negatively charged amino acid glutamic acid (E) is replaced by polar amino acid tryptophan (W), the number of positive charges is increased while the helix content of the polypeptide is not changed, and meanwhile, the tryptophan and the arginine are utilized to form an indole ring, so that the stability is improved. The sequence of the polypeptide after mutation is SEQ ID NO.1: YYRRLLRVLRRRW (SA-2), and the SA-2 contains 13 amino acids and 6 positive charges. Indicating that the positive charge of the polypeptide is increased. The full sequence is tyrosine-arginine-leucine-arginine-valine-leucine-arginine-tryptophan. The polypeptide SA-2 with antifungal activity is obtained by a solid phase synthesis method.

After obtaining the mutated polypeptide sequence, the polypeptide of the present invention is comprehensively evaluated in terms of in vitro antibacterial activity, polypeptide cytotoxicity and in vivo antibacterial activity.

In-vitro antibacterial test results show that the polypeptide SA-2 has better antibacterial activity on candida albicans and cryptococcus neoformans after positive charge is improved. The results of in vitro sterilization curves show that the polypeptide SA-2 has better sterilization effect on the drug-resistant strains of cryptococcus neoformans and can kill fungi in about 12 hours. In vitro toxicity tests show that the polypeptide SA-2 has lower hemolytic toxicity to sheep red blood cells, and simultaneously has lower cytotoxicity to lung epithelial cells and lung cancer cells A549 even at the concentration of 512 mu g/ml.

In a mouse lung infection model constructed by cryptococcus neoformans, the polypeptide SA-2 disclosed by the invention can be used for remarkably improving the survival rate of infected mice and has a certain protection effect on the body weight of the infected mice. In addition, the polypeptides of the invention can also significantly reduce the fungal burden in lung and brain tissue. The fungus is cryptococcus neoformans.

In conclusion, the antibacterial peptide SA-2 has a good killing effect on fungi, particularly cryptococcus neoformans under a low toxic concentration range, and is expected to become a novel medicine for treating cryptococcus neoformans lung infection. None of the reported cysteine protease inhibitor sequence SA, template sequence SA-1, and the present invention has achieved the above-described effect by replacing only the nonpolar amino acid alanine (A) in the sequence with the cationic amino acid arginine (R) or only the negatively charged amino acid glutamic acid (E) with the polar amino acid tryptophan (W).

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. compared with polypeptide SA or SA-1, the prepared antibacterial polypeptide SA-2 has better broad-spectrum antibacterial activity, not only can have obvious antibacterial activity on actinomycetes, but also has good antibacterial effect on fungi such as candida albicans and cryptococcus neoformans, and the cost is greatly reduced.

2. The prepared antibacterial polypeptide SA-2 has broad-spectrum antibacterial activity and obviously improved stability compared with SA-1.

3. The design and preparation method of the antibacterial polypeptide SA-2 is simple and convenient, the design is novel, the raw material source is easy to obtain, and the industrial production and application can be realized.

4. The antibacterial polypeptide SA-2 prepared by the invention can be applied to the preparation of antifungal infection resisting medicines and has good in-vivo and in-vitro antibacterial activity; in a mouse lung infection model constructed by cryptococcus neoformans, the polypeptide SA-2 disclosed by the invention can be used for remarkably improving the survival rate of an infected mouse and has a certain protection effect on the body weight of the infected mouse. In addition, the polypeptide can also obviously reduce the fungal load in lung and brain tissues, has better killing effect on fungi, particularly cryptococcus neoformans, and is expected to become a novel medicament for treating the pulmonary infection of the cryptococcus neoformans.

Drawings

FIG. 1 is a graph of the bactericidal profile of SA-2 against Candida albicans;

FIG. 2 is a graph of SA-2 sterilization of Cryptococcus neoformans;

FIG. 3 is the hemolytic activity of SA-2 on sheep erythrocytes;

FIG. 4 is the survival protection effect of SA-2 on cryptococcus neoformans infected mice;

FIG. 5 is the body weight protection effect of SA-2 on mice infected with Cryptococcus neoformans;

FIG. 6 is the effect of SA-2 on the lung tissue bacterial content of Cryptococcus neoformans infected mice;

FIG. 7 is a graph of the effect of SA-2 on brain tissue bacterial content of Cryptococcus neoformans infected mice;

FIG. 8 is an HPLC chromatogram of SA-2;

FIG. 9 is an MS map of SA-2.

Detailed Description

The present invention will be further described with reference to the following examples and the accompanying drawings.

Example 1

The polypeptide (SA-1) with the sequence of SEQ ID NO.2: YYRRLLRVLRARE is obtained by intercepting a sequence with strong hydrophobicity and high helicity in an SA protein sequence by using a cysteine protease inhibitor sequence (SA, NP-001313.1) as a polypeptide template. The nonpolar amino acid alanine (A) in the sequence is replaced by cationic amino acid arginine (R), the negatively charged amino acid glutamic acid (E) is replaced by polar amino acid tryptophan (W), and the mutated polypeptide sequence is SEQ ID NO.1: YYRRLLRVLRRRW (SA-2).

Synthesis of polypeptide SA-2 by solid phase synthesis

And (3) polypeptide synthesis: the polypeptide SA-2 is synthesized from the C end to the N end one by one. Soaking the Fmoc-Val-Wang Resin in dichloromethane for 15min, and after the Resin swells, extracting the dichloromethane; piperidine/DMF solution (10 ml per gram of resin) was added at a volume ratio of 1:4, the reaction was carried out 2 times for 5min and 15min with nitrogen bubbling, and the resin was washed 9 times with DMF after the reaction was completed. Taking 20-40 particles, adding 2-3 drops of each of the color testing agents ABC (liquid A: ninhydrin/absolute ethanol solution; liquid B: pyridine; liquid C: phenol/absolute ethanol solution), heating at 100 ℃ for 3min, and changing the color of the solution and the resin into blue to remove amino protection. An excess of Fmoc-Val-OH and HOBT, which reacted twice the moles, was added, dissolved in 10ml of DMF per gram of resin, DIC and Collidine, which reacted twice the moles, with nitrogen sparge for 1 h. After the reaction is finished, the resin is washed by DMF (dimethyl formamide) for 6 times, condensation reaction is repeatedly carried out, Fmoc protected amino acids are sequentially connected to complete the synthesis of a straight chain sequence, and the resin is soaked by dichloromethane and diethyl ether and then is drained. Adding TFA, reacting for 2h in a constant temperature shaker at a shaker rotation speed of 110r/min and a temperature of 25 ℃. Filtering to remove resin, adding anhydrous diethyl ether into the filtrate, centrifuging to obtain solid, adding anhydrous diethyl ether, washing, centrifuging, repeating for several times, and oven drying to obtain SA-2 crude polypeptide.

And (3) polypeptide purification: weighing a certain amount of crude product, adding a proper amount of acetonitrile, performing ultrasonic treatment until the mixture is clarified, and removing large-particle impurities by using a filter. Meanwhile, the samples are collected by stages through a preparative liquid chromatograph. And (4) performing gradient analysis by using an analytical chromatograph, and reserving a sample reaching the required purity. Then, freeze-drying treatment is carried out.

Purity determination (HPLC method) and mass spectrometry results of the polypeptide: and (3) after the polypeptide is synthesized, purifying to obtain a finished product, and identifying the finished product by high performance liquid chromatography and mass spectrometry.

Liquid chromatography conditions: c18 column (4.6X 250mm,5 μm); mobile phase a was acetonitrile containing 0.1% trifluoroacetic acid and mobile phase B was purified water containing 0.1% TFA. The detection wavelength is 220 nm; the flow rate is 1.0 ml/min; the sample was taken in an amount of 20. mu.l and gradient elution was carried out.

The polypeptide amino acid sequence is shown as SEQ ID NO: 1, purity greater than 98%. SA-2 has a molecular weight of 1906.3, and its HPLC and MS are shown in FIGS. 8 and 9, respectively, which are consistent with the theoretical values.

Example 2

Determination of in vitro antibacterial Activity of polypeptide SA-2 of the present invention

(1) Recovery and activation of bacterial strain

The experimental strains were transferred from glycerol tubes to the corresponding agar slants of saxose. Culturing at 28 deg.C for 48h, and storing in 4 deg.C refrigerator.

(2) Preparation of bacterial liquid

Scraping a small amount of thallus from the slant with inoculating loop, inoculating into 3ml Sabouraud's dextrose liquid culture medium, culturing at 28 deg.C on shaking table at 200rpm, diluting with liquid culture medium to 1 × 10⁵CFU/ml bacterial suspension for use.

(3) Preparation of drugs

The polypeptide prepared in example 1 was weighed, dissolved in pure water to prepare a mother liquor of 1024. mu.g/ml, filtered and sterilized by a 0.22 μm aqueous phase filter head, and stored at-20 ℃ for further use.

(4) Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of polypeptide SA-2 against test strains

Diluting polypeptide medicine with liquid culture medium to different concentration gradients, respectively placing 50 μ l in 96-well plate, inoculating with equal volume, diluting with liquid culture medium to 1 × 10⁵CFU/ml bacterial suspension, the final concentration of the medicine is 128, 64, 32, 16, 8, 4 and 2 mug/ml, and a blank control group without the medicine is set. The culture was incubated at 28 ℃ for 48h, and the results were observed and recorded. The minimum drug concentration of the non-growing bacteria is MIC.

Taking 100 mul of culture without colony growth in a 96-well plate for MIC determination, adding 900 mul of sterile culture medium for dilution, transferring to a sterile plate, pouring about 10ml of melted Sabouraud's dextrose agar culture medium, placing in an incubator at 28 ℃ for culturing for 48h, observing and recording the colony number in the plate, wherein the lowest drug concentration of the colony number smaller than 5 is the MBC value, and the result is shown in Table 1:

TABLE 1 MIC and MBC values of polypeptide SA-2 against Candida albicans and Cryptococcus neoformans

Note: candida albicans #1-3 as clinical strain, Cryptococcus neoformans #1-6 as clinical strain

As can be seen from Table 1, SA-2 has good antifungal activity, shows good antibacterial activity to various standard and clinical drug-resistant fungi, and has MIC value within the range of 8-32 mu g/ml, MBC value within the range of 16-128 mu g/ml, and MIC value to clinical drug-resistant cryptococcus neoformans and standard strains is only 8 mu g/ml. The polypeptide of the invention has better killing effect on fungi, especially cryptococcus neoformans.

(5) Sterilization curve of polypeptide SA-2 on Candida albicans and cryptococcus neoformans

Mixing the aboveThe test strains were diluted to about 1X 10 with Sabouraud's dextrose broth⁵CFU/ml, adding polypeptide SA-2 to make its concentration be 4 × MIC of corresponding strain, placing at 25 deg.C for constant temperature culture, simultaneously adding amphotericin B with 4 × MIC concentration of corresponding strain as positive control, and adding physiological saline as negative control. At 0, 1, 2, 4, 8 and 24h time, 200. mu.l of mixed culture was removed, and plate viable count was performed after gradient dilution. The sterilization curve was plotted with the drug action time as abscissa and the colony count value as ordinate, and the results are shown in fig. 1 and fig. 2:

FIGS. 1-2 show the bactericidal effect of SA-2 on fungi including Candida albicans and Cryptococcus neoformans. As can be seen from the figure, SA-2 can kill all Candida albicans and cryptococcus neoformans within 4h, so that the colony number is reduced to zero, and the SA-2 has a rapid bactericidal effect on the Candida albicans and the cryptococcus neoformans.

Comparative examples 1-5 for different polypeptides the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were tested using the method described above:

comparative example 1 is cystatin sequence SA;

comparative example 2 is polypeptide SA-1;

comparative example 3 the polypeptide was a cationic amino acid arginine (R) replacing only the nonpolar amino acid alanine (A) on the polypeptide SA-1 sequence;

comparative example 4 the polypeptide is an equivalent concentration of the polypeptide SA-1 sequence with only the negatively charged amino acid glutamic acid (E) replaced by the polar amino acid tryptophan (W);

comparative example 5 polypeptide in which the nonpolar amino acid alanine (a) on the sequence of the polypeptide SA-1 is replaced with the cationic amino acid lysine (K), the negatively charged amino acid glutamic acid (E) is replaced with the polar amino acid valine (V), and comparative examples 1 to 5 are subjected to the same in vitro antibacterial activity assay method as described above, wherein comparative examples 1 and 2 do not have any antibacterial and bactericidal activity against candida albicans and cryptococcus neoformans; the MICs of the comparative examples 3 and 4 to Candida albicans exceed 300 mug/ml, and the MICs to Cryptococcus neoformans exceed 500 mug/ml, which shows that the invention has obvious synergistic effect only by simultaneously replacing the nonpolar amino acid alanine (A) in the sequence with the cationic amino acid arginine (R) and replacing the negatively charged amino acid glutamic acid (E) with the polar amino acid tryptophan (W). In contrast, in comparative example 5, the nonpolar amino acid alanine (A) and the negatively charged amino acid glutamic acid (E) in the sequence of the polypeptide SA-1 are subjected to other substitutions, the MICs of Candida albicans and cryptococcus neoformans are both over 200 mu g/ml, and the effect is not as good as that of the polypeptide SA-2 of the invention, which shows that the mutation of the specific site of the invention can reach better antibacterial and bactericidal activity of the Candida albicans and cryptococcus neoformans.

Example 3

Protection effect of polypeptide SA-2 on lung infected mouse

The defibered sheep blood (3.0 ml) was aspirated by a sterile syringe, added to a sterile EP tube, centrifuged at 3000rpm for 10min in a refrigerated centrifuge, the supernatant was discarded, and washed 3 times with PBS (pH 7.4). After resuspension with PBS, a 3% (v/v) suspension of erythrocytes was prepared. 80 mul of erythrocyte suspension is respectively added into a 96-well plate, and then polypeptide medicines diluted in multiple proportion are added in equal volume, so that the final concentrations of the medicines are 512 mug/ml, 128 mug/ml, 32 mug/ml, 8 mug/ml and 2 mug/ml. Meanwhile, red blood cells treated by 1% Triton X-100 are used as a positive control group, red blood cells treated by PBS buffer are used as a negative control group, and 3 multiple wells are arranged in each group. The culture plate was incubated at 37 ℃ for 1 hour and then taken out, and centrifuged at 3000rpm in a refrigerated centrifuge at 4 ℃ for 10 min. Taking the supernatant, and determining OD with enzyme-linked immunosorbent assay₄₅₀Values, and calculating the hemolysis rate, the formula is as follows:

hemolysis rate (%) - (A-A)₀)/(A₁₀₀-A₀)]×100％

A represents the absorbance of the SA-2 polypeptide set. A. the₀Denotes the absorbance of the PBS group, A₁₀₀Absorbance values for the Triton X-100 panel are shown.

As can be seen from FIG. 3, even at the highest concentration of 512. mu.g/ml, the hemolytic rate of the polypeptide prepared according to the present invention on sheep-positive erythrocytes does not exceed 5%, and in combination with the range of MIC, it can be concluded that: the polypeptide SA-2 has no hemolytic toxicity basically under the bactericidal concentration.

Example 4

Protection effect of polypeptide SA-2 on lung infected mouse

Selecting four-week-old male ICR mice with weight of about 20-22 gRandomly divided into 6 groups of 8. The 5 groups of mice, except the blank control group, were administered cyclophosphamide (100mg/kg/d) for three consecutive days, then anesthetized with ether on the fourth day, and infected with 1X 10 by nasal drip⁸CFU/ml cryptococcus neoformans bacterial suspension (60 mu l/mouse) is used for establishing a cryptococcus neoformans lung infection model, and the mice in an infected group are treated by SA-2 polypeptide solutions with different concentrations immediately after infection, wherein the high, medium and low administration dose concentrations of the polypeptide are respectively 10 mg/kg/d, 5mg/kg/d and 2.5 mg/kg/d. The positive control group adopts the combination treatment of the administration of fluconazole (50mg/kg/d) through gastric gavage and the administration of amphotericin B (0.5mg/kg/d) through tail vein. The model group was injected with normal saline as a control. After 7 consecutive days of continued treatment, the mice were observed for 14 days and recorded for survival and weight change.

After two weeks of treatment, mice were sacrificed by dislocation of cervical vertebrae, lung and brain tissues were dissected under aseptic conditions and weighed, placed in a sterile EP tube, 9 volumes of physiological saline (V tissue: V physiological saline 1:9) was added, the tissues were cut with scissors, and the tissues were homogenized by an electric homogenizer. Diluting the homogenate with 10 times of normal saline, adding the diluted solution into a sterile plate, pouring a Sabouraud's dextrose agar culture medium, mixing uniformly, and culturing in an incubator at 28 ℃ for 48 h. The number of colonies on the plate was observed and recorded, and the number of fungi in lung and brain tissues was calculated. The results are shown in FIGS. 4-7.

As can be seen from FIG. 4, most of the mice in the model group died due to fungal infection, while the survival rates of the mice in the low and medium dose administration groups of the polypeptide increased to 25% and 37.5% respectively, compared with the model group, demonstrating that the polypeptide has a large degree of protection effect on the survival of the mice infected with the fungus, and the in vivo efficacy of the polypeptide varies in a dose-dependent manner in a certain concentration range (10 mg/kg).

As can be seen from FIG. 5, the body weight of the model group mice was largely reduced after infection with the fungus, and thereafter the body weight of the non-dead mice was slowly restored. In the polypeptide and positive drug treatment group, the weight of the mouse is recovered more quickly than that of the model group, which indicates that the polypeptide also has a certain protection effect on the weight of the mouse.

FIGS. 6 and 7 are graphs showing the fungal content in lung and brain tissue of mice, and the polypeptide high dose group reduced the fungal content in lung tissue of mice by 0.8 log compared to the model group; the dose group in the polypeptide reduces the fungal content of the brain tissues of the mice by about 0.5 log value; significant differences compared to model group (P < 0.05).

As can be seen from the comprehensive graphs of 4-7, the polypeptide SA-2 shows better fungus clearance, so that the survival rate of infected mice is improved, and the polypeptide SA-2 has the potential of treating cryptococcus neoformans lung infection. In addition, the polypeptide of the present invention may also protect mice infected with cryptococcus neoformans in brain tissue.

Sequence listing

<110> university of Chinese pharmacy

<120> antibacterial polypeptide SA-2, preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Tyr Tyr Arg Arg Leu Leu Arg Val Leu Arg Arg Arg Trp

1 5 10

<210> 2

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Tyr Tyr Arg Arg Leu Leu Arg Val Leu Arg Ala Arg Glu

1 5 10

Claims

1. An antibacterial polypeptide cystatin SA-2, characterized in that the amino acid sequence thereof is SEQ ID NO.1: YYRRLLRVLRRRW is added.

2. A method of producing the antibacterial cystatin SA-2 according to claim 1, comprising the steps of:

according to a cysteine protease inhibitor sequence, a sequence with strong hydrophobicity and high helicity in an SA protein sequence is selected as a polypeptide template by bioinformatics, the obtained polypeptide SA-1 with the sequence of SEQ ID NO.2: YYRRLLRVLRARE is obtained, a nonpolar amino acid alanine (A) on the SA-1 sequence is replaced by a cationic amino acid arginine (R), a negatively charged amino acid glutamic acid (E) is replaced by a polar amino acid tryptophan (W), and then the polypeptide SA-2 is synthesized by a solid phase synthesis method.

3. Use of the antimicrobial polypeptide cystatin SA-2 according to claim 1 for the manufacture of a medicament against infection by the pathogenic Candida albicans or Cryptococcus neoformans.

4. A pharmaceutical composition against pathogenic bacterial infections comprising the antibacterial pepstatin SA-2 of claim 1 and a pharmaceutically acceptable carrier therefor.