Expression Of Peptide Antibiotic HPAB-β In Pichia Pastoris And Its Biological Activity Against L-form Of Staphylococcus Aureus

Peptide antibiotics are small peptides that encoded by the genomic DNA of organisms. It is one of the important components of living creature's natural immunity system. Peptide antibiotics generally contain 13-60 residues with molecular weight of <5kDa, possess a net positive charge and an amphipathic structure. Compared with conventional antibiotics, which are often produced by Actinomycetes and Molds, peptide antibiotics exert activity against a broad spectrum of microorganisms including Gram-positive and Gram-negative bacteria, fungi, parasites, viruses and even some cancer cells. Peptide antibiotics kill bacteria mainly by membrane- targeting pore–forming mechanisms, so it is difficult for a bacterium to develop resistance. Since the bombinin, the first peptide antibiotic discovered, was isolated from Bombina variegata skin in 1962, more than one thousand of antimicrobial peptides have been identified from plants, insects, arthropods, amphibians, mammals, and humans. The emergence of multiple-drug-resistant bacteria, fungi and parasites in recent two decades has fueled considerable interest in using endogenous antibiotic peptides as alternatives to treat infections.Peptide antibiotic hPAB-βis a kind of mutants of human hBD-2, which maintain the similar biological activity with hBD-2. In our previous study, we successfully constructed the engineered strain pPIC9K-hPAB-β/GS115, which can express the hPAB-β. Based on the membrane-targeting pore–forming mechanism of peptide antibiotics actions, we suppose that they may have a better activity against the L-form of bacteria. The L-forms are cell wall-deficient bacteria which are able to grow as spheroplasts or protoplasts. Most L-forms maintain their pathogenicities, often cause chroinc and relapsing infections. Multi-organic chronic interstitial inflammation is a main manifestation of L-forms infection, which is different with that of its parental form and results in difficulty in clinical diagnose. The drug sensitivity of L-forms to chemical reagents is also different from its partental strain, so it is important to find a new effective reagent against L-forms of bacteria infection. In this study, we firstly expressed and purified the peptide antibiotic hPAB-βby recombinant Pichia pastoris, then the L-form of Staphylococcus aureus was induced and its sensitivity to hPAB-βwas evaluated. The main experimental methods and results are as following:1. High-density cell fermentation of Pichia pastoris for production of secreted hPAB-βThe high-cell density fermentation of pPIC9K-hPAB-β/GS115 strain You5 was performed in a 3.7 L fermentor. After the seeds grew 18-24h, added the fed growth medium to accelerate the growth of cell until the wet weight reached 200 ~ 250g/L, then added the methanol to induce the expression of the target gene. The fermentation was stopped 60h post induction and the total proteins in the fermentative supernatants were 664.0 mg/L, 781.8 mg/L and 721.3mg/L respectively (3 times of fermentation) determined by Brandford assay. The hPAB-βreached 33.4% of the supernatant total proteins analyzed by Quantity–one software (Bio-Rad) and the calculated amounts of expressed hPAB-βwere 241.2±29.5 mg/L.2. Purification and biological activity determination of recombinant hPAB-βThe aim of the purification is not only to get the pure products, but also to keep the bioactivity of the protein during the process of purification. The hPAB-βis a small cationic peptide with a pI of 9.001, so we designed a procedure of 10kDa membrane ultrafiltration, reverse phase chromatogryphy, cation exchange and gel filtration to purify hPAB-βfrom the fermentative supernatants of recombinant Pichia pastoris. The final recovery was 74.9±1.5 mg/L with a purity of 98%. The overall recovery rate was 31.5% after a 4-step purification process. The concentration of hPAB-βstock solution was 30.55 mg/ml (about 7.1 mmol/L) and it had high antibacterial activity against Staphylococcus aureus and Escherichia coli determined with agar diffusion method.3. Induction and identification of L-form of Staphylococcus aureusThe L-form of Staphylococcus aureus strain ATCC25923 was induced by oxacillin paper disk assay. The typical"fried egg"like colonies appeared in oxacillin inhibition zone 3 days post culture at 37℃. The short rod-shaped, filamentous and other pleomorphic Gram-negative cells were seen under the microscope after Gram staining. The L-form could permeated through a 0.45μm filter. After remove the inducer (oxacillin), the L-form of Staphylococcus aureus could reversed back to the wild type after 3~5 passages in high osmotic LB meidum. The most cell walls of the induced L-forms were partial absent and a few of them were completely lost observed under the transmission electron microscope (TEM). These results showed that the L-forms of Staphylococcus aureus were successfully induced with oxacillin.4. Analysis the antibacterial activity of hPAB-βagainst the L-form of Staphylococcus aureusThe antibacterial activity of hPAB-βagainst L-form of Staphylococcus aureus was performed by serial dilution method in a 96-well plate. Three other antibiotics (oxacillin, vancomycin and chloramphenicol) were chosen as controls. Among the four kinds of antibiotics, oxacillin exhibits the highest antibacterial activity against Staphylococcus aureus wild-type, hPAB-βranked the second and chloramphenicol held the last. When against the L-form, the activity ranked from strong to weak was chloramphenicol, peptide antibiotics hPAB-β, vancomycin and oxacillin. Our results shew that the sensitivity to antibiotics was shifted when a wild type of Staphylococcus aureus changed to a L-form. The minimum inhibitory concentration (MIC) of peptide antibiotics hPAB-βagainst the wild type was 8μmol/L (about 34μg/ml), while against the L-form was 32μmol/L (about 137μg/ml), which was 4 times higher than that against the wild type. The reason may be the effect of hypertonic medium (containing 40g/L NaCl in culturing the bacterial L-form) on the activity of peptide antibiotics.5. Establish the animal model infected by the L-form of Staphylococcus aureus.The morphological changes of mouse lung infected by L-form bacteria including larger volume, color dull red, congestion, pulmonary clearly visible and vascular congestion expansion. Through the inflammatory cell count, we can see that the number of WBC significantly increased. The number of neutrophil is increased in the infect group of wild-type bacteria while the L-form bacteria infect group didn't have obiviously change. The probable reason may be the lack of cell wall in the cells of L-form. After HE staining, the infected lung tissue indicated interstitial pneumonia lesions, interstitial alveolar hyperplasia, alveolar space narrowing, interstitial infiltration of lymphocytes and mononuclear cells, red blood cells effusion in alveolar cavity, vascular congestion expansion, small bronchial epithelial hyperplasia and intraluminal red blood cells leaking. The result showed that we successfully establish the animal model of mouse interstitial pneumonia.In conclusion, peptide antibiotic hPAB-βwas expressed in high-cell density fermentation of recombinant Pichia pastoris. The typical L-forms of Staphylococcus aureus strain ATCC25923 were induced by oxacillin paper disk diffusion method and their morphological characters, colonies formation, filtering features, ultra structure and reversion phenomenon were investigated. The antibacterial activity of peptide antibiotics hPAB-βagainst the L-form of Staphylococcus aureus was determined by serial dilution method and the minimum inhibitory concentration (MIC) was 32μmol/L (approximately 137μg/ml). A animal model of interstitial pneumonia lesions infectd by the L-form of Staphylococcus aureus was successfully established. Our study lay down a good foundation for futher evaluation of peptide antibiotics hPAB-βas a potential therapic agent against clinical infection of Staphylococcus aureus L-forms.