Characterization of a broad-spectrum antimicrobial peptide from Enterococcus mundtii active against bacteria associated with middle ear infections

Knoetze, Hendriette (Stellenbosch : University of Stellenbosch, 2006-12)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: Strain ST4SA, isolated from soya beans, was identified as Enterococcus mundtii. BacST4SA, a bacteriocin produced by strain ST4SA inhibited the growth of Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, and unidentified middle ear isolates A, BW, DW, F, G, and H. BacST4SA was active against Pseudomonas aeruginosa G, BG, I, J, B and E, although variable degrees of resistance were observed for some strains. BacST4SA is positively charged, hydrophobic, contains the YGNGV sequence in the N-terminal, a double-glycine processing site and a disulphide bridge, all of which is typical of a class IIa bacteriocin. The operon, which contains a structural-, ATP-dependent transporter- and immunity gene, is located on a 50-kb plasmid. The 58-amino acid prepeptide is homologous to mundticin KS, mundticin AT06 and bacteriocin QU 2, and differs from enterocin CRL35 by only two amino acids. The 674-amino acid ATP-dependent transporter, consisting of a peptidase C39B domain, an ABC-transporter and an ABC-DLP family domain, displayed 98.9% homology to mundticin KS and 99.25% to enterocin CRL35. The 98-amino acid immunity gene of bacST4SA is completely homologous to enterocin CRL35 and 96.9% to mundticin KS. BacST4SA is 3.950 kDa in size, based on electron spray mass spectrometry. The peptide was isolated from the cell-free supernatant, precipitated with 80% saturated ammonium sulphate, dialysed and freeze-dried to 1 638 400 AU (arbitrary units) per ml. No change in antimicrobial activity was recorded when bacST4SA was incubated in buffer ranging from pH 2 to 12, heated to 100 °C for 90 min and 121 °C for 20 min, and when incubated in the presence of Tween 20, Tween 80, Triton X-100, SDS, urea, EDTA, middle ear fluid and blood. Optimal levels of bacST4SA production (51 200 AU/ml) was recorded after 14 h of growth in MRS broth at 30°C. Maximum production (102 400 AU/ml) was recorded in modified MRS media supplemented with tryptone, yeast extract, a combination of tryptone and yeast extract, K2HPO4 (10.0 or 20.0 g/l), or with the addition of DL-6,8-thoictic acid, L-ascorbic acid, and thiamine, respectively. BacST4SA is bactericidal towards E. faecium HKLHS and bacteriostatic towards S. pneumoniae 40 and middle ear isolates F, BW and H. The peptide adsorbed maximal (94%) to S. pneumoniae 40, P. aeruginosa 25 and E. faecium HKLHS. BacST4SA forms pores in the cytoplasmic membrane of sensitive cells, leading to dissipation of the cell membrane and leakage of cytoplasmic material. BacST4SA was compared with various other antimicrobial treatment agents, and revealed similar to a higher activity towards a number of these agents. BacST4SA revealed a similar level of activity against E. faecium HKLHS and middle ear pathogens P. aeruginosa J and S. pneumoniae 27 when compared with tetracycline (30μg). However, bacST4SA revealed much higher activity when compared to nasal sprays, aminoglycosides, cephalosporins, fluoroquinolones, lincosamides, macrolides, nitroimidazole, penicillin, quinolones, sulfonamides, chloramphenicol, furanzolidone, fusidic acid, rifampicin, trimethoprim, trimethoprim-sulfamethoxazole and vancomycin when tested in vitro.

AFRIKAANSE OPSOMMING: Stam ST4SA, geïsoleer uit sojabone, is as Enterococcus mundtii geidentifiseer. BacST4SA, ‘n bakteriosien geproduseer deur stam ST4SA het die groei van Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae en ongeïdentifiseerde middeloor isolate A, BW, DW, F, G, en H geinhibeer. BacST4SA is aktief teen Pseudomonas aeruginosa stamme G, BG, I, J, B en E, alhoewel effense weerstand soms waargeneem is. BacST4SA het ‘n netto positiewe lading, is hidrofobies, bevat die YGNGV-volgorde in die N-terminaal, ‘n dubbel-glisien prosesserings setel en ‘n disulfied brug, kenmerkend van klas IIa bakteriosiene. Die operon, wat bestaan uit ‘n strukturele geen, ‘n ATP-afhanklike transport sisteem geen en ‘n immuniteits-geen, is op ‘n 50 kb plasmied gelokaliseer. Die voorloper peptied (58 aminosure lank), is homoloog aan mundticin KS, mundticin AT06 en bakteriosien QU 2 en verskil van enterocin CRL35 met slegs twee aminosure. Die ATP-afhanklike transporter (674 aminosure lank) bestaan uit ‘n peptidase C39B domein, ‘n ABC-transporter en ‘n ABC-DLP tipe domein en is 98.9% homoloog aan mundticin KS and 99.25% aan enterocin CRL35. Die immuniteits-geen (98 aminosure lank) van bacST4SA is ten volle homoloog aan enterocin CRL35 en 96.9% homoloog aan mundticin KS. BacST4SA is 3.950 kDa groot, gebaseer op elektrosproei-massa spektrometrie. Die peptied is uit selvrye supernatant geïsoleer, met 80% versadigde ammonium sulfaat gepresipiteer, gedialiseer en gevriesdroog tot ’n finale konsentrasie van 1 638 400 AE (arbitrêre eenhede) per ml. Geen verandering in antimikrobiese aktiwiteit is waargeneem tydens inkubasie van bacST4SA in buffer van pH 2 tot 12, tydens verhitting (100 °C vir 90 min en 121 °C vir 20 min) en tydens inkubasie in die teenwoordigheid van Tween 20, Tween 80, Triton X-100, SDS, ureum, EDTA, middeloor vloeistof en bloed. Optimale vlakke van bacST4SA produksie (51 200 AE/ml) is na 14 h groei in MRS media by 30°C waargeneem. Maksimale vlakke van die peptied (102 400 AE/ml) is geproduseer in gemodifiseerde MRS medium, aangevul met triptoon, gisekstrak, ‘n kombinasie van triptoon en gisekstrak, K2HPO4 (10.0 of 20.0 g/l), of met byvoeging van DL-6,8-thioktiensuur, L-askorbiensuur, en tiamien onderskeidelik. BacST4SA is bakteriosidies teenoor E. faecium HKLHS en bakteristaties teenoor S. pneumoniae 40 en middeloor isolate F, BW en H. Die peptied adsorbeer optimaal (94%) aan S. pneumoniae 40, P. aeruginosa 25 en E. faecium HKLHS. BacST4SA vorm porieë in die selmembraan van sensitiewe selle en lei tot vernietiging van die selmembraan en lekkasie van die sitoplasma inhoud. In vergelykende studies het bacST4SA ‘n soortgelyke en selfs hoër antimikrobiese aktiwiteit teenoor ‘n aantal bekende antimikrobiese middels getoon. Die aktiwiteit van bacST4SA is soortgelyk aan dié van tetrasiklien (30μg) in toetse teen E. faecium HKLHS en middeloor patogene P. aeruginosa J en S. pneumoniae 27. BacST4SA het egter in ’n in vitro vergelyking met neussproeie, aminoglisiedes, cephalosporiene, fluoroquinolone, lincosamides, makroliede, nitroimidazole, penisilien, quinolone, sulfonamide, chloramphenicol, furanzolidone, fusiensuur, rifampisien, trimethoprim, trimethoprim-sulfamethoxazool en vankomisien ‘n baie hoër aktiwiteit teen patogene getoon.

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