Characterization of an antimicrobial peptide from Enterococcus faecium active against bacteria associated with sexually transmitted diseases

De Kwaadsteniet, Michele (2006-04)

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

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Thesis

ENGLISH ABSTRACT : Urogenital tract infections are a major health problem among women. These infections include sexually transmitted diseases such as gonorrhoeae, chlamydia, syphilis and acquired immunity deficiency syndrome (AIDS) (Parratt and Hay, 2003), as well as opportunistic urogenital infections such as vaginosis and abnormal urinary tract infections (Foxman, 2003; Sobel and Chaim, 1996; Wang, 2000). Vaginosis and urinary tract infections affect more than 1 billion women worldwide annually (Reid and Bruce, 2003). Antibiotic resistance and the public demand for natural products have shifted the focus towards the application of probiotic lactic acid bacteria to try and combat these infections. Probiotics are marketed as microbial food supplements or capsules that may prevent diseases and clinical disorders (Schrezenmeir and de Vrese, 2001 ). Most of the well-known probiotics are lactic acid bacteria and include species such as Lactobacillus rhamnosus GR- 1 and Lactobacil/us fermentum RC-14 (Hoesl and Altwein, 2005; Stiles and Holfzapel, 1997). Many of these bacteria are natural inhabitants of the urogenital tract which they colonize to form a protective barrier against intestinal pathogens (Hoesl and Altwein, 2005; Song et al., 1999). Some probiotic strains produce antimicrobial peptides (bacteriocins ), usually with a narrow spectrum of activity, i.e. inhibition of only a certain group of pathogens (Klaenhammer, 1993). Bacteriocins are secreted either through ATP-binding cassettes (ABC) - transporters or via a general secretion pathway. Bacteriocins secreted by ABC-transporters have a doubleglycine- type leader sequence in their N-terminal, which serves as a recognition signal for protein procession and secretion (Haverstein et al., 1995). A sec-dependent leader signal peptide is characteristic of bacteriocins secreted through the general secretion pathway. The sec-dependent leader peptide is usually positively charged with a hydrophobic core and a specific cleavage region (von Heijne and Abrahmsen, 1989). In this study, lactic acid bacteria were screened for the production of bacteriocins active against uropathogenic strains of Enterococcus faecalis and Candida albicans. Enterococcus faecalis is usually associated with secondary bacteraemia of the urogenital tract and is linked to a large number of hospital-acquired infections. Human immunodefiency virus (HIV) patients are especially susceptible to E. faecalis infections. Candida albicans, on the other hand, is an opportunistic pathogen responsible for 80-90% of reported cases of candidal vaginosis, which is diagnosed in approximately 75% of women (Sobel, 1993). Approximate1y half of the patients suffering from candidal vulvovaginitis (CW) suffer from follow-up infections (Ferrer, 2000). Strain TB, isolated from vaginal secretions of children infected with HIV, inhibited the growth of uropathogenic strains of E. faeca/is and was identified as an Enterococcus faecium isolate. Two additional strains, Lactobacil/us plantarum 423 and Enterococcus mundtii ST15, isolated from sorghum beer and soy flour, respectively, also inhibited E. faecalis and were included in the study. None of the three strains produced hydrogen peroxide. All three strains produced bacteriocin-like peptides that inhibited E. faecalis. Plantaricin 423, produced by L. plantarum 423, has been described previously (van Reenen et al., 199B) and is similar to pentocin TV35b (Okkers et al., 1999). Bacteriocin ST15, produced by Enterococcus mundtii ST15, is similar to mundticiin ST 4SA produced by a different strain of E. mundtii (Knoetze, Department of Microbiology, University of Stellenbosch, personal communication). Bacteriocin TB, produced by E. faecium TB, is a class I la sec-dependent bacteriocin and may be considered a novel peptide produced by enterococci. The operon containing the structural gene, a potential immunity gene and mobilization genes is located on a 7 kb-plasmid. The operon was sequenced and the mode of activity of bacteriocin TB determined. Bacteriocin TB is 69% homologous to bacteriocin 31, produced by Enterococcus faecalis Y1717 (Tomita et al., 1996). The signal peptide of bacteriocin TB is only 27% homologous to the signal peptide of bacteriocin 31. The immunity peptide of bacteriocin TB displayed 50% homology to that of bacteriocin 31.

AFRIKAANS OPSOMMING: Urogenitale infeksies is 'n groat gesondheidsprobleem onder vroue. Hierdie infeksies sluit in seksuele oordraagbare siektes soos gonorree, chlamydia, sifilis en verworwe immuniteitsgebreks sindroom (VIGS) (Parratt en Hay, 2003), asook opportunistiese urogenitalie infeksies soos vaginose en abnormale urinekanaal infeksies (Foxman, 2003; Sobel en Chaim, 1996; Wang, 2000). Vaginose en urinekanaal infeksies affekteer meer as 1 biljoen vroue jaarliks wereldwyd (Reid en Bruce, 2003). Weerstand teen antibiotika en die publieke vraag na natuurlike produkte plaas die fokus op probiotika vir die bevegting van hierdie infeksies. Probiotika word bemark as mikrobiese voedsel-supplemente of kapsules wat siektes en kliniese kondisies kan voorkom (Schrezenmeir en de Vrese, 2001 ). Meeste van die bekende probiotika is melksuurbakteriee en sluit spesies soos Lactobacil/us rhamnosus GR-1 en Lactobacillus fermentum RC-14 in (Hoesl en Altwein, 2005; Stiles en Holfzapel, 1997). Baie van hierdie bakteriee is natuurlike inwoners van die urogenitale weg waar hulle koloniseer om 'n beskermende versperring te vorm (Hoesl en Altwein, 2005; Song et al., 1999). Sommige probiotiese stamme produseer antimikrobiese peptiede (bakteriosiene), maar gewoonlik met 'n beperkte spektrum van aktiwiteit, m.a.w. slegs sekere patogene word geinhibeer (Klaenhammer, 1993). Bakteriosiene word met behulp van ATP-bindende kaset (ABK)- vervoerders of 'n meer algemene sekresie-metode uitgeskei. Bakteriosiene wat deur ABK-vervoerders uitgeskei word besit 'n dubbel-glisien leiertipe volgorde in hul N-terminaal wat as 'n herkenningsetel vir prote'len-prosessering en sekresie dien (Haverstein et al., 1995). 'n Sec-afhanklike leierseinpeptied is kenmerkend van bakteriosiene wat deur die algemene sekresie-pad uitgeskei word. Die sec-afhanklike leier-peptied is gewoonlik positief gelaai en het 'n hidrofobiese kern en klewing area (von Heijne and Abrahmsen, 1989). In hierdie studie is melksuurbakteriee vir die produksie van bakteriosiene aktief teen patogene stamme van Enterococcus faecalis en Candida albicans geselekteer. Enterococcus faecalis is gewoonlik geassosieer met sekondere bakteraemia en is gekoppel aan 'n groot aantal hospitaal-verwante infeksies. Mens immuniteitsgebrek virus (MIV)pas'lente is veral vatbaar vir E. faecalis infeksies. C. albicans, aan die anderkant, is 'n opportunitiese patogeen wat vir 80 tot 90% van gerapporteerde gevalle van kandidale vaginose verantwoordelik is (Sobel, 1993). Simptome hiervan is in soveel as 75% van gevalle aangeteken. Omtrent helfte van die pas·iente wat aan kandidale vulvovaginitis lei ervaar verdere infeksies (Ferrer, 2000). Stam TB, ge'isoleer uit vaginale sekresies van kinders ge'infekteer met MIV, het die groei van patogene stamme van E. faecalis geinhibeer. Stam TB is as Enterococcus faecium geidentifiseer. Twee addisionele stamme, Lactobacillus plantarum 423 en Enterococcus mundtii ST15, ge·isoleer van sorgumbier en sojameel, onderskeidelik, het ook E. faecalis geinhibeer en is in hierdie studie ingesluit. Nie een van hierdie stamme produseer waterstofperoksied nie. Die drie stamme produseer bakteriosien-verwante peptiede wat die groei van E. Faecalis inhibeer. Plantaricin 423, geproduseer deur L. plantarum 423, is deur van Reenen et al. (199B) beskryf en is soortgelyk aan pentocin TV35b (Okkers et al., 1999). Bakteriosien ST15, geproduseer deur Enterococcus mundtii ST15, is soortgelyk aan mundticiin ST 4SA wat deur 'n ander E. mundtii stam geproduseer word (Knoetze, Departement Mikrobiologie, Universiteit van Stellenbosch, persoonlike kommunikasie). Bakteriosien TB, geproduseer deur Enterococcus faecium TB, is 'n nuwe klas Ila sec-afhanklike bakteriosien. Die operon wat die strukterele geen, 'n potensiele immuniteits-geen en mobiliseringsgene bevat, is op 'n 7 kb plasmied gelokaliseer. Die operon se DNA volgorde en die meganisme van werking van die bakteriosien is bepaal. Bakteriosien TB is 69% homoloog aan bakteriosien 31, geproduseer deur Enterococcus faecalis Y1717 (Tomita et al., 1996). Die sein-peptied en immuniteitsprote'ien van bakteriosien TB is onderskeidelik slegs 27% en 50% homoloog aan die ooreenstemmende peptiede van bakteriosien 31 .

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