Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618210
Title: Novel glycan-targeted extracellular proteases from divergent mucosal microbes
Author: Ndeh, Didier Akara
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2013
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Abstract:
Trillions of microorganisms inhabit mucosal surfaces of the human body. Despite increasing evidence of their impact on human health, many of the molecular mechanisms underlying hostmicrobial interactions (HMI) are poorly understood. To contribute to our understanding of HMI at mucosal surfaces, we investigated the novel family of M60-like/PF13402 domain-containing proteins and their putative functional partners. M60-like domains are shared by proteins from several mucosal microbes including two important human mucosal microbes; the bacterial mutualist Bacteroides thetaiotaomicron and the protist pathogen Trichomonas vaginalis, suggesting these proteins are important for interaction with the mucosal layer. We initially tested our hypothesis that these are glycoprotein-targeted metal dependent proteases in both these organisms. The three M60-like domains of B. thetaiotaomicron proteins (BT4244, BT3015 and BT4272) exhibited mucin protease activity. This proteolytic activity was shown to be inhibited in a mutant version of the protein (BT4244-FL-E575D) as well as in the presence of Ethylenediaminetetraacetic acid (EDTA), implying BT4244 and its relatives are metal dependent proteases. All M60-like proteins from B. thetaiotaomicron contained a carbohydrate binding module (CBM) from family 32 and these were shown to be capable of binding galacto-configured sugars that are common to mucin glycans, while in contrast the putative carbohydrate binding PA14 domain of the T. vaginalis TVAG339720 M60-like protein interacted with heparin and its sulphated derivatives. Mucins are glycoproteins and prominent components of the mucus secreted at mucosal surfaces while heparin is a close relative of heparan sulphate which typically exists as part of proteoglycans in the glycocalyx of mucosal epithelia. Although the actual target of the M60-like domain of TVAG339720 and its relatives in T. vaginalis are not currently known, the interaction of the TVAG339720 PA14 domain with heparin suggests that these may be proteases targeting proteoglycans and play a role in adhesion of the pathogen to the epithelial layer, a key initial step in pathogenesis. M60-like domain-containing proteins of B. thetaiotaomicron are also components of Sus-like systems. Sus-like systems are Bacteroidetes specific machinery that comprise a suite of cellenvelope located carbohydrate-active enzymes and sugar binding proteins that target complex glycans, with each Sus-like system tuned to the degradation of a specific glycan. The Sus-like system containing the BT4244 enzyme (BT4240-50), encoded by the polysaccharide locus (PUL) PULBT_4240-50 was characterised in this study. The results demonstrated that BT4244 is a surface protein and that its proteolytic activity is part of a concerted action of BT4240-50 components to utilise complex mucin glycoproteins containing the T (Galβ1-3GalNAc) and F (GalNAcα1-3GalNAc) antigens. Gene deletion studies revealed that PULBT_4240-50 provides a competitive advantage to the organism when grown on mucins, probably through its possession of the N-acetylgalactosamine (GalNAc) kinase BT4240, which was shown to be crucial for GalNAc utilisation. Finally, although variably conserved in closely related Bacteroides, the high frequency of PULBT_4240-50 components in this group of organisms suggests it may be an important evolutionary adaptation for survival at mucosal surfaces. Our findings not only set the stage for future functional studies on the novel M60-like/PF13402 family of proteins and their functional partners, but also further our understanding of host-microbial interactions at mucosal surfaces.
Supervisor: Not available Sponsor: Cameroonian Government ; Department for International Development (DFID) ; Newcastle University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.618210  DOI: Not available
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