Elucidation of the substrates of mycosin 3, an essential protease of Mycobacterium tuberculosis

Fang, Zhuo (2011-03)

Thesis (MScMedSc)--University of Stellenbosch, 2011.

Thesis

ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) veroorsaak, infekteer `n derde van die wêreld se bevolking en veroorsaak die dood van 1.7 miljoen mense per jaar. Die verhoogde voorkoms van multi- en ekstensiewe middelweerstandige stamme van M. tuberculosis beteken dat daar `n ernstige nodigheid is om nuwe anti-TB middels te ontwikkel. Die genoom van M. tuberculosis het vyf kopieë van die ESAT-6 geengroepe (ESX-1, -2, -3, -4 en -5), wat essensieel is vir die oorlewing (ESX-3) en patogenisiteit (ESX-1 and ESX-5) van die bakterium. Die ESX groepe enkodeer vir proteïene wat `n nuwe uitskeidingssisteem vorm wat bewys is om klein T-sel antigene van die esx geenfamilie, sowel as ander proteïene soos die PE en PPE proteïene uit te skei. Die mycosins is `n familie gene wat in die ESX geengroepe voorkom en wat waarskynlik enkodeer vir subtilisin-agtige serine proteases. Hierdie proteïene is die mees gekonserveerde proteïene in die vyf geengroepe. Mycosin-3 is `n essensiële protein wat spesifiek in die mikobakteriëe voorkom, sodat dit `n aantreklike teiken vir die ontwikkeling van middels is. Die identifisering van die substrate van mycosin-3 kan moontlik help om die funksie van die ensiem te verstaan en om nuwe inhibeerders vir die ensiem te ontdek, wat kan lei tot die onwikkeling van nuwe middels. Ons hipotese is dat die uitgeskeide proteïene van die ESX sisteem moontlik die substrate van die mycosin proteïene kan wees. Meer spesifiek, ons hipnotiseer dat die proteïene PE5, PPE4, esxG en esxH (wat almal in ESX-3 voorkom) die substrate vir mycosin-3 kan wees. Mycosin-3, PE5, PPE4, esxG en esxH is afsonderlik gekloneer, uitgedruk en gesuiwer. Die vier substrate is gebruik vir protease proewe met mycosin-3 as die protease. Die protease-substraat mengsel is hierna deur middel van 2-D SDS-PAGE geanaliseer om te kyk of daar enige kliewing van die vier substrate voorgekom het. Alhoewel al die teiken fusieproteïene suksesvol gekloneer, uitgedruk en gesuiwer is, het die protease proewe geen kliewing getoon vir enige van die vier potensiële substrate nie. Moontlike verklarings vir hierdie negatiewe resultaat is die volgende: (1) ensiem en substrate was moontlik onsuiwer; (2) bufferkondisies was moontlik nie korrek nie; (3) gehipotiseerde substrate mag moontlik nie substrate van mycosin-3 wees nie; en (4) nie-korrekte vouing of modifisering van die teiken proteïene kon moontlik plaasgevind het. Toekomstige navorsing sal daarop gemik wees om hierdie beperkinge aan te spreek om sodoende die funksie en substrate van mycosin-3 te kan ontdek en nuwe middels teen M. tuberculosis te ontwerp.

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