Elucidation of the structure and function of the Mycosins, a family of essential subtilisin-like serine proteases of Mycobacterium tuberculosis

Thesis (PhD)--Stellenbosch University, 2014.ENGLISH ABSTRACT: Mycobacterium tuberculosis is an ancient pathogen, which has been infecting humans for millennia. It remains globally spread, infecting one third of the world’s population and causing around two million fatal cases per year. The treatment of this infectious disease remains complex, time consuming, resource demanding and costly. In the absence of adherence, good quality drugs and appropriate treatment regimens, the pathogen is highly likely to develop resistance to the antibiotics used during treatment. Searching for new drug targets and developing new drugs are constantly in progress to address these issues. Type VII secretion system (T7SS) is a signature protein secretion system in mycobacteria, associated with virulence and nutrient acquisition. There are five copies of the T7SS in M. tuberculosis, namely ESX-1 to -5, with ESX-3 being essential for the bacterial growth in vitro. The importance of ESX-3 is supported by the fact that it influences two major iron uptake pathways in mycobacteria, namely mycobactin-mediated iron acquisition and heme acquisition. Mycosin-3 is the only membrane core component of ESX-3 that has a subtilisin-like serine protease signature and plays an essential role in ESX-3 secretion. Mycosin-3 has a unique catalytic specificity and its substrates are unknown. Elucidating the protein structure and determining the function of mycosin-3 will help the design of effective inhibitors to abolish the protease function providing for a potential therapeutic option. In this study, the mycosin-3 gene from the M. tuberculosis genome was cloned and expressed in Escherichia coli and the protein was purified in vitro, with the aim of conducting structural studies. However, the amount of soluble and stable mycosin-3 was insufficient to progress to X-ray crystallography for protein structure determination. According to the literature, this technical difficulty is not uncommon for M. tuberculosis genes because the gene transcription and protein production machineries in mycobacteria are distinct from the conventional protein production host E. coli. In vitro expression analysis suggested that mycosin-3 possibly exerts a toxic effect on the expression hosts: E. coli and Mycobacterium smegmatis. To overcome these complexities, M. smegmatis was used as a model organism for functional studies; the mycP3 gene was deleted from the genome, and the proteomes of the wild type and mycosin-3 deletion mutant were compared. No major phenotypic differences were observed between the wild type and mutant possibly because the model organism has an alternative exochelin-mediated iron acquisition pathway. Interestingly, in the absence of mycosin-3, one component of the mycobactin export system, MmpL5, was not detected in the whole cell lysate (containing both cytosolic and membrane fractions) by mass spectrometry although the mmpL5 gene was transcribed, suggesting rapid protein degradation. We hypothesize that mycosin-3 may plays a role in maintaining the integrity of the membrane protein MmpL5 prior to its secretion and in facilitating its localization on the membrane. The direct involvement of mycosin-3 in the posttranslational modification of MmpL5 is currently under investigation. This study provides evidence that mycosin-3 may be an attractive drug target - abolishing mycosin-3 could disable mycobactin export, with ensuring toxicity from intracellular mycobactin accumulation thereby eliminating M. tuberculosis.AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogeen wat mense reeds eeue infekteer en tuberkulose veroorsaak. Vandag kom tuberkulose steeds wêreldwyd voor en veroorsaak ongeveer twee miljoen sterftes jaarliks. Die behandeling van hierdie aansteeklike siekte bly kompleks, tydrowend, hulpbron-veeleisend en duur. Sonder toepaslike behandeling, goeie kwaliteit antibiotika en nakoming van behandelings regimente, is dit waarskynlik dat antibiotika weerstandigheid ontwikkel. Dit is gevolglik belangrik om nuwe antibiotika teikens te vind. Die Tipe VII sekresie sisteem (T7SS) is ‘n kenmerkende protein sekresie sisteem in mycobacteria en word met virulensie en die opname van voedingstowwe assosieer. Daar is vyf kopieë van T7SS in M. tuberculosis naamlik ESX-1 tot -5. ESX-3 is noodsaaklik vir bakteriële groei in vitro. Die belangrikheid van ESX-3 word beklemtoon deur die feit dat dit die twee hoof paaie vir ysteropname, naamlik mycobactin-bemiddelde-ysterverkryging en heme vekrygings paaie, in mycobacteria beïnvloed. Mycosin-3 is die enigste membraan kern komponent van ESX-3 wat ‘n subtilisin-agtige serien protease motief het en dit speel ‘n belangrike rol in ESX-3 sekresie. Mycosin-3 het unieke katalitiese spesifisiteit en die substrate daarvan is onbekend. Bepaling van die proteïen struktuur en funksie van mycosin-3 sal help met die ontwerp van effektiewe inhibeerders van die protease funksie. Dit kan gevolglik ‘n potensiële terapeutiese opsie verskaf. In hierdie studie is die mycosin-3-geen van die M. tuberculosis genoom gekloon en uitgedruk in Escherichia coli. Die in vitro proteïen is gesuiwer om strukturele studies te doen. Die hoeveelheid oplosbare en stabiele mycosin-3 was egter onvoldoende om te vorder tot X-straalkristallografie vir proteïen struktuur bepaling. Volgens die literatuur, is hierdie tegniese probleme nie ongewoon vir M. tuberculosis gene, aangesien die geen transkripsie en proteïen produksie masjinerie van mycobacteria uniek is en dus van die konvensionele masjinerie in die E. coli gasheer verskil. Analise van in vitro proteïen uitdrukking eksperimente stel voor dat mycosin-3 moontlik ‘n toksiese effek op die gasheer, E. coli en Mycobacterium smegmatis, kan hê. Om hierdie probleem te oorkom, is M. smegmatis as ‘n model organisme gebruik om funksionele studies te doen. Die mycP3 geen is uit die M. smegmatis genoom verwyder om ‘n delesie mutant te maak. Die proteome van die wilde-tipe en mycosin-3 geen delesie mutant is vergelyk. Daar was geen opmerklike verskille in die fenotipe van die wilde-tipe en mutant nie, moontlik omdat die model organisme ‘n alternatiewe exochelin-bemiddelde yster verkrygings pad het. Interessant genoeg, is een komponent van die mycobactin uitvoer sisteem, MmpL5, afwesig in die heel sel extrakte (van beide die sitosol en membraan fraksies) wanneer mycosin-3 afwesig is, volgens massa spektrofotometriese analise. Die mmpL5 geen is egter getranskribeer, wat daarop dui dat die proteïen vinnig degradeer. Ons hipotese is dat mycosin-3 moontlik ‘n rol speel in die integriteit van die membraan proteïen MmpL5 voordat dit uitgeskei word en in die fasilitering van MmpL5 se posisionering in die membraan. Die direkte betrokkenheid van mycosin-3 in die posttranslasionele wysiging van MmpL5 word tans ondersoek. Hierdie studie bewys dat mycosin-3 'n aantreklike antibiotika teiken kan wees aangesien die afwesigheid van mycosin-3 veroorsaak dat mycobactin uitvoer afgeskaf word en gevolglik tot toksiese opbou van mycobactin lei wat tot die dood van M. tuberculosis lei