Functional characterisation of a SufT homologue in mycobacterium smegmatis

Tamuhla, Tsaone (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of death globally and with drug resistant tuberculosis (TB) on the rise, there is an urgent need to find new anti-TB drugs and drug targets. Increasing our understanding of the physiology of M. tuberculosis can aid in elucidating novel essential pathways which can be used as new drug targets. One such pathway is the iron-sulphur (Fe-S) biogenesis pathway, which is encoded by the sufR-sufBsufD-sufC-csd-sufU-sufT operon (suf operon) in mycobacteria. Fe-S biogenesis is a vital process in cellular physiology yet the functioning of the Fe-S biogenesis machinery in mycobacteria is not fully understood. The last gene in the suf operon, sufT, encodes the only protein in the genome that contains a DUF59 domain. This study used targeted gene deletion and phenotypic characterisation of the resultant mutant to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. An M. smegmatis ΔsufT knockout mutant harbouring an unmarked deletion in sufT was generated using allelic exchange mutagenesis. SufT was confirmed to be dispensable for growth in standard aerobic culture. Loss of SufT significantly decreased the activity of the Fe-S containing enzyme succinate dehydrogenase (SDH) and is therefore proposed to be a putative Fe-S maturation protein. No decrease in aconitase (ACN) activity was observed, suggesting that its role could be limited to certain Fe-S cluster proteins. Loss of SufT did not impact the survival of M. smegmatis after exposure to oxidative stress induced by the redox cycler 2,3- dimethoxy-1,4-naphthoquinone (DMNQ), or the sensitivity of M. smegmatis to the anti-TB drugs isoniazid, clofazimine or rifampicin. The M. smegmatis ΔsufT mutant displayed a growth defect during planktonic growth under iron limiting conditions. This defect was characterised by an extended lag phase, which was observed for all iron concentrations below 2 µM. This suggests that SufT is needed for adaptation to growth under iron limitation. The exponential growth and final cell density achieved by the M. smegmatis ΔsufT mutant under iron limiting conditions was comparable to wild-type, suggesting that induction of a protein that compensates for the loss of sufT occurs. The study also confirmed that the cellular demand for iron during biofilm formation far exceeds that for planktonic growth, particularly during the maturation of the biofilms to form an extracellular matrix. This is the first study to functionally characterise SufT in mycobacteria providing a basis for further mechanistic studies.

AFRIAANSE OPSOMMING: Mycobacterium tuberculosis is een van die grootste oorsake van sterftes wêreldwyd en met antibiotika weerstandige tuberkulose wat aanhou toeneem, is dit noodsaaklik om nuwe antibiotika teen TB en antibiotika teikens te identifiseer. Deur ons kennis van die fisiologie van M. tuberculosis te verskerp kan nuwe noodsaaklike prosesse wat vir anti-TB antibiotika ontwikkeling gebruik kan word, gevind word. Een proses wat as potensiële teiken kan dien is die yster swawel (Fe-S) ko-faktor sintese proses, waarvoor die gene wat betrokke is in die proses in die sufR-sufB-sufD-sufC-csd-sufU-sufT (suf) operon enkodeer word in mycobacteria. Ten spyte van die balangrike rol wat Fe-S sintese in sellulêre fisiologie speel, word die proses nie ten volle verstaan in mycobacteria. Die laaste geen in die suf operon, sufT, enkodeer die enigste protein in die genoom wat ‘n DUF59 domein bevat. In hierdie studie word geteikende geen verwydering en fenotipiese karakterisering van die mutant gebruik om die rol van SufT in die fisiologie van mycobacteria te ondersoek. Mycobacterium smegmatis is as ‘n model organisme gebruik. ‘n M. smegmatis ΔsufT mutant waarvan die sufT geen verwyder is deur alleliese uitruil mutagenese is gegenereer. Die verlies van SufT het geen effek op die groei van die bakterie onder standaard aerobiese kondisies gehad nie. Die aktiwiteit van die Fe-S ko-faktor bevattende ensiem succinate dehydrogenase (SDH) is wel verlaag, wat aandui dat SufT ‘n Fe-S ko-faktor vorming proteïen is. Geen afname in aktiwiteit is waargeneem vir aconitase (ACN), wat aandui dat SufT slegs ‘n rol speel in die vorming van Fe-S ko-faktore vir sekere proteïene. Die verlies van SufT het geen invloed gehad op die oorlewing van M. smegmatis na blootstelling aan die redoks genereerder 2,3-dimethoxy-1,4- naphthoquinone (DMNQ) of die sensitiwiteit van M. smegmatis tot anti-TB antibiotikas isoniasied, rifampisien en clofazemien. Die ΔsufT mutant het ‘n groei defek gedurende planktoniese groei in yster beperkende media gehad. Hierdie defek is gekarakteriseer deur ‘n toename in die lag-groeifase en is in al die yster konsentrasies onder 2 µM waargeneem. Dit stel voor dat SufT benodig word vir die aanpassing tot groei in yster beperkende kondisies. Die eksponensiële groei van die bakterie en finale sel digtheid wat op die ou einde bereik is, was vergelykbaar tussen die mutant en die ongemuteerde bakterie. Dit kan aandui dat ‘n ander protein, met ‘n soortgelyke funksie as SufT moontlik kan intree en die funksie daarvan oorneem. In hierdie studie is daar ook gewys dat die sellulêre vraag na yster gedurende biofilm groei baie hoër is as die van planktoniese groei, veral wanneer dit kom by die vorming van die biofilm se ekstrasellulêre matriks. Hierdie studie is die eerste om SufT in mycobacteria te karakteriseer en bied die basis vir addisionele meganistiese studies.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105163
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