Differential expression of genes in clinical strains of mycobacterium tuberculosis in response to isoniazid

Seepe, Prudy Mashika (Stellenbosch : University of Stellenbosch, 2011-03)

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

Thesis

ENGLISH ABSTRACT: Isoniazid forms part of the first line anti-tuberculosis therapy and it is generally used to treat latent Mycobacterium tuberculosis infection. Isoniazid inhibits synthesis of long chain mycolic acids found in cell wall of Mycobacterium tuberculosis, which have proven vital for the survival of the bacterium. Mycolic acids are primarily synthesized by the fatty acid synthase enzyme (FAS) system found in mycobacteria as the FAS-I and FAS-II complex. Isoniazid kills the bacteria by blocking the FAS-II complex, required for extension of mycolates. It does this by entering the tubercle bacilli as a prodrug where isoniazid becomes activated by catalase peroxidase encoded by katG gene. The activated isoniazid then forms a complex with NAD+ which targets InhA (NADH-dependent enoyl-acyl carrier protein reductase) located in the FAS-II complex. Loss of catalase peroxidase, due to gene mutations or a complete katG gene deletion is one of the primary mechanisms conferring resistance to INH in Mycobacterium tuberculosis. In addition, four other genes (inhA, KasA, ndh and ahpC) are also associated with INH resistance. Nonetheless, mutations in these five genes are present in only 70-80% of INH resistant clinical isolates, implying that other mechanisms are involved in resistance of Mycobacterium tuberculosis to isoniazid. This study aims to quantify the expression level of genes induced by isoniazid in the mycolic acid pathway and drug transport in two closely related Mycobacterium tuberculosis Beijing cluster 208 isolates. These are the fully susceptible (K636) and isoniazid mono-resistance strains (R55), with minimum inhibitory concentrations of 0.1 and 4 µg/ml, respectively. Both these isolate had no isoniazid gene associated mutations. The isolates were cultured in the presence and absence of 0.1µg/ml isoniazid for 24 hours after which RNA was extracted followed by QRT-PCR analysis to identify differentially expressed genes. This result has shown that various genes were differentially expressed in response to low level INH exposure. The most significant up-regulation was observed in genes (acpM, fabD, Accd6 and fbpC) encoding the FAS-II complex and genes (efpA, iniA, iniB, and mmpl7) involved in drug transport. In addition, two genes (ndh and fbpC) were significantly down-regulated in the isoniazid mono-resistant isolate. Based on these findings, we propose a model whereby isoniazid exposure in the susceptible isolate inhibits FAS-II complex and with its associated accumulation in mycolates kills the bacterium. In contrast, we propose that in the resistance isolate the bacterium acquires additional resistance by the activation of efflux pumps in combination with disruption in INH-NAD+ complex formation that protect inhibition of InhA located in FAS-II complex.

AFRIKAANSE OPSOMMING: Isoniasied vorm deel van die eerste linie van behandeling teen tuberkulose en word algemeen gebruik om latente Mycobacterium tuberculosis infeksie te behandel. Isoniasied inhibeer die sintese van langketting mikolitiese sure wat in die selwand van Mycobacterium tuberculosis voorkom. Dit is bewys dat hierdie sure essensieel is vir die oorlewing van die bakterie. Mikolitiese sure word hoofsaaklik gesintetiseer deur die vetsuur sintase ensiem (FAS) sisteem wat in mikobakteriee voorkom as die FAS-I en FAS-II komplekse. Isoniasied dood die bakteriee deur die FAS-II kompleks, wat nodig is om die verlenging van mikoliete, te blokkeer. Dit word bewerkstellig deurdat 'n pro-vorm van die middel die tuberkulose bacilli binnedring, waarna isoniasied geaktiveer word deur katalase peroksidase, wat deur die katG geen geenkodeer word. Die geaktiveerde isoniasied vorm 'n kompleks met NAD+, wat InhA (NADH-afhanklike eno.asiel draer prote.enreduktase), gelee in die FAS-II kompleks teiken. Een van die primere meganismes wat weerstandigheid teen isoniasied bewerkstellig, is die verlies van katalase peroksidase weens geenmutasies of algehele delesie van die katG geen. 'n verdere vier gene (inhA, kasA, ndh en ahpC) word ook verbind met isoniasied weerstandigheid. Nietemin is mutasies in hierdie vyf gene teenwoordig in slegs 70-80% van isoniasied weerstandige kliniese isolate, wat impliseer dat ander meganismes ook betrokke is in die weerstandigheid van Mycobacterium tuberculosis teen isoniasied. Die doel van hierdie studie is om die vlak van uitdrukking van gene wat deur isoniasied in die mikolitiese suur biochemiese pad geïnduseer word, asook middel transport te kwantifiseer in twee naby verwante Mycobacterium tuberculosis isolate van Beijing groep 208. Die twee isolate is die volledig sensitiewe (K636) en isoniasied monoweerstandige (R55), met minimum inhiberende konsentrasies van onderskeidelik 0.1 en 4µg/ml. Mutasies wat geassosieer word met isoniasied weerstandigheid was afwesig in beide die isolate. Kulture is van die isolate gemaak met en sonder 0.1µg/ml isoniasied vir 24 uur, waarna RNA geekstraeer is deur middel van QRT-PCR analise om gene te identifiseer wat verskillend uitgedruk word. Die resultate toon dat verskeie gene verskillend uitgedruk is in reaksie op laevlak isoniasied blootstelling. Die mees prominente opregulering is waargeneem in die gene (acpM, fabD, accd6 en fbpC) wat die FAS-II kompleks enkodeer, asook die gene (efpA, iniA, iniB en mmpl7) wat betrokke is in middel transport. Beduidende afregulering van 'n verdere twee gene in die isoniasied monoweerstandige isolate, naamlik ndh en fbpC is waargeneem. Op grond van hierdie waarnemings, stel ons 'n model voor waarvolgens isoniasied blootstelling in die sensitiewe isolaat die FAS-II kompleks inhibeer, en met die gevolglike akkumulasie van mikoliete, dood dit die bakterium. In teenstelling stel ons voor dat addisionele weerstandigheid bekom word in die weerstandige isolaat deur die aktivering van uitvloeipompe, in kombinasie met die ontwrigting van die INH-NAD+ kompleksvorming wat die inhibisie van InhA binne die FAS-II kompleks beskerm.

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