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

[摘要] 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.

[发布机构] Stellenbosch University