Overexpression of Isocitrate Lyase in Mycobacterium Fortuitum in Vitro Studies

Abstract

Tuberculosis has been a health menace for the human race since time immemorial. Mycobacterium tuberculosis is the etiological agent of tuberculosis, whose preferred host cell is macrophage. In order to attenuate the survival of M. tuberculosis, the macrophage creates many stressful conditions like generation of ROI and RNI, antimicrobial peptides, hypoxia, nutrient deprivation, oxidative stress etc. But M. tuberculosis, being a prudent and an efficient organism has evolved many strategies to protect itself from the myriad of killing and trapping mechanisms of the macrophage. Isocitrate Lyase is an enzyme of glyoxylate cycle involved in host fatty acid degradation. Its role has been registered in persistence of M. tuberculosis inside the host cell, helping it to overcome the adversities like nutrient deprivation, hypoxic condition and maintaining antibiotic tolerance. Considering these vital functions, many ICL inhibitors had been designed but proved to be inefficient due to their toxicity and non specificity. Surplus studies on icl knockouts have already been done. However, the consequences of overexpression of this gene have not been elucidated yet. Therefore, by referring to the previous work on gene overexpression and its result on the survival of the organism, the present work is designed to have an idea of overexpression of icl on the bacterial growth under various stress conditions existing inside the host granuloma: acidic, nutrient deprivation, oxidative, detergent, heat, hypoxia. Over expression was done by using icl homologue of M. fortuitum (served as model organism here) in pMV261 vector. Surprisingly, the results showed diminished growth of M. fortuitum under hypoxic and acidic stress. It may be suggestive of the fact that the over expression may prove inhibitory to icl either by feedback inhibition mechanism or by regulation of genes responsible for survival in stress conditions. A lesson emerging from the gene knockout studies is that loss of function mutations is alone not enough to deduce the function of gene. In accordance with the other studies, this result signifies that the overexpression can also be disruptive to the organism. Hence, it should also be considered being studied to find out important drug target.