A study to perceive the role and function of Proteasome Accessory Factor-C (PafC) inMycobacterium tuberculosis

Abstract

Background: Tuberculosis (TB) is one of the major threats globally and the emergence of drug-resistant strains has further deteriorated the situation. Mycobacterium tuberculosis (Mtb) possess more than one mechanism to evade host defense machinery, thereby persisting inside the host for decades. The proteasome machinery is one such mechanism which plays an important role in its virulence and survival inside the host. The gene pafC has been hypothesized to specifically have a role in imparting intrinsic resistance against fluoroquinolones (FQs).

Objective: Identifying the role of Paf-C in the clinical isolates tuberculosis (PTB).

Methodology: The pafC gene was sub-cloned into pET28b and the recombinantly expressed protein was purified via Ni-NTA affinity chromatography. The protein was over-expressed in the non-pathogenic strain of Mtb for the comparative analysis of MICs of anti-mycobacterial drugs. The bacteria was subjected to various stress conditions. Secretory nature of PafC was analyzed by probing the purified protein against patient sera.

Result and Conclusion: The differential regulation of pafC gene has been observed by quantitative real-time PCR at the RNA level in the clinical samples of PTB. The over-expression and knock down clones expressing PafC were subjected for comparative profiling of primary and second-line anti-TB drugs. Protein expression of cultures that were subjected to stress was also measured. The data shows the probable role of PafC in intracellular survival. The absence of PafC in the culture filtrate and patient sera confirmed the non-secretory characteristic of the protein. The detailed results of the study will be discussed during the meeting.