Potential Role of Proteasome Accessory Factor-C in Resistance against Second Line Drugs in Mycobacteria.

Abstract

Objectives Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), can survive inside the host granuloma courtesy the various extrinsic and intrinsic factors involved. Continuous use or misuse of the anti TB drugs over the years has led to the development of resistance in MTB against antibiotics. Drug-resistant TB in particular has been a menace since treating it requires exposing the patient to drugs for a prolonged period of time. Multidrug-resistant (MDR) and extensively drug resistant TB cases have increased over the years mostly due to the exposure of MTB to suboptimal levels of drug. Proteasomes provide MTB its pathogenicity and hence helps it to survive inside the host even in the presence of drugs. Materials and Methods The recombinantly expressed proteasome accessory factor-C (PafC) protein was purified via Ni-NTA affinity chromatography and overexpressed in the nonpathogenic strain of mycobacteria (Mycobacterium smegmatis) for the comparative analysis of minimum inhibitory concentrations of antimycobacterial drugs. The bacteria were subjected to various stress conditions. Secretory nature of PafC was analyzed by probing the purified protein against patient sera. Quantitative mRNA analysis of paf C, lex A, and rec A was performed to check for their level under fluoroquinolone (FQ) presence. The data were validated in clinical samples of pulmonary TB patients. Results pafC , that forms one part of paf operon, is involved in providing MTB its resistance against FQs. Through a series of experiments, we established the fact that PafC is upregulated in mycobacteria upon exposure to FQs and it leads to the increased intracellular survival of mycobacteria under the stresses generated by FQs. The study also refers to the correlation of pafC to deoxyribonucleic acid (DNA) damage repair enzymes lexA and recA at transcriptional level. The results obtained in vitro corroborated when the pulmonary TB patients’ samples were subjected to the same molecular analysis. Statistical Analysis All experiments were conducted at least in triplicate. p -Value of <0.05 was considered to be statistically significant Conclusion PafC plays a significant role in providing resistance to mycobacteria against FQ class of drugs by increasing its intracellular survival through increased drug efflux and getting involved with DNA damage repair machinery.