Proteomic Analysis and Sequential Events During the in-vivo Acquisition of Drug Resistance in Clinical Isolates of <i>Mycobacterium tuberculosis</i>

Aim This study was undertaken to compare the proteomic profile of sequential isolates of Beijing lineage Mycobacterium tuberculosis (M. tuberculosis) from a patient who developed drugresistant tuberculosis (TB) in vivo during anti-tuberculosis therapy (ATT). Introduction Various studies have found the Beijing lineage of M. tuberculosis strongly associated with multidrug resistance (MDR) development. Objectives To identify and characterize the differentially expressed proteins during the in-vivo drug resistance conversion in M. tuberculosis Beijing lineage clinical isolates. Methods Drug-susceptible and drug-resistant M. tuberculosis isolates were confirmed as Beijing lineage. The isolates were grown in Middlebrook 7H9 medium for two weeks, and whole-cell lysate was prepared. Two-dimensional gel electrophoresis (2DGE) was used for proteomic analysis, and differentially expressed proteins were identified using MALDI-TOF-MS. Bioinformatics tools were used for molecular docking, phosphorylation, and pupylation site prediction. Results Seventeen proteins were found overexpressed in drug-resistant isolates as compared to drugsusceptible isolates, including the six proteins with unknown functions. Molecular docking showed that Isoniazid (INH) and Rifampicin (RIF) interacted with their conserved domains/active sites of these proteins. Discussion We characterized two paired clinical isolates from a patient, one being INH and RIF susceptible and other resistant. The comparative analysis of over expressed proteins showed that 5 of 17 proteins belonged to the cell wall and cell processes functional group, 3 to virulence, detoxification, adaptation functional group, and 3 to information pathways functional group, 2 proteins belonged to insertion sequences and phage functional group, and 1 each (Rv0242c, Rv2970c and Rv3208A) to lipid metabolism, intermediary metabolism & respiration and regulatory functional group. We found that the Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c proteins showed significant interaction in-silico with INH and RIF. Conclusions These over-expressed proteins probably play an important role in drug resistance development, and further studies on drug resistance mechanisms could provide more details. We also believe that these over-expressed proteins could be used as biomarkers for early prediction of in-vivo drug-resistance development.