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

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 drug-resistant tuberculosis (TB) in vivo during anti-tuberculosis therapy (ATT). Background: 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 drug-susceptible 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, detoxifica-tion, adaptation functional group, and 3 to information pathways functional group, 2 proteins be-longed 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 de-velopment, 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.