Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant.

Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Streptomycin induced protein expression analysis in Mycobacterium tuberculosis by two-dimensional gel electrophoresis & mass spectrometry.

Background & objectives: The resistance of Mycobacterium tuberculosis to streptomycin, a core drug for treatment of category II tuberculosis (TB) has posed a major challenge to the health providers as well as research workers worldwide and has severely compromised the therapeutic options. A significant proportion of streptomycin resistant M. tuberculosis isolates failed to show mutations in conventional targets like rpsL and rrs. Although efflux, permeability, etc. are also known to contribute, yet a substantial proportion of isolates remains resistant suggesting involvement of other unknown mechanism. A resistant isolate may show altered gene as well as protein expression under drug induced conditions and a whole cell proteome analysis under induced conditions might help in further understanding the mechanisms of drug resistance. The present study was therefore designed with the objective to identify proteins related to streptomycin resistance in M. tuberculosis isolate grown in presence and absence of streptomycin (SM). Methods: A clinical isolate of M. tuberculosis from Mycobacterial Repository Centre at the Institute (NJIL & OMD), Agra was grown in Sauton’s medium for 36 h with/without subinhibitory concentration of the drug (2 μg/ml) and the cell lysate of isolates was prepared by sonication and centrifugation. Two-dimensional (2D) gel electrophoresis was employed to study the protein profile. The selected proteins were finally identified by MALDI-TOF mass spectrometry. Results: Our study revealed eight inducible proteins (DnaK, fabG4, DNA-binding, hypothetical, two 14 kDa antigen and two 10 kDa chaperonin) that were upregulated in the presence of drug. Interpretation & conclusion: This preliminary study has thrown light on whether or not and how the resistant isolate responds to streptomycin at its non-toxic but sub-inhibitory concentration. An in-depth study of the upregulated proteins will give an insight into probable sites of drug action other than established primary sites.

A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis

Sample preparation for Two-dimensional gel electrophoresis (2DE) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of M. tuberculosis. High lipid content in mycobacteria limits the use of common methods as it can hinder the 2DE run. This study highlights the significance of SDS-TCA procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.

Analysis of mycobacterial strains by two-dimensional gel electrophoresis.

This study pertains to analysis of the protein profile of different mycobacterial strains by two-dimensional gel electrophoresis (2DE). The strains were selected as they exhibit different phenotypic behaviour. TCA-acetone precipitated proteins were resolved by 2DE using immobilized pH gradient (IPG) strips. This study demonstrates that 2DE may be used as a tool for characterization of mycobacterial strains. Visual examination of the electrophoretograms was sufficient for characterization. Detailed characterization of specific proteins might lead to development of novel targets, diagnostic probes or sub-unit vaccine(s) against tuberculosis.