Abstract
BackgroundTuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.ResultsOn comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.ConclusionsMass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.
Highlights
Tuberculosis (TB) is the most threatening infectious disease globally
2-DE of intracellular M.tuberculosis multidrug resistant (MDR) isolate proteins and their MALDI-MS analysis Comparison of composite 2-DE gels of broth-cultured M. tuberculosis MDR and sensitive isolates with intraphagosomally grown M.tuberculosis MDR isolate showed sixteen protein spots to be upregulated/ expressed by the intraphagosomal mycobacteria (Figure 1B)
MALDI-MS identification of the intracellular MDR and sensitive isolates revealed that majority of the common proteins upregulated/expressed in the intracellular state belonged to intermediary metabolism and respiration category
Summary
Tuberculosis (TB) is the most threatening infectious disease globally. progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. Different approaches have been used to study mycobacterial genes that play a role in the interaction with host cells and in virulence These include in vivo- induced antigen technology, subtractive hybridization, in vivo expression technology etc. Identification of mycobacterial proteins of drug resistant and sensitive isolates by two-dimensional electrophoresis (2-DE) and mass spectrometry has largely been applied to broth grown cultures, because abundant amounts of protein are available here for analysis and comparison. Such studies have been carried out by us [9] and other researchers [10,11]. Our knowledge regarding the proteomic profiles of drug resistant and sensitive M.tuberculosis during intracellular growth have been fragmentary
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