Differentially Expressed Proteins in Response to Resuscitation of Non-Culturable Cells of Mycobacterium tuberculosis H37Rv: Potential New Drug Targets

Abstract

The major obstacle in TB eradication is attributed to the dormant state of the TB bacilli, Mycobacterium tuberculosis and is characterized by asymptomatic, non-culturability and antibiotic resistant state of disease. Conventional antibiotic therapy is only applicable to the active form of the disease. These non-replicating cells can resume the growth in immunosuppression state and cause the symptomatic disease. The non-culturability (NC) can be generated in vitro in M. tuberculosis and their resuscitation to active growth has been demonstrated by the five resuscitation promoting factors (Rpfs) encoded by M. tuberculosis supporting the applicability of Rpf mediated resuscitation as a model for the study of dormancy and reactivation. In this study, M. tuberculosis H37Rv cells in replicating, non-culturable and resuscitation phase were generated in vitro and analysed by scanning electron microscopy for morphological changes that occurred during the transition of replicating bacilli to non-culturable to resuscitation phase. Two dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry was performed for proteins differentially expressed in three different stages. The analysis led to the identification of fourteen mycobacterial proteins that were differentially expressed during non-culturable and resuscitation phase compared to replicating. Five of the unique proteins identified in the resuscitation and NC phase by 2DE were also confirmed by western immunoblotting. The differential expression of 14 proteins in NC and resuscitation phase was also regulated at transcriptional level as revealed by real time RT-PCR analysis and could thus become potential drug targets