International Journal of Respiratory and Pulmonary Medicine Case Report: Open Access ClinMed International Library Citation: Gupta RK, Kumar B, Bisht D, Katoch K, Mitra K, et al. (2015) Differentially Expressed Proteins in Response to Resuscitation of Non-Culturable Cells of Mycobacterium Tuberculosis H37Rv: Potential New Drug Targets. Int J Respir Pulm Med 2:025 Received: April 24, 2015: Accepted: July 29, 2015: Published: August 03, 2015 Copyright: © 2015 Gupta RK. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Gupta et al. Int J Respir Pulm Med 2015, 2:3 ISSN: 2378-3516 Differentially Expressed Proteins in Response to Resuscitation of Non- Culturable Cells of Mycobacterium tuberculosis H37Rv: Potential New Drug Targets Ravi Kr. Gupta 1 , Bhavnesh Kumar 2 , Deepa Bisht 2 , Kiran Katoch 2 , Kalyan Mitra 3 and Ranjana Srivastava 1 * 1 Microbiology Division, Central Drug Research Institute, CSIR, India 2 Department of Biochemistry, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), India 3 Electron Microscopy Unit, Central Drug Research Institute, CSIR, India *Corresponding author: Dr. Ranjana Srivastava., Microbiology Division, Central Drug Research Institute, CSIR, Jankipuram Sector 10. Sitapur Road, Lucknow – 226031, India, Tel: +91-522-4007058, E-mail: ranjanasrivastava5@gmail.com 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 fve 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 identifcation of fourteen mycobacterial proteins that were differentially expressed during non-culturable and resuscitation phase compared to replicating. Five of the unique proteins identifed in the resuscitation and NC phase by 2DE were also confrmed 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 Keywords M. tuberculosis, Non-culturable cells, 2-DE, Scanning electron microscopy, Resuscitation promoting factors rest population bacillus may lie dormant in the body for decades in a latent stage. Te epidemiological data suggest that possibly up to one-third of the world’s population is latently infected with this microorganism [1]. Te latent stage of infection is normally associated with the bacteria surviving in the granuloma for years in a dormant state with low or no metabolic activity [2]. Tese bacteria appear to be resistant to standard chemotherapy and are capable of reactivating under immune suppression. Several in vitro and in vivo animal models have been established in an attempt to mimic the latent state and subsequent reactivation of M. tuberculosis and contributed signifcantly to our understanding of the biology of MTB [3-5]. Dormancy has been duplicated in vitro by growing tubercle bacilli to the extended stationary phase for 5-6 months or subjecting them to nutrient starvation or gradually decreasing the supply of oxygen which shif the actively replicating tubercle bacilli into the state of dormancy [4,5]. While latent bacilli can be reactivated in vivo under generalized immunosuppression, in vitro generated dormant cells can resume growth upon addition of a family of protein known as resuscitation promoting factors (Rpfs) isolated from Micrococcus luteus or Mycobacterium tuberculosis [6,7]. Mycobacterial Rpfs have been demonstrated to play an important role in reactivation of chronic infection and are important for in vivo persistence of M. tuberculosis in mice [8,9,10]. Te expression of rpf genes have been reported in human lung tissue and sputum of pulmonary TB patients. Te non-culturable latent bacilli present in the sputum of TB patients could be resuscitated by mycobacterial Rpfs [11]. Tis was the frst documentation of Rpf dependent resuscitation of non-culturable latent M. tb population in the sputum of TB patient [11]. Te in vitro model of non culturability and subsequent resuscitation by Rpf proteins thus is not only crucial for understanding the molecular mechanisms that controls the transition of replicating mycobacteria to a dormant state and vice versa but also may be examined for its applicability in latent infection and reactivation. Introduction Upon infection with Mycobacterium tuberculosis, only an estimated 10% of the people develop the active disease and in the