title: Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis sensitive and resistant Beijing type from a patient with pulmonary tuberculosis.
abstract: OBJECTIVE
  In India, tuberculosis (TB) is a foremost health problem, and the emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (M. tuberculosis) has further complicated the situation. Although various mechanisms have been proposed to elucidate the emergence of resistance, our knowledge remains insufficient. The formation of a very complex network and drugs of proteins are countered by their efflux/modification or target over-expression/modification. The analysis of the over-expressed proteins and their qualitative and phenotypic evaluation before and after the development of drug-resistance may be the most appropriate tool to understand the mechanisms of the mechanism of development of drug-resistance. Most studies are performed on distinct strains. Therefore, the objective of this study was to compare the proteomic information of sequential isolates of M. tuberculosis Beijing type from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy.
  
  
  METHODS
  In this study, a clinical isolate of M. tuberculosis was grown in Middlebrook 7H9 broth medium for 2weeks, and the cell lysate of isolates was prepared by sonication and centrifugation. We compared and analyzed the whole cell lysate proteins of M. tuberculosis sequential clinical isolate from a patient with pulmonary TB before and after the development of drug resistance using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and bioinformatics tools.
  
  
  RESULTS
  The genotypes of both isolates remained homologous, showing no re-infection. The first isolate (before treatment) was sensitive to all the first-line drugs, sequential isolate was found resistant to rifampicin (RIF) and isoniazid (INH) and developed mutations in rpoB, katG and inhA. The concentrations of 17 protein spots were found to be consistently over-expressed in RIF- and INH-resistant isolates. The most prominent and over-expressed proteins found during the development of drug resistance were wag31, Rv2714, GarA, SSB, FabG4, Probable lipase, Rv3924c, Rv3204A, Rv2031c, Rv3418c and GroES. The InterProScan and homology searches generated insights into the possible functions and essential domains of the proteins. Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c showed significant in silico interaction with RIF and INH; thus, the over-expression in the drug-resistant isolates could be compensating the inhibited/modulated molecules. Other proteins, which are over-expressed but do not unveil good binding with drug, might be indirectly associated with RIF and INH.
  
  
  CONCLUSIONS
  This proteomic study provides an understanding about the proteins that are over-expressed during the development of drug resistance. These over-expressed proteins, identified here, could prove useful as vaccine candidate, immunodiagnostic and possibly drug-resistant or chemotherapeutic markers in future.
authors.affiliation: Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India. Electronic address: amit4us@gmail.com.
authors.forename: Amit
authors.initials: A
authors.lastname: Singh
authors.affiliation: Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Gupta
authors.affiliation: Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India; Max Planck Institute for Infection Biology, Immunology, Berlin, Germany.
authors.forename: Krishnamoorthy
authors.initials: K
authors.lastname: Gopinath
authors.affiliation: National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
authors.forename: Divakar
authors.initials: D
authors.lastname: Sharma
authors.affiliation: National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Sharma
authors.affiliation: National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
authors.forename: Deepa
authors.initials: D
authors.lastname: Bisht
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Immunology Group, New Delhi, India,; NER-BPMC, Department of Biotechnology, New Delhi, India.
authors.forename: Pawan
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Sarman
authors.initials: S
authors.lastname: Singh
keywords: Beijing; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Multidrug-resistant tuberculosis; Proteomics; Tuberculosis