title: Proteome and phosphoproteome analysis of the serine/threonine protein kinase E mutant of Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Serine/threonine protein kinases (STPKs) have prominent roles in the survival mechanisms of Mycobacterium tuberculosis (M. tuberculosis). Previous studies from our laboratory underscored the role of PknE, an STPK in virulence, adaptation and the suppression of host cell apoptosis. In this study, two-dimensional gel electrophoresis was used to study the proteome and phosphoproteome profiles of wild type M. tuberculosis and its isogenic pknE deletion mutant (ΔpknE) during growth in Middlebrook 7H9 and nitric oxide stress.
  
  
  METHODS
  Wild-type M. tuberculosis and its isogenic pknE deletion mutant strain were grown in Middlebrook 7H9 as well as subjected to nitric oxide stress using sodium nitroprusside. Whole cell lysates were prepared and analyzed by 2D-gel electrophoresis. Phosphoproteomes were analyzed using phospho serine and phospho threonine antibodies after subjecting the 2D-gels to western blotting. Proteins of interest were identified using mass spectrometry.
  
  
  RESULTS
  Our analysis provides insights into the targets that impose pro-apoptotic as well as altered cellular phenotypes on ΔpknE, revealing novel substrates and functions for PknE.
  
  
  CONCLUSIONS
  For the first time, our proteome and phosphoproteome data decipher the function of PknE in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two-component systems and in the metabolic activity of M. tuberculosis.
authors: Department of Immunology, National Institute for Research in Tuberculosis, Chennai,India; Department of Immunology, International Centre for Genetic Engineering and Biotechnology, Aruna Asif Ali Marg, New Delhi, 110067,India.
authors: Dinesh Kumar
authors: DK
authors: Parandhaman
authors: Department of Biochemistry, National JALMA Institute for Leprosy and other Mycobacterial Diseases, Tajganj, Agra,India.
authors: Prashant
authors: P
authors: Sharma
authors: Department of Biochemistry, National JALMA Institute for Leprosy and other Mycobacterial Diseases, Tajganj, Agra,India.
authors: Deepa
authors: D
authors: Bisht
authors: Department of Immunology, National Institute for Research in Tuberculosis, Chennai,India. Electronic address: sujatha.sujatha36@gmail.com.
authors: Sujatha
authors: S
authors: Narayanan
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional; D017353:Gene Deletion; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D020543:Proteome; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D020543:Proteome; D009569:Nitric Oxide; D017346:Protein Serine-Threonine Kinases
keywords: 2D gel electrophoresis; Middlebrook 7H9; Mycobacterium tuberculosis; Nitrate stress; Phosphoproteome; PknE; Proteome; Sigma B; Western blotting