title: Proteomic analysis of Mycobacterium tuberculosis isolates resistant to kanamycin and amikacin.
abstract: Kanamycin (KM) and amikacin (AK) are the key aminoglycoside drugs against tuberculosis (TB) and resistance to them severely affects the options for treatment. Many explanations have been proposed for drug resistance to these drugs but still some mechanisms are unknown. Proteins are the functional moiety of the cell and manifest in most of the biological processes; so, these are potential foci for the development of new therapeutics, diagnostics and vaccine. We examined the KM and AK resistant isolates of Mycobacterium tuberculosis using proteomic analysis comprising of two dimensional gel electrophoresis (2DGE), matrix assisted laser desorption ionization time-of-flight/time-of flight (MALDI-TOF/TOF) and bioinformatic tools like BLASTP, InterProScan, KEGG motif scan and molecular docking. Proteins intensities of twelve spots were found to be consistently increased in KM and AK resistant isolates and these were identified as Rv3867, Rv1932, Rv3418c, Rv1876, Rv2031c, Rv0155, Rv0643c, Rv3224, Rv0952, and Rv0440. Among these, Rv3867 and Rv3224 were identified as proteins with unknown function. All the proteins identified were cellular proteins. Molecular docking shows the proper interaction of both drugs with these molecules. Also, Rv1876 and Rv3224 were found to be probably involved in iron regulation/metabolism indicating the role of iron in imparting resistance to second line drugs.
  
  
  CONCLUSIONS
  The study that was carried out shows that two dimensional electrophoresis along with mass spectrometry is still the best approach for proteomic analysis. To the best of our knowledge it is the first ever report on proteomic analysis of M. tuberculosis isolates resistant to second line drugs (kanamycin and amikacin). The major finding implicates that the genes/proteins involved in iron metabolism and the two hypothetical proteins (Rv3867 and Rv3224) might be playing some crucial role in contributing resistance to second line drugs. Further exploitation in this direction may lead to the development of newer therapeutics against tuberculosis.
authors.affiliation: Department of Biochemistry, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (Indian Council of Medical Research), Dr. Matsuki Miyazaki Road, Tajganj, PO Box No-1101, Agra, PIN-282004 India.
authors.forename: Bhavnesh
authors.initials: B
authors.lastname: Kumar
authors.forename: Divakar
authors.initials: D
authors.lastname: Sharma
authors.forename: Prashant
authors.initials: P
authors.lastname: Sharma
authors.forename: Vishwa Mohan
authors.initials: VM
authors.lastname: Katoch
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Venkatesan
authors.forename: Deepa
authors.initials: D
authors.lastname: Bisht
mesh_terms: D000583:Amikacin / Q000737:chemistry / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007612:Kanamycin / Q000737:chemistry / Q000494:pharmacology*; D062105:Molecular Docking Simulation; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D020543:Proteome; D007612:Kanamycin; D000583:Amikacin; D007501:Iron
keywords: 2D gel electrophoresis; Amikacin; Kanamycin; MALDI TOF/TOF; Mass spectrometry; Mycobacterium tuberculosis