title: jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol.
abstract: OBJECTIVE
  Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria.
  
  
  METHODS
  The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H(37)Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil.
  
  
  RESULTS
  In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA.
  
  
  CONCLUSIONS
  The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.
authors: Department of Microbiology & Molecular Biology, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
authors: Anuj Kumar
authors: AK
authors: Gupta
authors: Vineel P
authors: VP
authors: Reddy
authors: Mallika
authors: M
authors: Lavania
authors: D S
authors: DS
authors: Chauhan
authors: K
authors: K
authors: Venkatesan
authors: V D
authors: VD
authors: Sharma
authors: A K
authors: AK
authors: Tyagi
authors: V M
authors: VM
authors: Katoch
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004977:Ethambutol; D007538:Isoniazid; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D026901:Membrane Transport Proteins; D004977:Ethambutol; D007538:Isoniazid