title: Functional characterization of a novel outer membrane porin KpnO, regulated by PhoBR two-component system in Klebsiella pneumoniae NTUH-K2044.
abstract: BACKGROUND
  The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC(KP), to mediate antimicrobial resistance in K. pneumoniae.
  
  
  RESULTS
  In this study, functional characterization of the putative porin OmpC(KP) (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB(KP) isogenic mutant was constructed. The phoB(KP) mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB(KP) mutant compared to wild type. A regulatory role of PhoB(KP) for the expression of kpnO was further supported by the specific binding of PhoB(KP) to the putative promoter of kpnO.
  
  
  CONCLUSIONS
  Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.
authors: Council of Scientific Industrial Research, Institute of Microbial Technology, Sector 39 A, Chandigarh, India. vijirmohan@gmail.com
authors: Vijaya Bharathi
authors: VB
authors: Srinivasan
authors: Manjunath
authors: M
authors: Venkataramaiah
authors: Amitabha
authors: A
authors: Mondal
authors: Vasanth
authors: V
authors: Vaidyanathan
authors: Tanvi
authors: T
authors: Govil
authors: Govindan
authors: G
authors: Rajamohan
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000378:metabolism / Q000382:microbiology; D004195:Disease Models, Animal; D017353:Gene Deletion; D007710:Klebsiella Infections / Q000235:genetics / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D018272:Porins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000890:Anti-Infective Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D018272:Porins; C061278:PhoB protein, Bacteria; C061279:PhoR protein, Bacteria