Functional Characterization of a Novel Outer Membrane Porin KpnO, Regulated by PhoBR Two-Component System in Klebsiella pneumoniae NTUH-K2044

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 . Principal Findings 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, Δ kpnO in a clinical isolate of K. pneumoniae . Estimation of uronic acid content confirmed that Δ kpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The Δ kpnO 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 and Significance Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.