Abstract
BackgroundThe 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 OmpCKP, to mediate antimicrobial resistance in K. pneumoniae.Principal FindingsIn this study, functional characterization of the putative porin OmpCKP (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 PhoBKP for the expression of kpnO was further supported by the specific binding of PhoBKP to the putative promoter of kpnO.Conclusions and SignificanceLoss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.
Highlights
The cell envelope of Gram-negative bacteria consists of three principal layers: the outer membrane (OM), the peptidoglycan cell wall, and the inner membrane [1]
Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044
Bioinformatic Analysis of KpnO The nucleotide sequence deduced from 1098 bp DNA fragment obtained from K. pneumoniae NTUH-K2044 shared .80% identity with the OmpC porins from other Gram negative pathogens [20]
Summary
The cell envelope of Gram-negative bacteria consists of three principal layers: the outer membrane (OM), the peptidoglycan cell wall, and the inner membrane [1]. The OM of Gramnegative bacteria plays a significant role in a variety of functions; it serves as a diffusion barrier to extracellular solutes and interacts with the bacterial environment. Influx is largely controlled by porins that are represented in large amounts in the OM. They form water-filled open channels that span the OM and allow the passive penetration of small hydrophilic molecules Porins serve as receptors for bacteriophages and bacteriocins and, in conjunction with peptidoglycan and LPS, play a significant role in maintaining the integrity of bacterial cells. The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. We demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpCKP, to mediate antimicrobial resistance in K. pneumoniae
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