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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. 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, ?kpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ?kpnO produced ?2.0 fold lesser capsular polysaccharide than the wild-type. The ?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.

SUBMITTER: Srinivasan VB

PROVIDER: S-EPMC3405095 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Functional characterization of a novel outer membrane porin KpnO, regulated by PhoBR two-component system in Klebsiella pneumoniae NTUH-K2044.

Srinivasan Vijaya Bharathi VB Venkataramaiah Manjunath M Mondal Amitabha A Vaidyanathan Vasanth V Govil Tanvi T Rajamohan Govindan G

PloS one 20120725 7

<h4>Background</h4>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.<h4>Principal findings</h4>In this study, functional characterization of the putative porin OmpC(KP) (denoted kpnO) with respect to antimicrobial s ...[more]

PMID: 22848515

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Project description:BackgroundKlebsiella pneumoniae is a gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.Principal findingsIn the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR(KP) to promoter region of ompC(KP) in a concentration dependent manner.Conclusions and significanceThe Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.

Dataset Information

Functional characterization of a novel outer membrane porin KpnO, regulated by PhoBR two-component system in Klebsiella pneumoniae NTUH-K2044.

Publications

Functional characterization of a novel outer membrane porin KpnO, regulated by PhoBR two-component system in Klebsiella pneumoniae NTUH-K2044.

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