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, 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.

Nosocomial infection and its molecular mechanisms of antibiotic resistance

“…These porins play a significant physiological role in the transport of sugars, amino acids, and phosphates, and so on (63,64). Some hydrophilic antibiotics, such as β-lactams, aminoglycosides, tetracyclines, and some fluoroquinolones, have been shown to pass through the outer membrane porins (65)(66)(67)(68). Thus, deficiency of porins can diminish susceptibility of P. aeruginosa to some antibiotics.…”

AbuO, a TolC-Like Outer Membrane Protein of Acinetobacter baumannii, Is Involved in Antimicrobial and Oxidative Stress Resistance

“…ABAYE2390 (soxR; 453 bp, 150 amino acids [aa] and 17.01 kDa) was amplified using gene-specific primers, which contained NdeI and BamHI sites of the pET28C vector to generate an N-terminal His 6 -SoxR fusion protein. The ability of SoxR to bind the abuO promoter was deciphered by electrophoretic mobility shift assays (EMSAs) as described previously (33). To confirm that the interaction between SoxR and the promoter region of abuO was specific, experiments with competitive [specific, 10-fold excess of cold promoter; nonspecific, poly(dI-dC)] and noncompetitive (bovine serum albumin [BSA]) inhibitors were also performed.…”

“…The growth profiles of the WT, ⌬abuO, and ⌬abuO ⍀abuO strains were monitored in LB at different pHs (5.0, 6.0, 7.0, 8.0, 10.0, and 12.0) for 18 h at 37°C with shaking using Bioscreen C automated growth analysis system (Labsystems, Helsinki, Finland) at the optical density at 600 nm (OD 600 ). The growth inhibition assay was performed as before with slight modifications using ciprofloxacin (0.005 g/ml), ethidium bromide (EtBr [4 g/ml]), and chlorhexidine (1.6 g/ ml) (33). The impact of abuO inactivation on motility behavior and biofilm formation was examined as described before (34).…”

“…Studies to decipher the impact of oxidative stress-inducing agent hydrogen peroxide (H 2 O 2 ) and nitrosative stress-inducing agents (sodium nitroprusside [SNP] and acidified nitrite) on the WT, ⌬abuO, and ⌬abuO ⍀abuO strains were performed as described before (33). The WT, ⌬abuO, and ⌬abuO ⍀abuO strains were exposed to hostile stress agents at different concentrations, such as bile salt deoxycholate, sodium chloride (NaCl), EtBr, acridine orange, acriflavine, rhodamine, safranin, ampicillin, neomycin, ciprofloxacin, chloramphenicol, tetracycline, benzalkonium chloride, chlorhexidine, and triclosan, and survival capability was determined as described before (33).…”

“…Antibiotic susceptibility and MIC were examined using commercial discs and Estrips (Hi Media, Mumbai, India), and data were analyzed according to the interpretation criteria recommended by the CLSI (35). Accumulation assays using fluorescent substrates EtBr and ciprofloxacin and purification of OMPs from the WT, ⌬abuO, and ⌬abuO ⍀abuO strains were done as described before (33).…”