Klebsiella pneumoniae TolC contributes to antimicrobial resistance, exopolysaccharide production, and virulence

Abstract

Klebsiella pneumoniae is a Gram-negative bacterium that causes a variety of human diseases, ranging from pneumonia to urinary tract infections and invasive diseases. The emergence of K. pneumoniae strains that are resistant to multiple antibiotics has made treatment more complex and led to K. pneumoniae becoming a global health threat. Addressing this threat necessitates the development of new therapeutic strategies to combat this pathogen, including strategies to overcome antimicrobial resistance and therapeutics for novel targets such as antivirulence. Here, we investigated the function of TolC, an outer membrane protein essential for the function of tripartite transporters, in K. pneumoniae. Mutation of tolC rendered K. pneumoniae hypersensitive to multiple antibiotics. Moreover, the tolC mutation impaired capsule production and affected the expression of key capsule biosynthetic genes, indicating a regulatory role for TolC in capsule biosynthesis. Additionally, TolC was essential for growth under iron-limiting conditions, suggesting its involvement in iron acquisition. The tolC mutant exhibited increased adherence to human enterocytes and enhanced serum sensitivity. In the Galleria mellonella infection model, the tolC mutant displayed reduced virulence compared to the wild type. Our findings highlight the pleiotropic role of TolC in K. pneumoniae pathobiology, influencing antimicrobial resistance, capsule production, iron homeostasis, adherence to host cells, and virulence. Understanding the multifaceted role of TolC in K. pneumoniae may guide the development of new therapeutic strategies against this pathogen. .

Keywords: Klebsiella pneumoniae; antimicrobial resistance; capsule; hypermucoviscosity; pathogenesis; virulence.

FIG 1

Growth of the tolC mutant in iron-limiting conditions. WT and ΔtolC were inoculated in triplicate wells of a microtiter plate containing (A) LB medium or (B) LB medium containing 100 μM 2,2′-dipyridyl or 100 μM 2,2′-dipyridyl plus 100 μM FeCl₃ as described in the Materials and Methods. The cultures were then incubated at 37°C in a microplate reader, and growth was monitored by the change in OD₆₃₀ over (A) 18 or (B) 12 h. The results are the average of triplicate replicates and representative of three independent experiments. Statistical significance was determined by a (A) t-test or (B) one-way analysis of variance of the area under the curve. *, P < 0.05; **, P < 0.001 relative to all other samples.

FIG 2

Mucoviscosity, uronic acid production, and capsule gene expression in K. pneumoniae ΔtolC. (A) The optical density at 600 nm of hypermucoviscous cells that remain in suspension after low-speed centrifugation of 1 mL of a saturated overnight LB culture of the test strains that were normalized by their optical density at 600 nm prior to centrifugation. (B) The mean capsular polysaccharide (CPS) production of K. pneumoniae wild-type, ΔtolC, and ΔtolC complemented with pBAD33 or pBAD33-tolC was quantified in triplicate by measuring uronic acid content and normalized to the optical density at 600 nm. The data represent the mean and standard deviation between WT and the tolC mutants. (C) Relative expression of capsule genes in the ΔtolC mutant relative to expression in the WT strain following growth in LB medium to middle logarithmic state (OD₆₀₀ ~1.0). The data are the means and standard deviations from three independent experiments. Statistical significance was determined using a (A) paired t-test or (B) one-way analysis of variance with Dunnett’s test, comparing each mean value to that of the WT or (C) a t-test relative to a hypothetical value of 100%. *, P < 0.05.

FIG 3

TolC contributes to biofilm production. Bacterial cells attached to the wall of borosilicate glass test tubes following growth for 72 h at room temperature were stained with crystal violet and photographed (A and B) before the biofilm-associated crystal violet was dissolved in DMSO and quantified by measuring the absorbance at 550 nm (C and D) as described in the Materials and Methods. (A) Photograph of crystal violet-stained biofilms produced by KPPR1 and the isogenic ΔtolC mutant and (B) quantification of biofilms following destaining with DMSO. The arrow in panel A indicates the location of the air-liquid interface. Complementation of ΔtolC (B and D). The indicated strains bearing pBAD33 or pBAD33-tolC were cultured at room temperature for 72 h in LB media containing chloramphenicol and the indicated concentrations of arabinose before being (B) stained with crystal violet and photographed and (D) quantified after dissolving the dye with DMSO. Statistical significance was determined relative to WT using a t-test. *, P < 0.05 relative to WT; **, P < 0.05 relative to WT and ΔtolC.

FIG 4

Serum bactericidal activity. K. pneumoniae KPPR1 wild-type, ΔtolC, and ΔtolC bearing pBAD33 or pBAD33-tolC were incubated with normal human serum for 60 min, when the number of viable cells was determined by dilution plating on LB agar. Cell survival is expressed as the number of surviving cells in the serum-treated samples versus the total number of input cells. The results are the mean and standard deviation of three independent experiments. Statistical significance was determined using a one-way analysis of variance with Dunnett’s test. *, P < 0.05 relative to WT; **, P < 0.05 relative to each of the other groups.

FIG 5

K. pneumoniae ΔtolC adherence to HT29 and HT29-MTX cells. HT29 (black bars) and HT29-MTX (green bars) cell monolayers were incubated with washed K. pneumoniae KPPR1 (WT), an isogenic ΔtolC mutant, and the pXB506-complemented ΔtolC mutant at 37°C for 2 h. Monolayers were then washed five times with phosphate buffered saline. The monolayer cells were then collected, and the adherent bacteria were enumerated by plating serial dilutions on LB agar plates. Adherence is expressed as the percentage of attached bacteria relative to the total number of input bacteria. The presented data are the means and standard deviations of three independent experiments. Statistical significance was determined using a one-way analysis of variance with Dunnett’s test, comparing each mean value to that of the WT. *, P < 0.05.

FIG 6

K. pneumoniae ΔtolC virulence in G. mellonella infection model. Kaplan-Meier killing curves for G. mellonella larva. G. mellonella larvae (n = 13–15 larvae per test strain) were challenged with 2 × 10⁵ CFU of K. pneumoniae wild-type strain XBK14, ΔtolC strain XBK17, or XBK17 bearing pXB506 (pBAD33-tolC) as described in the Materials and Methods. The infected larvae were then incubated at room temperature, and larval survival was monitored every 12 h over 3 days. Statistical significance relative to the WT was determined using a log-rank (Mantel-Cox) test in GraphPad Prism version 9.4.1. *, P < 0.05; **, not significant.