H-NS Nucleoid Protein Controls Virulence Features of Klebsiella pneumoniae by Regulating the Expression of Type 3 Pili and the Capsule Polysaccharide

Abstract

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae.

Keywords: H-NS; K. pneumoniae; T3P; adherence; capsule; phagocytosis; virulence.

Figure 1

H-NS protein in K. pneumoniae. (A) Alignment of amino acid sequences of H-NS proteins from several enterobacteria: K. pneumoniae (MGH, 342 and NTUH-K2044), Shigella dysenteriae (Sd197), Shigella boydii (Sb227), Shigella flexneri (2a str. 2457T), Enterohemorragic E. coli (EDL933), Enteropathogenic E. coli (E2348/69), E. coli K-12 (MG1655), Salmonella enterica serovar Typhimurium (LT2), Salmonella enterica serovar Typhi (CT18), Yersinia pestis (KIM 10), Yersinia pseudotuberculosis (IP 32953) and Yersinia enterocolitica (8081). Analysis was performed using the ClustalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Growth kinetics of wild-type K. pneumoniae, and the isogenic mutants at 37°C (B) and 25°C (C). Bacterial cultures were grown for 8 h in LB medium.

Figure 2

H-NS positively regulates mrkA expression. (A) Fold-change expression (qRT-PCR) of the pilin genes and their regulators in the Δhns mutant as compared to the K. pneumoniae wild-type strain. (B) Quantification of biofilm formation by measuring Crystal Violet uptake. (C) Quantification of biofilm formation by measuring Crystal Violet uptake overexpressing the MrkH activator protein (0.1% arabinose) in both wild-type and hns background. Results shown represent mean and standard deviations of 3 independent experiments performed. ns, not significant; statistically significant with respect to the wild-type strain ^***p < 0.001; ^**p < 0.01; ^*p < 0.05.

Figure 3

H-NS represses capsular polysaccharide in K. pneumoniae. (A,B) Mucoviscosity of K. pneumoniae wild-type, Δhns mutant, complemented Δhns mutant, ΔmrkA mutant, Δcps mutant, and Δhns Δcps mutant. The mucoviscosity was determined by low speed centrifugation and is expressed as OD₆₀₀ of the supernatant. (C) Capsule quantification of K. pneumoniae strains. The glucuronic acid concentration in each strain was determined from capsular polysaccharides extracted of 0.5 ml bacterial cultures. (D) Transcriptional expression (qRT-PCR) of the rcsA, galF, wzi, and manC genes in the WT K. pneumoniae strain, Δhns mutant and complemented Δhns mutant. Data represent the mean of at least three independent experiments (mean ± SD). Statistically significant with respect to the wild-type strain ^***p < 0.001; ^**p < 0.01; ^*p < 0.05.

Figure 4

H-NS thermoregulates both fimbrial and capsular genes. Fold-change expression (qRT-PCR) of the fimbrial (A) and capsular genes (B) in the Δhns mutant as compared to the K. pneumoniae wild-type strain. Data represent the mean of at least three independent experiments (mean ± SD). Statistically significant with respect to the wild-type strain ^***p < 0.001; ^**p < 0.01.

Figure 5

Adherence and phagocytosis of the K. pneumoniae Δhns mutant. (A) Comparison of adherence levels of K. pneumoniae wild-type, Δhns mutant, complemented Δhns mutant, ΔmrkA mutant, Δcps mutant, and Δhns Δcps mutant to HeLa and A549 cells. (B) Adherence levels of wild-type strain and isogenic Δhns mutant to HeLa cells, overexpressing the MrkH activator protein (0.1% arabinose). (C) Comparison of phagocytic uptake of indicated K. pneumoniae strains by RAW264.7 and THP-1 macrophages. (D) Adherence levels of K. pneumoniae wild-type strain and isogenic mutants to RAW264.7 and THP-1 macrophages. Results represent means and standard deviations of the results obtained from the 3 experiments performed in triplicates. ns, not significant; statistically significant with respect to the wild-type strain ^***p < 0.001; ^**1p < 0.01; ^*p < 0.05.