Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

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

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.