Navais, R. | Search bioRxiv

bioRxiv2022

RpoN is required for a functional type III secretion system in Yersinia pseudotuberculosis

Pathogenic bacteria use a broad range of virulence factors to thrive within their host. Yersinia pseudotuberculosis, a gram-negative enteropathogen in humans, utilises a Type III secretion system to overcome the hosts innate immune response. A global regulator previously shown to be essential for virulence in Y. pseudotuberculosis is RpoN. We show here that a strain lacking RpoN has a severely reduced capacity to secrete Yop effectors. This strain has a substantially reduced expression of genes encoding structural components of the secretion apparatus, the ysc operons, while expression of genes encoding effectors and translocators is less affected. We show that RpoN regulates a complex network, where one part is suggested to contribute to inducing Type III secretion via the enhancer-binding protein GlrR/RpoN regulon. By analogy, during inducement of Type III secretion, RpoN has a positive effect on expression of the sigma factor RpoE, which also is known to act downstream of GlrR. Interestingly, we found putative RpoE binding sites upstream of the ysc operons, and also a partial rescue of Yop secretion in the rpoN mutant strain by overexpression of RpoE. Our findings suggest that the RpoN-mediated effect on expression of type III genes involves a sigma factor hierarchy, where RpoN via RpoE contributes to inducing Type III secretion.

molecular biology↗

bioRxiv2020

Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis

RpoN, an alternative sigma factor commonly known as sigma 54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN-binding motif located at 103 inter- and intragenic sites on both sense and anti-sense strands. Deletion of rpoN had a large impact on gene expression, including down-regulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the anti-sense strand commonly reduced expression of genes encoded by the corresponding sense strand. IMPORTANCEThe alternative sigma factor, RpoN ({sigma} 54), which is widely distributed in eubacteria have been implicated to control gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first of its kind study of RpoN in Y. pseudotuberculosis revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA providing important information about RpoN regulation in enterobacteria.

microbiology↗