The Helicobacter pylori HrcA repressor: Study of the Global Transcriptional Response during Heat Shock.

The ability to respond quickly to environmental changes and thus modulate gene expression is a crucial skill exploited by bacteria for their survival. In this context, the widespread human pathogen Helicobacter pylori, under stress conditions induces the synthesis of a class of highly conserved proteins, called Heat Shock Proteins. In this bacterium, the major heat-shock genes are negatively regulated by two transcriptional repressors, HspR and HrcA. Although the heat-shock regulatory circuit is well-studied, not enough is known about the global heat shock response in H. pylori. In order to identify other potential cellular process regulated by HrcA, we performed differential transcriptional analysis of heat shock regulation by RNA-seq, comparing the transcriptome of the H. pylori G27 wild type strain subjected and not subjected to heat shock stress versus the HrcA isogenic G27 mutant strain. This analysis showed that most of the differentially expressed genes were deregulated only under heat shock treatment. Moreover, several non-heat shock responsive genes were deregulated in the HrcA mutant. To further characterize the HrcA regulon in H. pylori we attempted setting up a ChIP-seq experiment. However, the HrcA protein appears to be poorly immunogenic, for this reason we used a strategy in which an epitope (3XFLAG), recognized by commercial antibodies, was fused to the N-terminal region of the HrcA protein. In addition, three levels of expression (weak, intermediate and strong) of the 3XFLA-HrcA were obtained by using three known H. pylori promoters. The immunoprecipitation assays performed showed a poor enrichment of DNA fragments bound by HrcA and hindered the identification of novel in vivo HrcA binding sites. Thereafter, we further analyzed the HrcA-DNA interactions on some putative targets through DNaseI footprinting assays on novel putative target promoters binding of the regulator could not be detected and possibly transcriptional regulation by HrcA of these genes is indirect

Helicobacter pylori Stress-Response: Definition of the HrcA Regulon

Bacteria respond to different environmental stresses by reprogramming the transcription of specific genes whose proper expression is critical for their survival. In this regard, the heat-shock response, a widespread protective mechanism, triggers a sudden increase in the cellular concentration of different proteins, including molecular chaperones and proteases, to preserve protein folding and maintain cellular homeostasis. In the medically important gastric pathogen Helicobacter pylori the regulation of the principal heat-shock genes is under the transcriptional control of two repressor proteins named HspR and HrcA. To define the HrcA regulon, we carried out whole transcriptome analysis through RNA-sequencing, comparing the transcriptome of the H. pylori G27 wild type strain to that of the isogenic hrcA-knockout strain. Overall, differential gene expression analysis outlined 49 genes to be deregulated upon hrcA gene inactivation. Interestingly, besides controlling the transcription of genes coding for molecular chaperones and stress-related mediators, HrcA is involved in regulating the expression of proteins whose function is linked to several cellular processes crucial for bacterial survival and virulence. These include cell motility, membrane transporters, Lipopolysaccharide modifiers and adhesins. The role of HrcA as a central regulator of H. pylori transcriptome, as well as its interconnections with the HspR regulon are here analyzed and discussed. As the HrcA protein acts as a pleiotropic regulator, influencing the expression of several stress-unrelated genes, it may be considered a promising target for the design of new antimicrobial strategies