Abstract
The ability of pathogens to perceive environmental conditions and modulate gene expression accordingly is a crucial feature for bacterial survival. In this respect, the heat-shock response, a universal cellular response, allows cells to adapt to hostile environmental conditions and to survive during stress. In the major human pathogen Helicobacter pylori the expression of chaperone-encoding operons is under control of two auto-regulated transcriptional repressors, HrcA and HspR, with the latter acting as the master regulator of the regulatory circuit. To further characterize the HspR regulon in H. pylori, we used global transcriptome analysis (RNA-sequencing) in combination with Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-sequencing) of HspR genomic binding sites. Intriguingly, these analyses showed that HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. Moreover, we further characterized HspR-DNA interactions through hydroxyl-radical footprinting assays. This analysis in combination with a nucleotide sequence alignment of HspR binding sites, revealed a peculiar pattern of DNA protection and highlighted sequence conservation with the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.). Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA. This finding is compatible with a model in which possibly a dimer of HspR recognizes the HAIR motif overlapping its promoter for binding and in turn cooperatively recruits two additional dimers on both sides of the HAIR motif.
Highlights
Helicobacter pylori represents one of the most widespread human pathogens, recognized as the principal causative agent of different gastrointestinal severe diseases such as atrophic gastritis, peptic ulcer, MALT-lymphoma and gastric adenocarcinoma (Gisbert and Calvet, 2011; Salama et al, 2013)
To define the HspR contribution to the heat-shock response, we performed a strand-specific whole transcriptome analysis of the wild type H. pylori G27 strain and of hspR mutant both grown to the exponential growth phase at 37◦C and of the wild type strain subjected to 30 min heat-shock at 42◦C (Supplementary Table S2, Materials and Methods)
We defined the role of HspR in standard growth conditions by comparing the transcriptome of the hspR mutant to that of the H. pylori wild type strain, both grown at 37◦C
Summary
Helicobacter pylori represents one of the most widespread human pathogens, recognized as the principal causative agent of different gastrointestinal severe diseases such as atrophic gastritis, peptic ulcer, MALT-lymphoma and gastric adenocarcinoma (Gisbert and Calvet, 2011; Salama et al, 2013). Helicobacter pylori transcriptional regulators appear to be arranged in different regulatory modules, transducing separate environmental inputs Such regulatory modules are constituted by a master regulator, followed by intermediate regulators and regulatory interactions resulting into a coordinated output of target gene’s expressions (Danielli et al, 2010). Several lines of evidence show that the heat-shock proteins of H. pylori play non-canonical roles and some of them seem to have undertaken additional functions during the interaction with the host (Evans et al, 1992; Huesca et al, 1996; Phadnis et al, 1996; Dunn et al, 1997; Kao et al, 2016) Because of these important functions in the cell, H. pylori has developed regulatory strategies to tightly modulate HSPs expression level in response to environmental signals
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