Abstract
Helicobacter pylori plays an essential role in the pathogenesis of gastritis, peptic ulcer disease, and gastric cancer. The serine protease HtrA, an important secreted virulence factor, disrupts the gastric epithelium, which enables H. pylori to transmigrate across the epithelium and inject the oncogenic CagA protein into host cells. The function of periplasmic HtrA for the H. pylori cell is unknown, mainly due to unavailability of the htrA mutants. In fact, htrA has been described as an essential gene in this bacterium. We have screened 100 worldwide H. pylori isolates and show that only in the N6 strain it was possible to delete htrA or mutate the htrA gene to produce proteolytically inactive HtrA. We have sequenced the wild-type and mutant chromosomes and we found that inactivation of htrA is associated with mutations in SecA – a component of the Sec translocon apparatus used to translocate proteins from the cytoplasm into the periplasm. The cooperation of SecA and HtrA has been already suggested in Streptococcus pneumonia, in which these two proteins co-localize. Hence, our results pinpointing a potential functional relationship between HtrA and the Sec translocon in H. pylori possibly indicate for the more general mechanism responsible to maintain bacterial periplasmic homeostasis.
Highlights
HtrA (High Temperature Requirement A) proteins are a family of evolutionarily well preserved serine proteases, identified in the majority of the examined organisms
The analysis indicated that no htrA gene was present anymore in six H. pylori htrA deletion (N6 ΔhtrA) clones, while htrA was detected in the H. pylori strain with serine 221 into alanine (S221A) mutation (N6 htrAS221A) (Fig. 1AB and Fig. S2)
Using two independent clones of each mutant type, we performed Southern blotting, which confirmed that recombination occurred at the desired locus in both N6 ΔhtrA and N6 htrAS221A strains and that no additional copy of htrA was present in the H. pylori wild-type or mutant strains (Fig. 1D)
Summary
HtrA (High Temperature Requirement A) proteins are a family of evolutionarily well preserved serine proteases, identified in the majority of the examined organisms In addition to their proteolytic activity, several HtrAs exhibit chaperone-like properties. Most of HtrAs are involved in the protein quality control and are responsible for the removal of improperly folded proteins from the cellular envelope using their both, proteolytic and chaperone-like, activities[2] Aside from their housekeeping functions, certain HtrA homologs play regulatory roles, e.g., regulate the σE-dependent stress response[3], others are involved in maturation and secretion of surface proteins, including several virulence factors[4]. In the Gram-positive bacteria, Streptococcus pyogenes and Streptococcus pneumoniae, HtrA co-localizes to the Sec machinery on the cell surface[5,6,7] This fact raises the possibility that HtrA is involved in processing/maturation and/or in quality control of the exported proteins in streptococci. Our work led to construction of a highly valuable new tool for further studies on the role of HtrA in H. pylori physiology and virulence, and indicated for the possible interdependence between Sec translocon and HtrA in H. pylori in maintaining proper homeostasis in the H. pylori periplasm
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