Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA_Hp) belongs to the well conserved family of serine proteases. HtrA_Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA_Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrA_Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA_Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA_Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (T_m) values of above 85^∘C. Moreover, HtrA_Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA_Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Background: Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. Methods: The in vitro study of H. pylori HtrA (HtrA_Hp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrA_Hp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrA_Hp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. Results: We demonstrate that HtrA_Hp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrA_Hp variants during infection of human epithelial cells and for E-cadherin cleavage. Conclusion: Here we identified the chaperone activity of the HtrA_Hp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrA_Hp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

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.

Background: An increasing resistance of bacteria to the commonly used antimicrobials forces to search for alternative or supportive ways to cure infections. Targeting virulence factors is one of such approaches. The bacterial HtrA proteins are strongly involved in virulence and the lack of functional HtrA in many cases impairs invasiveness of pathogens. HtrAs act by protecting the cells under stressful conditions as well as they take direct part in invasion of the host. The latter function is played predominantly by the recently identified extracellular fraction of HtrA. This review aims to evaluate HtrAs as therapeutic targets, including design of chemical inhibitors and vaccines. Methods: We undertook a thorough search of bibliographic databases for peer-reviewed scientific literature. Results: One hundred and sixty-four papers were included in the review. First, we briefly summarized key structural and functional properties of known HtrA proteins with the special focus on the extracellular HtrA fraction. Then we provided an overview of efforts and advancements to target HtrAs of pathogenic bacteria as a promising antimicrobial therapy. In some cases, encouraging results were obtained and application of HtrAspecific inhibitors protected tissues from damage and killed bacteria. Also promising reports concerning the use of HtrA as a protective antigen in several disease models have recently been published. Conclusion: The findings of this review suggest that the exported HtrA proteins are very attractive therapeutic targets due to their accessibility, significance in virulence and immunogenicity. However, further extensive studies are still needed to develop a safe antimicrobial treatment.