Extracellular secretion of protease HtrA fromCampylobacter jejuniis highly efficient and independent of its protease activity and flagellum

Abstract

The serine protease HtrA of C. jejuni has been identified as a novel secreted virulence factor which opens cell-to-cell junctions by cleaving E-cadherin. Efficient C. jejuni transmigration across polarized human epithelial cells requires the intact flagellum and HtrA; however, the mechanism of HtrA secretion into the supernatant is unknown. Here we show that HtrA secretion is highly efficient and does not require its proteolytic activity because the protease-inactive S197A mutant is secreted like wild-type HtrA. In addition, the flagellar mutants ΔflaA/B, ΔfliI, ΔflgH, ΔflhA, ΔflhB, and ΔflgS were also able to secrete HtrA in high amounts, while they were strongly attenuated in secreting the well-known invasion antigen CiaB. We also tested several culture media and cell lines of different origin such as human, mouse, hamster, dog, and chicken for their ability to influence HtrA secretion. Interestingly, HtrA was effectively secreted in the presence of most but not all cell lines and media, albeit at different levels, but secretion was significantly higher when fetal calf serum (FCS) was added. These results demonstrate that HtrA secretion by Campylobacter proceeds independent of HtrA's protease activity, the flagellum and origin of cell lines, but can be strongly enhanced by molecular compound(s) present in FCS.

Campylobacter jejuniserine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

“…Commonly, HtrAs exhibit no sequence homology to typical autotransporters, which process themselves by autoproteolysis. This is in line with our observation that the HtrA protease activity is not required for secretion [39]. Thus, HtrA very likely requires the assistance of other bacterial factors for delivery.…”

“…We have generated several ΔhtrA deletion mutants in the well-known C. jejuni strains 81-176 and NCTC11168 [29,39]. Next aim was to develop a fast and reliable genetic complementation system of the wt htrA gene for further investigation of the importance of this factor both during conventional growth and infection processes.…”

“…Finally, we aimed to analyze if the complemented C. jejuni strains can restore the transmigration defect of ΔhtrA mutants across polarized epithelial cells reported recently [26,39]. To test this important function of HtrA, polarized MKN-28 cells were seeded and differentiated over 14 days in a transwell fi lter system.…”

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens

“…During infection with H. pylori and C. jejuni , HtrA is secreted into the microenvironment [25, 26] and was detected in outer membrane vesicles (OMVs) [27, 28]. H. pylori and C. jejuni HtrAs cleave-off the extracellular domain of the cell adhesion protein E-cadherin on epithelial cells [15, 27, 29, 30].…”

Bacterial serine protease HtrA as a promising new target for antimicrobial therapy?

“…Consequently, this observation leads to the question if such a factor might also serve as a potent macromolecular drug target? In fact, targeting HtrA offers some potential advantages:it is secreted into the extracellular micro-milieu or presented on the bacterial cell surface and therefore accessible to drug compounds [43, 44],it has a defined enzymatic active site and substrate recognition [19, 20, 45, 46],it cleaves E-cadherin, proteoglycans and fibronectin as host factors with important functions for bacterial pathogenesis [19–21, 47], andit is an essential enzyme in H. pylori physiology [40, 41]. …”