Campylobacter jejuniouter membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin

Abstract

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.

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

“…E-cadherin shedding can be induced by host proteases [30, 41] or by bacterial proteases, such as HtrA proteins [15, 27, 30]. To evaluate if HtrAs of S. Typhimurium, Y. enterocolitica , or P. mirabilis are expressed and capable of E-cadherin cleavage, we analyzed the expression of proteolytic active proteases by casein zymography in a first step.…”

“…Furthermore, HtrA proteins are also implicated in the modulation of pathogen-host interaction by processing of surface-presented virulence factors or adhesins [14, 16, 17]. Another important function was observed for secreted or outer-membrane vesicle-associated HtrA from H. pylori and C. jejuni , which directly cleaves the extracellular domain of E-cadherin on host cells [15, 27, 29, 30]. HtrA-mediated E-cadherin cleavage opens intercellular adherens junctions allowing bacterial transmigration across the epithelial barrier [15, 29, 30, 33].…”

“…E-cadherin cleavage during infection has been described for H. pylori , C. jejuni , EPEC and S. flexneri [15, 27, 29]. In this study, we added S. Typhimurium, Y. enterocolitica , and P. mirabilis to the collection of E-cadherin-targeting 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]. E-cadherin is an important key molecule in the establishment and maintenance of an intact epithelial barrier.…”

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

“…Campylobacter jejuni and its close relative Helicobacter pylori actively secrete HtrA proteins in the extracellular environment, where they target host cell factors [19–21]. HtrA was also identified in outer membrane vesicles released by C. jejuni , H. pylori , Vibrio cholera , Chlamydia muridarum or Borrelia burgdorferi [22–26]. Infection experiments with polarized cell monolayers in vitro suggested that H. pylori and C. jejuni HtrA can disrupt the epithelial barrier by opening cell-to-cell junctions.…”