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Abstract
BackgroundThe serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function.MethodsE-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine—to—alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments.ResultsInfection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro.ConclusionsThese data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0153-y) contains supplementary material, which is available to authorized users.
Highlights
The serine proteases High temperature requirement A (HtrA)/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions
We investigated E-cadherin shedding in response to infection with the Gram-negative gastrointestinal pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Y. enterocolitica, S. enterica subsp
Typhimurium and Y. enterocolitica express DegP, DegS and DegQ, and the genome of P. mirabilis contains DegQ and DegS. To analyze their capacity to induce E-cadherin ectodomain shedding during infection, epithelial cells were colonized with selected pathogens and E-cadherin cleavage was investigated through detection of the loss of full length E-cadherin (E-cadFL) in whole cell lysates and the formation of the soluble Nterminal fragment (E-cadNTF) in the supernatants of infected cells
Summary
The serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function. The serine proteases HtrA/DegP are important key players in protein quality control and stress response through refolding and degrading misfolded proteins in the periplasm of bacteria [1, 2]. DegS is considered as a regulatory protease targeting the antisigma factor RseA in the periplasm, which is implicated in sensing protein folding stress. After detecting misfolded outer membrane proteins, DegS processes the anti-sigma factor RseA, which is followed by RseP cleavage. As a regulated intramembrane proteolysis cascade, this leads to the sigma-E-mediated expression of factors involved in protein folding stress in the periplasm and assembly of outer membrane proteins [3, 12]
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