Journal of Cellular Immunology and Serum Biology
Expression of Helicobacter pylori Serine Protease HtrA in Campylobacter jejuni Reveals a Crucial Function in Oxygen Stress Resistance, Heat Tolerance and Epithelial Barrier Disruption
- 1Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen/Nuremberg, Staudtstr 5, D-91058 Erlangen, Germany
- 2Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland
Steffen Backert Ph.D., Friedrich Alexander University Erlangen/Nuremberg Department of Biology, Division of Microbiology, Staudtstr 5, D-91058 Erlangen, Germany, Tel: 49 9131 8528588; Fax: 49 9131 8528082; E-mail: email@example.com
Backert, S., et al. Expression of Helicobacter pylori Serine Protease HtrA in Campylobacter jejuni Reveals a Crucial Function in Oxygen Stress Resistance, Heat Tolerance and Epithelial Barrier Disruption. (2017) Cell Immunol Serum Biol Sci 3(2): 105- 114
© 2017 Backert, S. This is an Open access article distributed under the terms of Creative Commons Attribution 4.0 International License.
KeywordsC. jejuni; H. pylori; HtrA; Adherens junctions; Tight junctions; Secretion; Chaperone; E-cadherin; Molecular pathogenesis; Cellular invasion; Signaling; transwell; Paracellular; Virulence; Stress response
Aim: The gastrointestinal pathogens Helicobacter pylori and Campylobacter jejuni secrete serine protease HtrA into the extracellular space, where it cleaves the adherens junction protein E-cadherin. However, while htrA can be mutagenized in C. jejuni, htrA is an essential gene in H. pylori and rapid research progress on HtrA functions is hindered by the nonexistence of ΔhtrA deletion mutants in these bacteria. To bypass this drawback and to study the function of HtrA further, we aimed to clone H. pylori htrA (Hp HtrA) in two C. jejuni strains and study various physiological and virulence properties of the bacteria.
Materials and methods: We developed a genetic complementation system to express Hp HtrA in C. jejuni. We have then used numerous approaches to study the growth of the bacteria under various temperature and oxidative stress conditions. We obtained bacteria-free supernatants of cultured bacteria by biochemical fractionation and utilized Western blotting to evaluate the protein content of cellular fractions. Casein zymography was used to determine the proteolytic activity of HtrA. Polarized human Caco-2 and MKN-28 cells grown in a transwell filter system were applied to study the transmigration of C. jejuni. The transepithelial electrical resistance (TER) was measured to characterize the functionality of tight and adherens junctions over time. Immunofluorescence microscopy was employed to visualize the bacteria and E-cadherin during the infection process. Using gentamicin protection assay we quantified the numbers of cell-bound and invasive C. jejuni during infection of INT-407 epithelial cells.
Results: HtrA is a central regulator of stress responses and protein quality control in bacteria. Secreted HtrA is implicated in disrupting cell-to-cell junctions, allowing paracellular transmigration of the microbes across polarized epithelial host cell monolayers. Introduction of Hp HtrA in C. jejuni ΔhtrA knockout mutants restored its defects with the following phenotypes: (i) expression of proteolytically active HtrA, (ii) secretion of HtrA into the supernatant, (iii) bacterial growth at high temperature (44°C), (iv) growth at high oxygen stress conditions, (v) bacterial attachment and invasion of host cells, and even substantially enhanced the (vi) disruption of E-cadherin-based cell-to-cell junctions as well as (vii) paracellular transmigration of the bacteria across polarized Caco-2 and MKN-28 epithelial cells.
Conclusions: These results establish a genetic complementation system for Hp HtrA in C. jejuni, approve important functional features of the H. pylori protease and allow further detailed examination of htrA functions in vitro and in vivo.