Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori

Abstract

BackgroundThe serine protease HtrA is an important factor for regulating stress responses and protein quality control in bacteria. In recent studies, we have demonstrated that the gastric pathogen Helicobacter pylori can secrete HtrA into the extracellular environment, where it cleaves-off the ectodomain of the tumor suppressor and adherens junction protein E-cadherin on gastric epithelial cells.ResultsE-cadherin cleavage opens cell-to-cell junctions, allowing paracellular transmigration of the bacteria across polarized monolayers of MKN-28 and Caco-2 epithelial cells. However, rapid research progress on HtrA function is mainly hampered by the lack of ΔhtrA knockout mutants, suggesting that htrA may represent an essential gene in H. pylori. To circumvent this major handicap and to investigate the role of HtrA further, we overexpressed HtrA by introducing a second functional htrA gene copy in the chromosome and studied various virulence properties of the bacteria. The resulting data demonstrate that overexpression of HtrA in H. pylori gives rise to elevated rates of HtrA secretion, cleavage of E-cadherin, bacterial transmigration and delivery of the type IV secretion system (T4SS) effector protein CagA into polarized epithelial cells, but did not affect IL-8 chemokine production or the secretion of vacuolating cytotoxin VacA and γ-glutamyl-transpeptidase GGT.ConclusionsThese data provide for the first time genetic evidence in H. pylori that HtrA is a novel major virulence factor controlling multiple pathogenic activities of this important microbe.

Pathogenesis of Helicobacter pylori infection

“…An exciting new study showed a critical role for HtrA in cleaving E‐cadherin to disrupt cell‐to‐cell junctions, which may allow H. pylori to migrate to the basolateral side of epithelial cells and interact with the α5β1 receptor for CagA translocation . The H. pylori migration and CagA translocation activity were proportional to the expression of HtrA . In addition, the HtrA activity was affected by the variation on the amino‐terminal between H46/D47 and K50/D51.…”

“…The amino‐terminal variations may affect the oligomerization, secretion, and regulatory activities . The overexpression of HtrA did not affect the expression of other virulence proteins, such as VacA and γ‐glutamyl‐transpeptidase (GGT) …”

“…21 The H. pylori migration and CagA translocation activity were proportional to the expression of HtrA. 22 In addition, the HtrA activity was affected by the variation on the amino-terminal between H46/D47 and K50/D51. The amino-terminal variations may affect the oligomerization, secretion, and regulatory activities.…”

“…23 The overexpression of HtrA did not affect the expression of other virulence proteins, such as VacA and γ-glutamyl-transpeptidase (GGT). 22 A structural similarity analysis using a DALI server, a protein structure comparison server, 24 showed that Lpp20 is very similar to the TNFα inducing protein (Tipα), with an additional short α-helix at the N-terminal. 25 Cell culture experiments showed that Lpp20 promotes migration and proliferation of gastric cancer cells (AGS) by inducing formation of actin-containing filament around the cell surfaces.…”

How many protein molecules are secreted by single Helicobacter pylori cells: Quantification of serine protease HtrA

“…This enables H. pylori to transmigrate via the paracellular route to enter the basolateral side of the epithelium, where it injects CagA into the host cell by means of T4SS and integrin receptors (Backert et al, ; Hoy et al, ). Indeed, the latter process is enhanced when H. pylori overexpresses HtrA by introduction of a second htrA gene copy in the genome (Harrer, Boehm, Backert, & Tegtmeyer, ). The gene coding for HtrA is highly conserved between H. pylori strains, and because viable knockout mutants could not be created until today, htrA can be considered as an essential gene (Tegtmeyer et al, ).…”