- Open Access
Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori
© The Author(s) 2017
- Received: 18 March 2017
- Accepted: 11 July 2017
- Published: 25 July 2017
The 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.
E-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.
These 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.
- Adherens junction
- Tight junction
- Helicobacter pylori
- Type IV secretion system
Helicobacter pylori is a Gram-negative, flagellated pathogen, which persistently colonizes the human stomach [1, 2]. About 50% of the world population carries these bacteria, and infections are associated with chronic, often asymptomatic gastritis in all infected individuals. However, more severe gastric diseases such as peptic ulceration, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma can arise in a subset of patients [3, 4]. The clinical outcome of H. pylori infection is regulated by several key elements including the genetic predisposition of the host, the bacterial genotype and environmental factors [5–7]. Dozens of bacterial determinants have been described to impact H. pylori pathogenicity. Two classical virulence factors are known, the vacuolating cytotoxin (VacA) and the cytotoxin-associated genes pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) for transport of the oncoprotein CagA across the bacterial membranes into host target cells [8, 9]. Upon delivery, CagA undergoes phosphorylation at C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) sequence repeats by the c-Src and c-Abl family of tyrosine kinases [10–12]. Translocated CagA binds to and activates or inactivates a series of signaling factors in a phosphorylation-dependent and phosphorylation-independent fashion [13, 14]. The T4SS can also induce profound pro-inflammatory responses such as the release of chemokine interleukin-8 (IL-8) via transcription factor NF-κB, which proceeds widely independently of CagA delivery [15–17]. On the other hand, VacA is an autotransporter and secreted into the extracellular space, where it induces multiple responses including cell vacuolation, alteration of endo-lysosomal trafficking, immune cell inhibition and apoptosis [5, 18]. Other pathogenicity-associated processes comprise urease-triggered neutralization of acidic pH, flagella-mediated motility, expression of multiple adhesins (BabA/B, SabA, AlpA/B, HopQ, HopZ, OipA and others), inhibition of T cell proliferation by secreted γ-glutamyl-transpeptidase GGT, and secretion of proteases such as HtrA [3, 19–21].
High temperature requirement protein A (HtrA ) family members comprise a set of evolutionarily related serine proteases and chaperones, which are found in most prokaryotes and eukaryotes [22–24]. HtrA proteases are generally transported into the periplasm, where they form proteolytically active oligomers with important function in protein quality control [25, 26]. Its chief role is to remove damaged, misfolded or mislocalized proteins in the periplasm. HtrA proteins contain no regulatory components or ATP binding domains . Thus, they are commonly referred to as ATP-independent chaperone-proteases. Bacterial HtrA proteases commonly comprise an N-terminal signal sequence, followed by a trypsin-like serine protease domain and one or two PDZ modules at the C-terminus, which permit protein–protein interactions [23, 27–29]. Inactivation of the htrA gene by mutation regularly results in high temperature sensitivity of many bacteria [30–35]. For a long time it was supposed that HtrA proteases are strictly functioning only inside the bacterial periplasm. However, we have previously introduced a new characteristic for the HtrAs of Campylobacter jejuni and H. pylori. These HtrA proteins can be actively secreted into the extracellular environment, where they cleave host cell factors [36–41]. It has been demonstrated that secreted HtrA from both species can open the adherens junctions in cultured polarized epithelial cells in vitro by cleaving the extracellular NTF (N-terminal fragment)-domain of E-cadherin, a well-known cell-to-cell adhesion factor [37, 39, 42]. Inactivation of C. jejuni htrA results in downregulated E-cadherin cleavage and bacterial transmigration across polarized cell monolayers in vitro [35, 39], and reduced apoptosis and immunopathology in the gut of infected mice in vivo [43, 44]. Similarly, HtrA is fundamental for the virulence of various other pathogens including Yersinia enterocolitica, Klebsiella pneumoniae, Chlamydia trachomatis, Salmonella enterica, Listeria monocytogenes, Legionella pneumophila, Shigella flexneri, Burkholderia cenocepacia and Borrelia burgdorferi [31, 32, 34, 45–50]. However, an htrA knockout strain in H. pylori is not yet available because the generation of mutants was unsuccessful in a broad collection of worldwide strains, suggesting that htrA may represent an essential gene in H. pylori [51, 52]. To study the role of HtrA further, we aimed to overexpress HtrA in H. pylori and examine various virulence properties of the bacteria. Our results show that overexpression of HtrA in H. pylori results in elevated secretion rates of the protease, cleavage of E-cadherin, bacterial transmigration and delivery of CagA into polarized epithelial cells.
Introduction and expression of a second htrA gene copy in H. pylori
Overexpression of HtrA in H. pylori enhances its proteolytic activity
Induction of HtrA leads to higher secretion levels of HtrA, but not VacA and GGT
Overexpression of HtrA does not affect host cell binding and IL-8 secretion by H. pylori
Overexpression of HtrA enhances disruption of cell-to-cell junctions by H. pylori
Overexpression of HtrA enhances bacterial transmigration across polarized cells
Overexpression of HtrA results in elevated E-cadherin cleavage and CagA phosphorylation
Diverse pathogens encode proteases with crucial functions during infection, but knowledge on secreted proteases and their activities in H. pylori is very limited. In many bacteria, HtrA is a well-recognized factor in the periplasm, which contains chaperone and proteolytic functions with important roles in protein quality control involved in stress tolerance and bacterial survival [23, 25–29]. In addition, it was demonstrated that HtrA has a significant impact on the virulence of multiple bacterial pathogens including Borrelia, Burkholderia, Campylobacter, Chlamydia, Klebsiella, Legionella, Listeria, Salmonella, Shigella and Yersinia species. Interestingly, htrA does not appear as an essential gene in each of these bacteria because ΔhtrA knockout mutation has been described [31, 32, 34, 43–50]. In contrast, inactivation of the htrA gene in H. pylori has been unsuccessful in more than one hundred worldwide isolates, but the reasons for this failure are still unclear [37, 51, 52]. Remarkably, it was also demonstrated that pharmacological inhibition of HtrA protease activity effectively killed H. pylori, while it did not affect the growth and viability of other Gram-negative pathogens including Salmonella and Shigella .
Research progress on H. pylori HtrA is mainly hampered by the lack of ΔhtrA knockout mutants. Thus, other genetic manipulation strategies are required to study HtrA function during the infection process. Here we developed a genetic approach to overexpress HtrA in two clinical isolates, P12 and 26695. For this purpose, a second htrA gene copy was introduced into the H. pylori chromosome and placed under an IPTG-inducible promotor . Once the HtrA proteins are translated by the bacteria they are delivered into the periplasm and subsequently secreted into the extracellular environment. This important new aspect seems to be conserved among a wide range of worldwide H. pylori isolates . We could show here that overexpression of HtrA enhanced not only its proteolytic activity by up to ~2.5-fold, but also the secretion of the protease by ~1.8-fold. Interestingly, the secretion of other well-known bacterial virulence determinants, VacA and GGT, was not affected by HtrA overexpression, suggesting that the secretion of these factors follow different, non-linked pathways. In addition, we could demonstrate that various virulence-associated properties of H. pylori were also not affected including bacterial attachment to the epithelial cells and induction of pro-inflammatory responses such as the secretion of IL-8. In contrast, the transepithelial migration of H. pylori overexpressing HtrA increasing significantly up to ~2.2-fold compared to the control bacteria. This phenotype was accompanied by significantly enhanced damage to the adherens junction protein E-cadherin. Our Western blotting data demonstrated that HtrA-mediated cleavage of full-length E-cadherin was enhanced, leading to elevated levels of the 90 kDa E-cadherin NTF-fragment in the supernatants of infected cells. Immunofluorescence microscopy confirmed these observations and showed that the cell-to-cell junctions of infected Caco-2 cells were significantly more disrupted after 24 h compared to the wild-type control infection, explaining why higher numbers of bacteria can cross the epithelial barrier and reach basolateral compartments. Finally, we observed that the levels of CagA translocation and phosphorylation increased up to ~twofold in HtrA-overexpressing H. pylori compared to the control bacteria. These observations can be explained by reports showing that CagA delivery into host cells requires a receptor, which was identified as the basolateral integrin member α5β1 [56–62]. Integrins are well-known mammalian cell adhesion receptors, which facilitate anchoring of host cells to the extracellular matrix and which are absent at apical surfaces [63, 64]. These findings let us to suggest a novel mechanism how the T4SS of H. pylori works in polarized epithelial cells by cooperating with the secreted serine protease HtrA, which opens cell-to-cell junctions. Using an inducible genetic system to overexpress HtrA, we could enhance the proteolytic activity of HtrA, necessary for elevated paracellular transmigration of H. pylori across the polarized epithelial cells to reach basolateral membranes and inject CagA in an integrin-dependent fashion. Extensive research has shown in recent years that the above discussed features basically resemble a phenotype, called epithelial-mesenchymal transition (EMT). Gastric cancerogenesis is known for its aggressiveness and tendency to metastasize. EMT is the initial step in metastasis, orchestrated by various cellular factors . We proposed that the activity of secreted HtrA is maybe the initial step in a signaling cascade, followed by CagA and probably others, that triggers EMT in gastric epithelial cells. Translocated CagA can then deregulate cell polarity and scattering, by various pathways including the interaction with partioning kinase Par1b changing cell polarity  and by stabilizing Snail, a transcriptional repressor of E-cadherin expression . Taken together, these data provide for the first time genetic evidence that HtrA is a major novel virulence factor of H. pylori, controlling multiple pathogenic activities of this important microbe.
MKN-28 and Caco-2 cell culture and H. pylori infection
Human MKN-28 cells (JCRB, #0253) were originally isolated from gastric adenocarcinoma. The Caco-2 cells (ATCC HTB-37) were obtained from a human colon adenocarcinoma. Both cell lines have been extensively used over the last twenty years as models for studying the gastrointestinal barrier. Cells were cultured in 6-well plates with RPMI1640 or DMEM medium, respectively, containing 4 mM glutamine (Invitrogen, Karlsruhe/Germany), and 10% FCS (Invitrogen, Karlsruhe/Germany) . H. pylori strains 26695, P12 and their mutants were grown on horse serum GC agar plates supplemented with nystatin (1 μg/mL), vancomycin (10 μg/mL) and trimethoprim (5 μg/mL), and if necessary with 4 μg/chloramphenicol per mL. Growth was performed for 2 days at 37 °C in anaerobic chambers containing a CampyGen gas mix (Oxoid, Wesel/Germany) at 37 °C . H. pylori was harvested and resuspended in phosphate buffered saline (PBS, pH 7.4) using sterile cotton swabs (Carl Roth, Karlsruhe/Germany). The bacterial concentration was measured in a spectrophotometer as optical density (OD) at 600 nm (Eppendorf, Hamburg/Germany). Infections were carried out at a multiplicity of infection (MOI) of 50 . All infection assays were done in triplicates.
H. pylori mutagenesis
To introduce a second htrA gene copy in the H. pylori chromosome, we made use of the previously generated IPTG-inducible LacIq pTac system for lacZ gene expression as cloned in vector pILL2150 . In this system, the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor, which is present in two copies (Fig. 1a, top). Expression of the lacZ reporter gene was driven by the pTac promotor as described . We replaced the lacZ gene of pILL2150 by the htrA gene of strain 26695 using the NdeI and BamHI restriction sites. Then the complete cassette shown in Fig. 1a (top) was introduced in the chromosomal plasticity region of H. pylori strains P12 and 26695 (between ORFs HP0999 and HP1000) as shown in Fig. 1a (bottom) using established transformation methods [71, 72]. At the 3′ end, a chloramphenicol resistance gene cassette (CAT) was added to select clones. The correct integration and expression of the htrA gene was verified by PCR and Western blotting, respectively.
HtrA, VacA and GGT secretion assays
Wild-type and mutant H. pylori strains were suspended in BHI medium supplemented with 1% β-cyclodextrin (Sigma Aldrich) . The optical density was determined and adjusted to OD600 = 0.2. To allow bacterial protein secretion in the culture supernatant, H. pylori was grown for 24 h under shaking at 160 rpm in the presence or absence of IPTG (Sigma Aldrich). The cell pellets and the supernatants were prepared by centrifugation at 4000 rpm. The supernatants were transferred through 0.21 μm sterile filters (Sigma Aldrich) to remove remnant bacterial cells. Lack of live bacteria in the supernatant was verified by the absence of bacterial growth after 5 days of incubation on GC agar plates. The resulting bacterial pellets and supernatants were then analysed by Western blotting as described below.
Transwell infection studies
MKN-28 and Caco-2 cells were cultured on 0.33 cm2 cell culture inserts with 3 μm pore size (Corning Life Sciences, Schiphol/Netherlands). The cells were grown to confluent monolayers, and then incubated for another 14 days to allow cell polarization . TER was measured with an Electrical Resistance System (ERS) (World Precision Instruments, Berlin/Germany). Maximal TER values indicated that the monolayers reached maximal cell polarity . The cells were infected in the apical compartment at MOI of 50 and the numbers of transmigrated bacteria were quantified in aliquots taken from the basal chambers and counting colony forming units (CFU) on GC agar plates after 5 days of incubation .
Cell binding assay
Infection of MKN-28 and Caco-2 cell monolayers was performed at a density of 3.5 × 105 cells in 6-well plates as described previously . After infection, infected cells were washed three times with 1 mL of pre-warmed culture medium per well to remove non-adherent bacteria. To determine the total CFU corresponding to cell-associated bacteria, the infected monolayers were incubated with 1 mL of 0.1% saponin in PBS at 37 °C for 15 min. The resulting suspensions were diluted and plated on GC agar plates. The CFUs were counted after 5 days of incubation.
Undiluted aliquots of the bacteria were loaded onto 10% SDS–PAGE gels containing 0.1% casein (Carl Roth, Karlsruhe/Germany) and separated by electrophoresis under non-reducing conditions. After protein separation, the gel was renatured in 2.5% Triton X-100 solution at room temperature for 60 min with gentle agitation, equilibrated in developing buffer (50 mM Tris–HCl, pH 7.4, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij35) at room temperature for 30 min with gentle agitation, and incubated overnight in fresh developing buffer at 37 °C. Transparent HtrA bands with caseinolytic activity were visualized by staining with 0.5% Coomassie Blue R250 as described [35, 39].
The following antibodies were purchased: rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA), monoclonal pan-phosphotyrosine α-PY99 (Santa Cruz, Santa Cruz, CA/USA), rabbit α-GAPDH (Santa Cruz), rabbit α-H. pylori (Dako, Glostrup/Denmark) and two monoclonal antibodies directed against the extracellular domain of E-cadherin, H-108 (Santa Cruz) and CD324 (BD Biosciences, San Jose, CA/USA). HtrA proteins were detected by rabbit polyclonal α-HtrA antiserum raised against purified recombinant HtrA (Biogenes, Berlin/Germany). Rabbit polyclonal α-FlaA and α-GGT antibodies were described previously [74, 75]. The α-VacA antibody (#123) was kindly provided by Timothy Cover (Nashville, TN/USA).
Immunofluorescence staining and microscopy
Immunofluorescence staining with different antibodies as shown in each experiment was performed as described . Briefly, cell samples were fixed with methanol at −20 °C for 10 min followed by permeabilization with 0.5% Triton-X100 for 1 min and blocking with 1% BSA, 0.1% Tween-20 in PBS for 30 min. Proteins were stained with the above mentioned α-E-cadherin (BD) and α-H. pylori antibodies. As secondary antibodies, we used TRITC (tetramethylrhodamine isothiocyanate)-conjugated goat anti-rabbit and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse (Thermo Fisher Scientific, Darmstadt/Germany). Samples were analysed using a Leica DMI4000B fluorescence microscope and different lasers (Leica Microsystems, Wetzlar/Germany). Images were obtained via LAS AF computer software (Leica Microsystems) and E-cadherin staining was quantified as “fold change” using the ImageJ Software (version 2.0). The mock control was set as “1”.
SDS–PAGE, dot blots and immunoblotting
Bacterial pellets, cell-free supernatants or infected cells were mixed with equal amounts of 2× SDS–PAGE buffer and boiled for 5 min. Proteins were separated by SDS–PAGE on 8% polyacrylamide gels and blotted onto PVDF membranes (Immobilon-P, Merck Millipore) as described . Before addition of the antibodies, membranes were blocked in TBST buffer (140 mM NaCl, 25 mM Tris–HCl pH 7.4, 0.1% Tween-20) with 3% BSA or 5% skim milk for 1 h at room temperature . As secondary antibodies, horseradish peroxidase-conjugated α-mouse or α-rabbit polyvalent rabbit and pig immunoglobulin, respectively, were used (Life Technologies, Darmstadt/Germany). Antibody detection was performed with the ECL Plus chemiluminescence Western Blot kit (GE Healthcare Life Sciences, Munich/Germany) .
Quantification of IL-8 chemokines by ELISA
MKN-28 cells were incubated for 8 h with H. pylori, and mock cells with medium served as negative control. The culture supernatants were collected and stored at −80 °C until assayed. IL-8 concentrations in the supernatants were determined by standard ELISA according to manufacturer’s procedures (Becton–Dickinson, Heidelberg/Germany) .
Quantification of band intensities in Western blots and casein gels
Quantification of band signals on immunoblots was performed by densitometric analysis using the Image Lab software (BioRad, Munich/Germany) and indicated the “fold change” of protein expression or phosphorylation level per sample. As shown in the corresponding figures, the control band on each gel was set as “1”.
All data were evaluated via Student’s t test with SigmaPlot statistical software (version 13.0). Statistical significance was defined by p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***).
AH, MB, SB and NT designed and performed the experiments. NT, the senior/corresponding author, supervised the experiments and wrote the manuscript together with SB. All authors read and approved the final manuscript.
We thank Drs. Ivo Boneca (Institute Pasteur, Paris, France) for providing plasmid pILL2150, Benjamin Hoy/Silja Wessler (University of Salzburg, Austria) for cloning of the htrA gene, Timothy Cover (Vanderbilt University Nashville, TN/USA) for the α-VacA antibody and Benjamin Schmid (OICE Erlangen, Germany) for help with the IFM signal quantification. We also thank Wilhelm Brill and Vanessa Schmidt for excellent technical assistance. We acknowledge support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) within the funding pogramme Open Access Publishing.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
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Ethics approval and consent to participate
This work was supported by the German Science Foundation to NT (project TE776/3-1), to MB (project BO4724/1-1) and SB (project B10 in CRC-796 and A04 in CRC-1181).
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- Robinson K, Letley DP, Kaneko K. The human stomach in health and disease: infection strategies by Helicobacter pylori. Curr Top Microbiol Immunol. 2017;400:1–26.PubMedGoogle Scholar
- Mejías-Luque R, Gerhard M. Immune evasion strategies and persistence of Helicobacter pylori. Curr Top Microbiol Immunol. 2017;400:53–71.PubMedGoogle Scholar
- Salama NR, Hartung ML, Müller A. Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol. 2013;11:385–99.View ArticlePubMedPubMed CentralGoogle Scholar
- Figueiredo C, Camargo CM, Leite M, Fuentes-Pananá EM, Rabkin CS, Machado JC. Pathogenesis of gastric cancer: genetics and molecular classification. Curr Top Microbiol Immunol. 2017;400:277–304.PubMedGoogle Scholar
- Cover TL, Peek RM Jr. Diet, microbial virulence, and Helicobacter pylori-induced gastric cancer. Gut Microbes. 2013;4:482–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Backert S, Blaser MJ. The role of CagA in the gastric biology of Helicobacter pylori. Cancer Res. 2016;76:4028–31.View ArticlePubMedGoogle Scholar
- Gobert AP, Wilson KT. Human and Helicobacter pylori interactions determine the outcome of gastric diseases. Curr Top Microbiol Immunol. 2017;400:27–52.PubMedPubMed CentralGoogle Scholar
- Backert S, Tegtmeyer N, Fischer W. Composition, structure and function of the Helicobacter pylori cag pathogenicity island encoded type IV secretion system. Future Microbiol. 2015;10:955–65.View ArticlePubMedPubMed CentralGoogle Scholar
- Bergé C, Terradot L. Structural insights into Helicobacter pylori Cag protein interactions with host cell factors. Curr Top Microbiol Immunol. 2017;400:129–47.PubMedGoogle Scholar
- Backert S, Feller SM, Wessler S. Emerging roles of Abl family tyrosine kinases in microbial pathogenesis. Trends Biochem Sci. 2008;33:80–90.View ArticlePubMedGoogle Scholar
- Lind J, Backert S, Pfleiderer K, Berg DE, Yamaoka Y, Sticht H, et al. Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter pylori strains. PLoS ONE. 2014;9:e96488.View ArticlePubMedPubMed CentralGoogle Scholar
- Lind J, Backert S, Hoffmann R, Eichler J, Yamaoka Y, Perez-Perez GI, et al. Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains. BMC Microbiol. 2016;16:201.View ArticlePubMedPubMed CentralGoogle Scholar
- Mueller D, Tegtmeyer N, Brandt S, Yamaoka Y, De Poire E, Sgouras D, et al. c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in western and East Asian Helicobacter pylori strains. J Clin Invest. 2012;122:1553–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Hatakeyama M. Helicobacter pylori CagA and gastric cancer: a paradigm for hit-and-run carcinogenesis. Cell Host Microbe. 2014;15:306–16.View ArticlePubMedGoogle Scholar
- Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, et al. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol. 2004;5:1166–74.View ArticlePubMedGoogle Scholar
- Brandt S, Kwok T, Hartig R, König W, Backert S. NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci USA. 2005;102:9300–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Backert S, Naumann M. What a disorder: proinflammatory signaling pathways induced by Helicobacter pylori. Trends Microbiol. 2010;18:479–86.View ArticlePubMedGoogle Scholar
- Posselt G, Backert S, Wessler S. The functional interplay of Helicobacter pylori factors with gastric epithelial cells induces a multi-step process in pathogenesis. Cell Commun Signal. 2013;11:77.View ArticlePubMedPubMed CentralGoogle Scholar
- Rad R, Gerhard M, Lang R, Schöniger M, Rösch T, Schepp W, et al. The Helicobacter pylori blood group antigen-binding adhesin facilitates bacterial colonization and augments a nonspecific immune response. J Immunol. 2002;168:3033–41.View ArticlePubMedGoogle Scholar
- Backert S, Clyne M, Tegtmeyer N. Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori. Cell Commun Signal. 2011;9:28.View ArticlePubMedPubMed CentralGoogle Scholar
- Ricci V, Giannouli M, Romano M, Zarrilli R. Helicobacter pylori gamma-glutamyl transpeptidase and its pathogenic role. World J Gastroenterol. 2014;20:630–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Frees D, Brøndsted L, Ingmer H. Bacterial proteases and virulence. Subcell Biochem. 2013;66:161–92.View ArticlePubMedGoogle Scholar
- Skórko-Glonek J, Figaj D, Zarzecka U, Przepiora T, Renke J, Lipinska B. The extracellular bacterial HtrA proteins as potential therapeutic targets and vaccine candidates. Curr Med Chem. 2016 (Epub ahead of print).Google Scholar
- Wessler S, Schneider G, Backert S. Bacterial serine protease HtrA as a promising new target for antimicrobial therapy? Cell Commun Signal. 2017;15:4.View ArticlePubMedPubMed CentralGoogle Scholar
- Gottesman S, Wickner S, Maurizi MR. Protein quality control: triage by chaperone and proteases. Genes Dev. 1997;11:815–23.View ArticlePubMedGoogle Scholar
- Ingmer H, Brøndsted L. Proteases in bacterial pathogenesis. Res Microbiol. 2009;160:704–10.View ArticlePubMedGoogle Scholar
- Clausen T, Southan C, Ehrmann M. The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell. 2002;10:443–55.View ArticlePubMedGoogle Scholar
- Kim DY, Kim KK. Structure and function of HtrA family proteins, the key players in protein quality control. J Biochem Mol Biol. 2005;38:266–74.PubMedGoogle Scholar
- Backert S, Boehm M, Wessler S, Tegtmeyer N. Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both? Cell Commun Signal. 2013;11:72.View ArticlePubMedPubMed CentralGoogle Scholar
- Lipinska B, Fayet O, Baird L, Georgopoulos CJ. Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. Bacteriol. 1989;171(3):1574–84.View ArticleGoogle Scholar
- Pedersen LL, Radulic M, Doric M, Kwaik YA. HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells. Infect Immun. 2001;69:2569–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Cortés G, de Astorza B, Benedí VJ, Albertí S. Role of the htrA gene in Klebsiella pneumoniae virulence. Infect Immun. 2002;70:4772–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Mo E, Peters SE, Willers C, Maskell DJ, Charles IG. Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents. Infect Immun. 2007;75(4):1679–89.View ArticleGoogle Scholar
- Flannagan RS, Aubert D, Kooi C, Sokol PA, Valvano MA. Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. Infect Immun. 2007;75:1679–89.View ArticlePubMedPubMed CentralGoogle Scholar
- Boehm M, Lind J, Backert S, Tegtmeyer N. Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration. Eur J Microbiol Immunol (Bp). 2015;5:68–80.View ArticleGoogle Scholar
- Löwer M, Weydig C, Metzler D, Reuter A, Starzinski-Powitz A, Wessler S, et al. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA. PLoS ONE. 2008;3:e3510.View ArticlePubMedPubMed CentralGoogle Scholar
- Hoy B, Löwer M, Weydig C, Carra G, Tegtmeyer N, Geppert T, et al. Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep. 2010;11:798–804.View ArticlePubMedPubMed CentralGoogle Scholar
- Hoy B, Geppert T, Boehm M, Reisen F, Plattner P, Gadermaier G, et al. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin. J Biol Chem. 2012;23:10115–20.View ArticleGoogle Scholar
- Boehm M, Hoy B, Rohde M, Tegtmeyer N, Bæk KT, Oyarzabal OA, et al. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin. Gut Pathog. 2012;4:3.View ArticlePubMedPubMed CentralGoogle Scholar
- Schmidt TP, Perna AM, Fugmann T, Boehm M, Hiss Jan, Haller S, et al. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA. Sci Rep. 2016;6:23264.View ArticlePubMedPubMed CentralGoogle Scholar
- Abfalter CM, Schubert M, Götz C, Schmidt TP, Posselt G, Wessler S. HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens. Cell Commun Signal. 2016;14(1):30.View ArticlePubMedPubMed CentralGoogle Scholar
- Backert S, Schmidt TP, Harrer A, Wessler S. Exploiting the gastric epithelial barrier: Helicobacter pylori’s attack on tight and adherens junctions. Curr Top Microbiol Immunol. 2017;400:195–226.PubMedGoogle Scholar
- Heimesaat MM, Alutis M, Grundmann U, Fischer A, Tegtmeyer N, Boehm M, et al. The role of serine protease HtrA in acute ulcerative enterocolitis and extra-intestinal immune responses during Campylobacter jejuni infection of gnotobiotic IL-10 deficient mice. Front Cell Infect Microbiol. 2014;4:77.View ArticlePubMedPubMed CentralGoogle Scholar
- Heimesaat MM, Fischer A, Alutis M, Grundmann U, Boehm M, Tegtmeyer N, et al. The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice. Gut Pathog. 2014;6:16.View ArticlePubMedPubMed CentralGoogle Scholar
- Li SR, Dorrell N, Everest PH, Dougan G, Wren BW. Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant. Infect Immun. 1996;64:2088–94.PubMedPubMed CentralGoogle Scholar
- Humphreys S, Stevenson A, Bacon A, Weinhardt AB, Roberts M. The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella typhimurium. Infect Immun. 1999;67:1560–8.PubMedPubMed CentralGoogle Scholar
- Purdy GE, Hong M, Payne SM. Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread. Infect Immun. 2002;70:6355–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Wilson RL, Brown LL, Kirkwood-watts D, Warren TK, Lund SA, King DS, et al. Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence. Microbiology. 2006;74:765–8.Google Scholar
- Gloeckl S, Ong VA, Patel P, Tyndall JD, Timms P, Beagley KW, et al. Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis. Mol Microbiol. 2013;89:676–89.View ArticlePubMedGoogle Scholar
- Ye M, Sharma K, Thakur M, Smith AA, Buyuktanir O, Xiang X, et al. HtrA, a temperature- and stationary phase-activated protease involved in maturation of a key microbial virulence determinant, facilitates Borrelia burgdorferi infection in mammalian hosts. Infect Immun. 2016;84(8):2372–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Salama NR, Shepherd B, Falkow S. Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J Bacteriol. 2004;186:7926–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Tegtmeyer N, Moodley Y, Yamaoka Y, Pernitzsch SR, Schmidt V, Traverso FR, et al. Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA. Mol Microbiol. 2016;99(5):925–44.View ArticlePubMedGoogle Scholar
- Boneca IG, Ecobichon C, Chaput C, Mathieu A, Guadagnini S, Prévost MC, et al. Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori. Appl Environ Microbiol. 2008;74(7):2095–102.View ArticlePubMedPubMed CentralGoogle Scholar
- Backert S, Ziska E, Brinkmann V, Zimny-Arndt U, Fauconnier A, Jungblut PR, et al. Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol. 2000;2(2):155–64.View ArticlePubMedGoogle Scholar
- Kwok T, Backert S, Schwarz H, Berger J, Meyer TF. Specific entry of Helicobacter pylori into cultured gastric epithelial cells via a zipper-like mechanism. Infect Immun. 2002;70(4):2108–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, et al. Helicobacter exploits integrin for type IV secretion and kinase activation. Nature. 2007;449(7164):862–6.View ArticlePubMedGoogle Scholar
- Jiménez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E, Kapp U, et al. Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel RGD-independent manner. PLoS Pathog. 2009;5:e1000684.View ArticlePubMedPubMed CentralGoogle Scholar
- Saha A, Backert S, Hammond CE, Gooz M, Smolka AJ. Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H, K-ATPase alpha subunit. Gastroenterology. 2010;139(1):239–48.View ArticlePubMedPubMed CentralGoogle Scholar
- Conradi J, Huber S, Gaus K, Mertink F, Royo Gracia S, Strijowski U, et al. Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1. Amino Acids. 2012;43:219–32.View ArticlePubMedGoogle Scholar
- Conradi J, Tegtmeyer N, Woźna M, Wissbrock M, Michalek C, Gagell C, et al. An RGD helper sequence in CagL of Helicobacter pylori assists in interactions with integrins and injection of CagA. Front Cell Infect Microbiol. 2012;2:70.View ArticlePubMedPubMed CentralGoogle Scholar
- Barden S, Lange S, Tegtmeyer N, Conradi J, Sewald N, Backert S, et al. A helical RGD motif promoting cell adhesion: crystal structures of the Helicobacter pylori type IV secretion system pilus protein CagL. Structure. 2013;21:1931–41.View ArticlePubMedGoogle Scholar
- Tegtmeyer N, Lind J, Schmid B, Backert S. Helicobacter pylori CagL Y58/E59 mutation turns-off type IV secretion-dependent delivery of CagA into host cells. PLoS ONE. 2014;9(6):e97782.View ArticlePubMedPubMed CentralGoogle Scholar
- Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol. 2004;5(10):816–26.View ArticlePubMedGoogle Scholar
- Horton ER, Humphries JD, James J, Jones MC, Askari JA, Humphries MJ. The integrin adhesome network at a glance. J Cell Sci. 2016;129:4159–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Li B, Huang C. Regulation of EMT by STAT3 in gastrointestinal cancer. Int J Oncol. 2017;50:753–67.PubMedGoogle Scholar
- Saadat I, Higashi H, Obuse C, Murata-Kamiya N, Umeda M, Saito Y, et al. Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature. 2007;447:330–3.View ArticlePubMedGoogle Scholar
- Lee DG, Kim HS, Lee YS, Cha SY, Kim S, Ota I, et al. H. pylori CagA promotes snail-mediated epithelial-mesenchymal transition by reducing GSK-3 activity. Nat Commun. 2014;5:4423.PubMedGoogle Scholar
- Zhang YM, Noto JM, Hammond CE, Barth JL, Argraves WS, Backert S, et al. Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA. Am J Physiol Gastrointest Liver Physiol. 2014;306:G606–13.View ArticlePubMedPubMed CentralGoogle Scholar
- Wiedemann T, Hofbaur S, Tegtmeyer N, Huber S, Sewald N, Wessler S, et al. Helicobacter pylori CagL dependent induction of gastrin expression via a novel αvβ5-integrin-integrin linked kinase signaling complex. Gut. 2012;61:986–96.View ArticlePubMedGoogle Scholar
- Kim DJ, Park JH, Franchi L, Backert S, Núñez G. The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL-1β production in Helicobacter pylori infected dendritic cells. Eur J Immunol. 2013;43:2650–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Patel SR, Smith K, Letley DP, Cook KW, Memon AA, Ingram RJ, et al. Helicobacter pylori downregulates expression of human β-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion. Cell Microbiol. 2013;15:2080–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Tenguria S, Ansari SA, Khan N, Ranjan A, Devi S, Tegtmeyer N, et al. Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in mouse macrophages and acts as auto-phosphorylating tyrosine kinase. Int J Med Microbiol. 2014;304:1066–76.View ArticlePubMedGoogle Scholar
- Bumann D, Aksu S, Wendland M, Janek K, Zimny-Arndt U, Sabarth N, et al. Proteome analysis of secreted proteins of the gastric pathogen H. pylori. Infect Immun. 2002;70(7):3396–403.View ArticlePubMedPubMed CentralGoogle Scholar
- Roure S, Bonis M, Chaput C, Ecobichon C, Mattox A, Barrière C, et al. Peptidoglycan maturation enzymes affect flagellar functionality in bacteria. Mol Microbiol. 2012;86:845–56.View ArticlePubMedGoogle Scholar
- Tegtmeyer N, Rivas Traverso F, Rohde M, Oyarzabal OA, Lehn N, Schneider-Brachert W, et al. Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger. PLoS ONE. 2013;8:e71220.View ArticlePubMedPubMed CentralGoogle Scholar
- Tegtmeyer N, Wittelsberger R, Hartig R, Wessler S, Martinez-Quiles N, Backert S. Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori. Cell Host Microbe. 2011;9(6):520–31.View ArticlePubMedGoogle Scholar
- Boehm M, Krause-Gruszczynska M, Rohde M, Tegtmeyer N, Takahashi S, Oyarzabal OA, et al. Major host factors involved in epithelial cell invasion of Campylobacter jejuni: role of fibronectin, integrin beta1, FAK, Tiam-1, and DOCK180 in activating Rho GTPase Rac1. Front Cell Infect Microbiol. 2011;12:17.Google Scholar
- Zhang XS, Tegtmeyer N, Traube L, Jindal S, Perez-Perez G, Sticht H, et al. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions. PLoS Pathog. 2015;11(2):e1004621.View ArticlePubMedPubMed CentralGoogle Scholar
- Hirsch C, Tegtmeyer N, Rohde M, Rowland M, Oyarzabal OA, Backert S. Live Helicobacter pylori in the root canal of endodontic-infected deciduous teeth. J Gastroenterol. 2012;47:936–40.View ArticlePubMedGoogle Scholar
- Kumar Pachathundikandi S, Brandt S, Madassery J, Backert S. Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signaling leading to the secretion of IL-8 and TNF-α. PLoS ONE. 2011;6:e19614.View ArticlePubMedPubMed CentralGoogle Scholar