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.
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.
Results and discussion
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.
Consent for publication
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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