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Background High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity. Results Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17. Conclusions Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions.

Persistent infections with the human pathogen Helicobacter pylori (H. pylori) have been closely associated with the induction and progression of a wide range of gastric disorders, including acute and chronic gastritis, ulceration in the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. The pathogenesis of H. pylori is determined by a complicated network of manifold mechanisms of pathogen–host interactions, which involves a coordinated interplay of H. pylori pathogenicity and virulence factors with host cells. While these molecular and cellular mechanisms have been intensively investigated to date, the knowledge about outer membrane vesicles (OMVs) derived from H. pylori and their implication in bacterial pathogenesis is not well developed. In this review, we summarize the current knowledge on H. pylori-derived OMVs.

Additional file 1 Figure S1. H. pylori induces maturation of human DCs. Monocytes were isolated from human PBMCs and differentiated into moDCs in the presence of IL-4 and GMCSF (50 ng/mL each). Immature DCs were re-plated in fresh medium on day 7, H. pylori strain P12 was harvested in PBS and added to the cells at increasing MOIs of 0.2, 2 and 20. Surface marker expression (A) and cytokine secretion (B) were analyzed 48 h post-infection. Data represent mean + SD of one experiment comprising three individual donors (N = 3). For statistical analysis, one-way ANOVA with Dunnett’s post-hoc test was performed. *p

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections. (VLID)5311015

Helicobacter pylori (H. pylori) is a stomach pathogen that persistently colonizes the gastric mucosa, often leading to chronic inflammation and gastric pathologies. Although infection with H. pylori is the primary risk factor for gastric cancer, the underlying mechanisms of pathogen persistence and consequential chronic inflammation are still not well understood. Conventional dendritic cells (cDCs), which are among the first immune cells to encounter H. pylori in the gastric lining, and the cytokines and chemokines they secrete, contribute to both acute and chronic inflammation. Therefore, this study aimed to unravel the contributions of specific signaling pathways within human CD1c+ cDCs (cDC2s) to the composition of secreted cytokines and chemokines in H. pylori infection. Here, we show that the type IV secretion system (T4SS) plays only a minor role in H. pylori-induced activation of cDC2s. In contrast, Toll-like receptor 4 (TLR4) signaling drives the secretion of inflammatory mediators, including IL-12 and IL-18, while signaling via TLR10 attenuates the release of IL-1 and other inflammatory cytokines upon H. pylori infection. The TLR2 pathway significantly blocks the release of CXCL1 and CXCL8, while it promotes the secretion of TNF and GM-CSF. Taken together, these results highlight how specific TLR-signaling pathways in human cDC2s shape the H. pylori-induced cytokine and chemokine milieu, which plays a pivotal role in the onset of an effective immune response. (VLID)5166249

(VLID)5468879

Supplementary Information. (DOCX 32 kb)

Figure S2. Tyrosine phosphorylation, but not threonine phosphorylation of c-Abl depends on CagL. (A) AGS cells were transfected with pSGT-Ablwt and remained uninfected or were infected with isogenic Hp wt, ΔCagL, or ΔCagL/CagL strains for 6 h. Whole cell lysates were subjected to Western blotting to analyze pAblT735, pAblY245 and pAblY412. c-Abl and β-actin were shown as loading controls. Infections were further analyzed for pCagA and CagA. (B) Quantification of pAblT735, pAblY245 and pAblY412 was performed by Western blot densitometry, which was normalized to corresponding β-actin levels. Graphs show mean ± SD of three independent experiments. (C) Cells were infected with Hp wt, ΔRfaE or ΔPAI. pAblT735, AblY245, pCagA, CagA and GAPDH were detected using specific antibodies. (DOCX 2290 kb)

Figure S5. Gleevec decreases Hp pathology. C57BL/6 mice were infected with Hp PMSS1 for two months, were supplied with STI-571 or remained untreated (control). Representative sections of the gastric tissues are shown. (DOCX 261 kb)

Background High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail. Results In this study, we cloned LmHtrA wildtype (LmHtrAwt) and generated a proteolytic inactive LmHtrASA mutant. Recombinant LmHtrAwt and LmHtrASA were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures. Conclusions Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future. (VLID)4599705

Figure S4. Generation of stable AGS cell lines. (A) Untreated AGS cells and AGS cells transfected with TAP-Ablwt or TAP-AblTA were either left untreated (mock) or infected with Hp at a MOI 100 for 6 h and analyzed by Western blot for pAblT735 and c-Abl. β-actin served as loading control. (B) Untreated AGS cells and AGS cells expressing TAP-Ablwt or TAP-AblTA were either left untreated (mock) or infected with Hp at a MOI 100. The scattering phenotype was documented using phase contrast microscopy. (C) Untreated AGS cells and AGS cells stably transfected with control shRNA (shCtrl) or c-Abl shRNA (shAbl) were lysed and analyzed by Western blotting for c-Abl and GAPDH expression (D) AGS cells stably transfected with control shRNA (shCtrl) or c-Abl shRNA (shAbl) were either left untreated (mock) or infected with Hp at a MOI 100 for 6 h. Scattering phenotype was documented using phase contrast microscopy. (E) AGS cells stably transfected with control (shCtr) or Abl shRNA (shAbl) were left untreated (−) or infected with Hp wt for 48 h. Percent apoptosis was calculated by analyzing annexin single-positive and annexin/7AAD positive cells. (DOCX 276 kb)

Helicobacter pylori (H. pylori) has been identified as a leading cause of gastric cancer, which is one of the most frequent and malignant types of tumor. It is characterized by its rapid progression, distant metastases, and resistance to conventional chemotherapy. A number of receptor tyrosine kinases and non-receptor tyrosine kinases have been implicated in H. pylori-mediated pathogenesis and tumorigenesis. In this review, recent findings of deregulated EGFR, c-Met, JAK, FAK, Src, and c-Abl and their functions in H. pylori pathogenesis are summarized. (VLID)4588600

Figure S3. Differential phosphorylation patterns in c-Abl mutants. (A) AGS cell were transfected with pSGT-Ablwt, pSGT-AblTA, pSGT-AblPP, pSGT-AblKD, pSGT-AblY245F, pSGT-cAblY412F, or empty vector (ut) and either left untreated, infected with Hp wt or stimulated with H2O2/vanadate (H/V, left panel) or PMA (right panel) for 6 h. Whole cell lysates were analyzed by Western blotting for pAblT735, pAblY245 or pAblY412, pCagA, CagA, GAPDH and β-actin. Quantification of pAblT735 (B) pAblY245 (C) and pAblY412 (D) were performed by blot densitometry and normalized to the corresponding β-actin levels. Graphs present mean ± SD of three independent experiments. (E) Transfected AGS cells were pretreated with 10 μM STI-571 and infected with Hp for 6 h as indicated. Whole cell lysates were analyzed by Western blotting for pAblT735, pAblY245, Abl and GAPDH. (F) AGS cells were transfected with pSGT-Ablwt or pSGT-AblTA and then infected with Hp for 4 h. Nuclear and cytoplasmic localization was quantified from four independent experiments. (G) AGS stably transfected with pNTAP Ablwt were pretreated with a 14–3-3 inhibitor (BV02) or vehicle control (DMSO) and infected with Hp for 8 h. Cell elongation was determined by measuring the largest cell diameter of individual cells from three independent experiments. (DOCX 310 kb)

Figure S1. c-Abl threonine phosphorylation in AGS, MKN28 and MCF-7 cells. (A) AGS cells were transfected with pSGT-Ablwt and either left untreated or infected with Hp wt for the indicated periods of time. Levels of pAblT735 (white bars) and total Abl (black bars) were quantified by blot densitometry and normalized to GAPDH. Fold changes compared to uninfected cells are shown. (B) MKN28 and (C) MCF-7 cells were transfected with pSGT-Abl and either left untreated or infected with Hp for the indicated periods of time. Levels of pAblT735, total c-Abl and GAPDH are shown. (D) Lysates of uninfected (â ) and Hp-infected (+) MKN28 cells were subjected to immunoprecipitation (IP) using a specific c-Abl antibody. Lysates before IP (pre IP), the precipitates (IP) and lysates after IP (post IP) were analyzed by Western blotting to detect pAblT735 and c-Abl. (E) Transfected AGS cells were infected with several Western (P12, P1, Hp26695 and B8) and East Asian Hp isolates (42GX and 48GX) and analyzed by Western blotting to detect pAblT735, c-Abl and GAPDH. (DOCX 180 kb)