Bacterial Proteases in Helicobacter pylori Infections and Gastric Disease.

Helicobacter pylori (H. pylori) proteases have become a major focus of research in recent years, because they not only have an important function in bacterial physiology, but also directly alter host cell functions. In this review, we summarize recent findings on extracellular H. pylori proteases that target host-derived substrates to facilitate bacterial pathogenesis. In particular, the secreted H. pylori collagenase (Hp0169), the metalloprotease Hp1012, or the serine protease High temperature requirement A (HtrA) are of great interest. Specifically, various host cell-derived substrates were identified for HtrA that directly interfere with the gastric epithelial barrier allowing full pathogenesis. In light of increasing antibiotic resistance, the development of inhibitory compounds for extracellular proteases as potential targets is an innovative field that offers alternatives to existing therapies.

Proteolytic Landscapes in Gastric Pathology and Cancerogenesis.

Gastric cancer is a leading cause of cancer-related death, and a large proportion of cases are inseparably linked to infections with the bacterial pathogen and type I carcinogen Helicobacter pylori. The development of gastric cancer follows a cascade of transformative tissue events in an inflammatory environment. Proteases of host origin as well as H. pylori-derived proteases contribute to disease progression at every stage, from chronic gastritis to gastric cancer. In the present article, we discuss the importance of (metallo-)proteases in colonization, epithelial inflammation, and barrier disruption in tissue transformation, deregulation of cell proliferation and cell death, as well as tumor metastasis and neoangiogenesis. Proteases of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase domain-containing protein (ADAM) families, caspases, calpain, and the H. pylori proteases HtrA, Hp1012, and Hp0169 cleave substrates including extracellular matrix molecules, chemokines, and cytokines, as well as their cognate receptors, and thus shape the pathogenic microenvironment. This review aims to summarize the current understanding of how proteases contribute to disease progression in the gastric compartment.

Identification of Desmoglein-2 as a novel target of Helicobacter pylori HtrA in epithelial cells.

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. Video Abstract.

Morphing of Amphipathic Helices to Explore the Activity and Selectivity of Membranolytic Antimicrobial Peptides.

Naturally occurring membranolytic antimicrobial peptides (AMPs) are rarely cell-type selective and highly potent at the same time. Template-based peptide design can be used to generate AMPs with improved properties de novo. Following this approach, 18 linear peptides were obtained by computationally morphing the natural AMP Aurein 2.2d2 GLFDIVKKVVGALG into the synthetic model AMP KLLKLLKKLLKLLK. Eleven of the 18 chimeric designs inhibited the growth of Staphylococcus aureus, and six peptides were tested and found to be active against one resistant pathogenic strain or more. One of the peptides was broadly active against bacterial and fungal pathogens without exhibiting toxicity to certain human cell lines. Solution nuclear magnetic resonance and molecular dynamics simulation suggested an oblique-oriented membrane insertion mechanism of this helical de novo peptide. Temperature-resolved circular dichroism spectroscopy pointed to conformational flexibility as an essential feature of cell-type selective AMPs.

H. pylori modulates DC functions via T4SS/TNFα/p38-dependent SOCS3 expression.

BACKGROUND: Helicobacter pylori (H. pylori) is a gram-negative bacterium that chronically infects approximately 50% of the world's human population. While in most cases the infection remains asymptomatic, 10% of infected individuals develop gastric pathologies and 1-3% progress to gastric cancer. Although H. pylori induces severe inflammatory responses, the host's immune system fails to clear the pathogen and H. pylori can persist in the human stomach for decades. As suppressor of cytokine signaling (SOCS) proteins are important feedback regulators limiting inflammatory responses, we hypothesized that H. pylori could modulate the host's immune responses by inducing SOCS expression. METHODS: The phenotype of human monocyte-derived DCs (moDCs) infected with H. pylori was analyzed by flow cytometry and multiplex technology. SOCS expression levels were monitored by qPCR and signaling studies were conducted by means of Western blot. For functional studies, RNA interference-based silencing of SOCS1-3 and co-cultures with CD4+ T cells were performed. RESULTS: We show that H. pylori positive gastritis patients express significantly higher SOCS3, but not SOCS1 and SOCS2, levels compared to H. pylori negative patients. Moreover, infection of human moDCs with H. pylori rapidly induces SOCS3 expression, which requires the type IV secretion system (T4SS), release of TNFα, and signaling via the MAP kinase p38, but appears to be independent of TLR2, TLR4, MEK1/2 and STAT proteins. Silencing of SOCS3 expression in moDCs prior to H. pylori infection resulted in increased release of both pro- and anti-inflammatory cytokines, upregulation of PD-L1, and decreased T-cell proliferation. CONCLUSIONS: This study shows that H. pylori induces SOCS3 via an autocrine loop involving the T4SS and TNFα and p38 signaling. Moreover, we demonstrate that high levels of SOCS3 in DCs dampen PD-L1 expression on DCs, which in turn drives T-cell proliferation. Video Abstract.

TLR2, TLR4 and TLR10 Shape the Cytokine and Chemokine Release of H. pylori-Infected Human DCs.

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-1ß 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 TNFα 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.

The proteolytic activity of Listeria monocytogenes HtrA.

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.

Helicobacter pylori-controlled c-Abl localization promotes cell migration and limits apoptosis.

BACKGROUND: Deregulated c-Abl activity has been intensively studied in a variety of solid tumors and leukemia. The class-I carcinogen Helicobacter pylori (Hp) activates the non-receptor tyrosine kinase c-Abl to phosphorylate the oncoprotein cytotoxin-associated gene A (CagA). The role of c-Abl in CagA-dependent pathways is well established; however, the knowledge of CagA-independent c-Abl processes is scarce. METHODS: c-Abl phosphorylation and localization were analyzed by immunostaining and immunofluorescence. Interaction partners were identified by tandem-affinity purification. Cell elongation and migration were analyzed in transwell-filter experiments. Apoptosis and cell survival were examined by FACS analyses and MTT assays. In mice experiments and human biopsies, the involvement of c-Abl in Hp pathogenesis was investigated. RESULTS: Here, we investigated the activity and subcellular localization of c-Abl in vitro and in vivo and unraveled the contribution of c-Abl in CagA-dependent and -independent pathways to gastric Hp pathogenesis. We report a novel mechanism and identified strong c-Abl threonine 735 phosphorylation (pAblT735) mediated by the type-IV secretion system (T4SS) effector D-glycero-ß-D-manno-heptose-1,7-bisphosphate (ßHBP) and protein kinase C (PKC) as a new c-Abl kinase. pAblT735 interacted with 14-3-3 proteins, which caused cytoplasmic retention of c-Abl, where it potentiated Hp-mediated cell elongation and migration. Further, the nuclear exclusion of pAblT735 attenuated caspase-8 and caspase-9-dependent apoptosis. Importantly, in human patients suffering from Hp-mediated gastritis c-Abl expression and pAblT735 phosphorylation were drastically enhanced as compared to type C gastritis patients or healthy individuals. Pharmacological inhibition using the selective c-Abl kinase inhibitor Gleevec confirmed that c-Abl plays an important role in Hp pathogenesis in a murine in vivo model. CONCLUSIONS: In this study, we identified a novel regulatory mechanism in Hp-infected gastric epithelial cells by which Hp determines the subcellular localization of activated c-Abl to control Hp-mediated EMT-like processes while decreasing cell death.

Rational Design of Membrane-Pore-Forming Peptides.

Specific interactions of peptides with lipid membranes are essential for cellular communication and constitute a central aspect of the innate host defense against pathogens. A computational method for generating innovative membrane-pore-forming peptides inspired by natural templates is presented. Peptide representation in terms of sequence- and topology-dependent hydrophobic moments is introduced. This design concept proves to be appropriate for the de novo generation of first-in-class membrane-active peptides with the anticipated mode of action. The designed peptides outperform the natural template in terms of their antibacterial activity. They form a kinked helical structure and self-assemble in the membrane by an entropy-driven mechanism to form dynamically growing pores that are dependent on the lipid composition. The results of this study demonstrate the unique potential of natural template-based peptide design for chemical biology and medicinal chemistry.

Multi-Approach Analysis for the Identification of Proteases within Birch Pollen.

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.

CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families.

CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma.

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens.

BACKGROUND: The serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function. METHODS: E-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine-to-alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments. RESULTS: Infection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro. CONCLUSIONS: These data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.

Dendritic cells activated by IFN-γ/STAT1 express IL-31 receptor and release proinflammatory mediators upon IL-31 treatment.

IL-31 is a T cell-derived cytokine that signals via a heterodimeric receptor composed of IL-31Rα and oncostatin M receptor ß. Although several studies have aimed to investigate IL-31-mediated effects, the biological functions of this cytokine are currently not well understood. IL-31 expression correlates with the expression of IL-4 and IL-13 and is associated with atopic dermatitis in humans, indicating that IL-31 is involved in Th2-mediated skin inflammation. Because dendritic cells are the main activators of Th cell responses, we posed the question of whether dendritic cells express the IL-31R complex and govern immune responses triggered by IL-31. In the current study, we report that primary human CD1c(+) as well as monocyte-derived dendritic cells significantly upregulate the IL-31Rα receptor chain upon stimulation with IFN-γ. EMSAs, chromatin immunoprecipitation assays, and small interfering RNA-based silencing assays revealed that STAT1 is the main transcription factor involved in IFN-γ-dependent IL-31Rα expression. Subsequent IL-31 stimulation resulted in a dose-dependent release of proinflammatory mediators, including TNF-α, IL-6, CXCL8, CCL2, CCL5, and CCL22. Because these cytokines are crucially involved in skin inflammation, we hypothesize that IL-31-specific activation of dendritic cells may be part of a positive feedback loop driving the progression of inflammatory skin diseases.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Background: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.

The functional interplay of Helicobacter pylori factors with gastric epithelial cells induces a multi-step process in pathogenesis.

Infections with the human pathogen Helicobacter pylori (H. pylori) can lead to severe gastric diseases ranging from chronic gastritis and ulceration to neoplastic changes in the stomach. Development and progress of H. pylori-associated disorders are determined by multifarious bacterial factors. Many of them interact directly with host cells or require specific receptors, while others enter the host cytoplasm to derail cellular functions. Several adhesins (e.g. BabA, SabA, AlpA/B, or OipA) establish close contact with the gastric epithelium as an important first step in persistent colonization. Soluble H. pylori factors (e.g. urease, VacA, or HtrA) have been suggested to alter cell survival and intercellular adhesions. Via a type IV secretion system (T4SS), H. pylori also translocates the effector cytotoxin-associated gene A (CagA) and peptidoglycan directly into the host cytoplasm, where cancer- and inflammation-associated signal transduction pathways can be deregulated. Through these manifold possibilities of interaction with host cells, H. pylori interferes with the complex signal transduction networks in its host and mediates a multi-step pathogenesis.

Suppressor of cytokine signaling 2 is a feedback inhibitor of TLR-induced activation in human monocyte-derived dendritic cells.

Dendritic cells (DCs) are key players in initiating and directing the immune response. Therefore, their activation state and functional differentiation need to be tightly controlled. The activating stimuli and their signaling networks have long been an area of focus in DC research. Recent investigations have also shed light on the mechanisms of counterregulation and fine-tuning of DC functions. One class of proteins involved in these processes is the family of suppressors of cytokine signaling (SOCS), whose members were originally described as feedback inhibitors of cytokine-induced JAK/STAT signaling. Essential roles in DC function have been assigned to SOCS1 and SOCS3. In this article, we show that SOCS2 also is involved in DC regulation. In human and in murine DCs, SOCS2 is a highly TLR-responsive gene, which is expressed in a time-delayed fashion beginning 8 h after TLR ligation. Functionally, silencing of SOCS2 in DCs results in hyperphosphorylation of STAT3 at later time points. As a consequence, SOCS2-deficient DCs secrete increased amounts of the cytokines IL-1ß and IL-10, both being transcriptional targets of STAT3. We propose a model in which SOCS2 acts as a negative regulator of TLR-induced DC activation. The delayed expression of SOCS2 provides a mechanism of late-phase counterregulation and limitation of inflammation-driving DC activity.

Inhibition of suppressive T cell factor 1 (TCF-1) isoforms in naive CD4+ T cells is mediated by IL-4/STAT6 signaling.

The Wnt pathway transcription factor T cell factor 1 (TCF-1) plays essential roles in the control of several developmental processes, including T cell development in the thymus. Although previously regarded as being required only during early T cell development, recent studies demonstrate an important role for TCF-1 in T helper 2 (Th2) cell polarization. TCF-1 was shown to activate expression of the Th2 transcription factor GATA-binding protein 3 (GATA3) and thus to promote the development of IL-4-producing Th2 cells independent of STAT6 signaling. In this study, we show that TCF-1 is down-regulated in human naive CD4(+) T cells cultured under Th2-polarizing conditions. The down-regulation is largely due to the polarizing cytokine IL-4 because IL-4 alone is sufficient to substantially inhibit TCF-1 expression. The IL-4-induced suppression of TCF-1 is mediated by STAT6, as shown by electrophoretic mobility shift assays, chromatin immunoprecipitation, and STAT6 knockdown experiments. Moreover, we found that IL-4/STAT6 predominantly inhibits the shorter, dominant-negative TCF-1 isoforms, which were reported to inhibit IL-4 transcription. Thus, this study provides a model for an IL-4/STAT6-dependent fine tuning mechanism of TCF-1-driven T helper cell polarization.

Beyond the gastric epithelium - the paradox of Helicobacter pylori-induced immune responses.

Chronic infections are typically characterized by an ineffective immune response to the inducing pathogen. While failing to clear the infectious microbe, the provoked inflammatory processes may cause severe tissue damage culminating in functional impairment of the affected organ. The human pathogen Helicobacter pylori is a uniquely successful Gram-negative microorganism inhabiting the gastric mucosa in approximately 50% of the world's population. This bacterial species has evolved spectacular means of evading immune surveillance and influencing host immunity, leading to a fragile equilibrium between proinflammatory and anti-inflammatory signals, the breakdown of which can have serious consequences for the host, including gastric ulceration and cancer. This review highlights novel insights into this delicate interaction between host and pathogen from an immunological perspective.

E-Cadherin Orthologues as Substrates for the Serine Protease High Temperature Requirement A (HtrA).

Helicobacter pylori (H. pylori) expresses the serine protease and chaperone High temperature requirement A (HtrA) that is involved in periplasmic unfolded protein stress response. Additionally, H. pylori-secreted HtrA directly cleaves the human cell adhesion molecule E-cadherin leading to a local disruption of intercellular adhesions during pathogenesis. HtrA-mediated E-cadherin cleavage has been observed in response to a broad range of pathogens, implying that it is a prevalent mechanism in humans. However, less is known whether E-cadherin orthologues serve as substrates for bacterial HtrA. Here, we compared HtrA-mediated cleavage of human E-cadherin with murine, canine, and simian E-cadherin in vitro and during bacterial infection. We found that HtrA targeted mouse and dog E-cadherin equally well, whereas macaque E-cadherin was less fragmented in vitro. We stably re-expressed orthologous E-cadherin (Cdh1) in a CRISPR/Cas9-mediated cdh1 knockout cell line to investigate E-cadherin shedding upon infection using H. pylori wildtype, an isogenic htrA deletion mutant, or complemented mutants as bacterial paradigms. In Western blot analyses and super-resolution microscopy, we demonstrated that H. pylori efficiently cleaved E-cadherin orthologues in an HtrA-dependent manner. These data extend previous knowledge to HtrA-mediated E-cadherin release in mammals, which may shed new light on bacterial infections in non-human organisms.

Discovery and Characterization of Synthesized and FDA-Approved Inhibitors of Clostridial and Bacillary Collagenases.

In view of the worldwide antimicrobial resistance (AMR) threat, new bacterial targets and anti-infective agents are needed. Since important roles in bacterial pathogenesis have been demonstrated for the collagenase H and G (ColH and ColG) from Clostridium histolyticum, collagenase Q1 and A (ColQ1 and ColA) from Bacillus cereus represent attractive antivirulence targets. Furthermore, repurposing FDA-approved drugs may assist to tackle the AMR crisis and was addressed in this work. Here, we report on the discovery of two potent and chemically stable bacterial collagenase inhibitors: synthesized and FDA-approved diphosphonates and hydroxamates. Both classes showed high in vitro activity against the clostridial and bacillary collagenases. The potent diphosphonates reduced B. cereus-mediated detachment and death of cells and Galleria mellonella larvae. The hydroxamates were also tested in a similar manner; they did not have an effect in infection models. This might be due to their fast binding kinetics to bacterial collagenases.