Targeting focal adhesions:Helicobacter pylori-host communication in cell migration

Highly dynamic integrin-based focal adhesions provide an important structural basis for anchoring the cellular actin cytoskeleton to the surrounding extracellular matrix. The human pathogen Helicobacter pylori (H. pylori) directly targets integrins with drastic consequences on the epithelial cell morphology and migration, which might contribute to the disruption of the gastric epithelium in vivo. In this review, we summarize the recent findings concerning the complex mechanism through which H. pylori interferes with host integrin signaling thereby deregulating focal adhesions and the actin cytoskeleton of motile epithelial cells

Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies

Inhibitors of Helicobacter pylori Protease HtrA Found by ‘Virtual Ligand’ Screening Combat Bacterial Invasion of Epithelia

Background: The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings: We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance: This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection

Untersuchungen zum Mechanismus der Helicobacter pylori- induzierten Auflösung epithelialer Zelladhäsion

Helicobacter pylori (H. pylori) ist ein weit verbreitetes Humanpathogen, welches den menschlichen Magen besiedelt und zu schwerwiegenden entzündlichen Erkrankungen des gastralen Traktes führen kann. Bereits 1994 wurde das Bakterium als ein Klasse 1 Karzinogen deklariert, da H. pylori im erwiesenen Maße mit der Entstehung von hochinvasivem Magenkrebs in Verbindung gebracht wird. In vitro induziert H. pylori eine starke Migration der infizierten Epithelzellen, die unter anderem mit der Auflösung der Zellkontakte einhergeht. Die zugrunde liegenden molekularen Zusammenhänge konnten bisher noch nicht vollständig aufgeklärt werden. Die Mechanismen der Auflösung der Zelladhäsion wurden in der vorliegenden Arbeit untersucht, um einen tieferen Einblick in die H. pylori vermittelten Virulenz zu erhalten. So konnte eine H. pylori-induzierte Dissoziation des E-Cadherin Komplexes, bestehend aus p120, 􀄮- und 􀈕-Catenin beobachtet werden, der in einem Verlust der Zelladhäsion resultierte. Es konnte darüber hinaus eine Spaltung der extrazellulären Domäne von ECadherin detektiert werden, die wahrscheinlich zu einer Destabilisierung und somit zur Auflösung des gesamten E-Cadherin Komplexes führte. Durch den Zerfall der Adhärenzverbindungen wurden Catenine in den zytoplasmatischen Pool freigegeben, von denen p120 in den Zellkern translozierte und die Transaktivierung von Zielgenen auslöste, die in diesem Zusammenhang mit Hilfe von Reportergenanalysen quantifiziert wurden. Diese Prozesse zeigten sich von dem Pathogenitätsfaktor CagA (cytotoxin associated gene A) unabhängig, der über das bakterielle Typ IV Sekretionssystem in die Wirtszellen transloziert wird und krebsassoziierte Signaltransduktionswege aktivieren kann. In weiteren Untersuchungen wurden deshalb die Auswirkungen löslicher H. pylori Faktoren auf die Spaltung von E-Cadherin und folglich auf die Zellmotilität und Morphologie epithelialer Zellen analysiert. Aufgrund dieser Beobachtungen wurden in weiteren Experimenten proteolytische Aktivitäten von H. pylori untersucht. Dabei konnte erstmalig die hypothetische Protease HtrA (high temperature requirement protein A) von H. pylori durch massenspektrometrischen Analysen als eine caseinolytisch aktive Protease gefunden werden. Nach der Klonierung und Aufreinigung von HtrA konnte darüber hinaus auch E-Cadherin als spezifisches biologisches Substrat der Wirtszellen für HtrA identifiziert werden. Diese selektive Fragmentierung von E-Cadherin durch HtrA fügt sich als ein neues Element in das Modell der H. pylori Pathogenese, die einen initialen Schritt in der frühen Phase der Infektion darstellen könnte

Identification of <i>H. pylori</i> proteases.

<p>(A) For a preparative analyses, 18×10⁹ bacteria were lysed and analyzed by zymography. The upper (1) and lower (2) negatively stained protease bands were excised, proteins were eluated and separated by SDS PAGE and Coomassie staining (B). Indicated protein bands were analyzed by mass-spectrometry.</p

Proteolytic activity of the Hp1018/19 protein.

<p>(A) For the construction of the GST-Hp1018/19Δsp fusion protein, the re-sequenced Hp1018/19 gene was amplified without the putative signal peptide and cloned into the pGEX-6P-1 vector. (B) The <i>gst-hp1018/19Δsp</i> construct was transformed in <i>E. coli</i> for overexpression and total protein extracts from untreated (lane 1) and IPTG-induced <i>E. coli</i> (lane 2) were separated by SDS PAGE. Overexpressed GST-Hp1018/19Δsp was precipitated using glutathione sepharose and released by three eluation steps (lanes 3–5). To remove the GST tag, GST-Hp1018/19Δsp bound to glutathione sepharose were treated with PreScission protease and 30 µg protein of the supernatant containing the Hp1018/19 (lane 6) were loaded on a SDS PAGE followed by Coomassie staining. (C) Three µg of purified GST-Hp1018/19Δsp^S205A (lane 1), GST-Hp1018/19Δsp (lane 3), PreScission protease-treated Hp1018/19Δsp^S205A (lane 2) and Hp1018/19Δsp (lane 4) were analyzed by casein zymography for proteolytic activity.</p

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion

Hoy B, Loewer M, Weydig C, et al. Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion. EMBO REPORTS. 2010;11(10):798-804.Mammalian and prokaryotic high-temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell-adhesion protein E-cadherin. E-cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small-molecule inhibitor that efficiently blocks HtrA activity, E-cadherin cleavage and intercellular entry of H. pylori