Benjamin Hoy

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

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.

Distinct Roles of Secreted HtrA Proteases from Gram-negative Pathogens in Cleaving the Junctional Protein and Tumor Suppressor E-cadherin

Background: The function of HtrA proteases in bacterial infections is widely unknown. Results: Secreted HtrA from various bacterial pathogens exhibits a conserved specificity for cleavage of E-cadherin. Conclusion: HtrA-mediated E-cadherin cleavage is a prevalent novel mechanism in bacterial pathogenesis. Significance: HtrA activity plays a direct role in the pathogenesis of different bacteria. The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple Gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections.

Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

BackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

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.

Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells

ABSTRACT Helicobacter pylori is an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer. H. pylori pathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms of H. pylori were performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative analysis of 14 different human gastric epithelial cell lines after colonization with H. pylori. We found remarkable differences in host cell morphology, extent of CagA tyrosine phosphorylation, adhesion to host cells, vacuolization, and interleukin-8 (IL-8) secretion. These data might help in the selection of suitable cell lines to study host cell responses to H. pylori in vitro, and they imply that different host cell factors are involved in the determination of H. pylori pathogenesis. A better understanding of H. pylori-directed cellular responses can provide novel and more balanced insights into the molecular mechanisms of H. pylori-dependent pathogenesis in vivo and may lead to new therapeutic approaches.

Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

Extracellular secretion of protease HtrA from Campylobacter jejuni is highly efficient and independent of its protease activity and flagellum.

The serine protease HtrA of C. jejuni has been identified as a novel secreted virulence factor which opens cell-to-cell junctions by cleaving E-cadherin. Efficient C. jejuni transmigration across polarized human epithelial cells requires the intact flagellum and HtrA; however, the mechanism of HtrA secretion into the supernatant is unknown. Here we show that HtrA secretion is highly efficient and does not require its proteolytic activity because the protease-inactive S197A mutant is secreted like wild-type HtrA. In addition, the flagellar mutants ΔflaA/B, ΔfliI, ΔflgH, ΔflhA, ΔflhB, and ΔflgS were also able to secrete HtrA in high amounts, while they were strongly attenuated in secreting the well-known invasion antigen CiaB. We also tested several culture media and cell lines of different origin such as human, mouse, hamster, dog, and chicken for their ability to influence HtrA secretion. Interestingly, HtrA was effectively secreted in the presence of most but not all cell lines and media, albeit at different levels, but secretion was significantly higher when fetal calf serum (FCS) was added. These results demonstrate that HtrA secretion by Campylobacter proceeds independent of HtrAs protease activity, the flagellum and origin of cell lines, but can be strongly enhanced by molecular compound(s) present in FCS.

The stability and activity of recombinant Helicobacter pylori HtrA under stress conditions

The bifunctional protein HtrA displays chaperone and protease activities, enabling bacteria to cope with environmental stress conditions such as heat shock or extreme pH by orchestrating protein folding or degradation. Recently, we added a novel aspect to HtrA functions by identifying HtrA of the human pathogen and class I carcinogen Helicobacter pylori (Hp) as a secreted virulence factor that cleaves the cell adhesion molecule and tumor suppressor E‐cadherin. In this study, we analyzed the structural integrity and activity of oligomeric HtrA from Hp under stress conditions. Examining different parameters, HtrA oligomers were investigated by casein zymography and HtrA activity was further analyzed in in vitro cleavage assays using E‐cadherin as a substrate. HtrA showed temperature‐dependent disintegration of oligomers. Denaturing agents targeting hydrogen bonds within HtrA destabilized HtrA oligomers while reducing agents disrupting disulfide bonds had no effect. Optimal proteolytic activity was dependent on a neutral pH; however, addition of mono‐ and divalent salts or reducing agents did not interfere with proteolytic activity. These data indicate the HtrA is active under stress conditions which might support Hp colonizing in the gastric environment.

From Virtual Screening to Bioactive Compounds by Visualizing and Clustering of Chemical Space

Identification and visualization of ‘activity islands’ in chemical space is presented as a straightforward method for rapid automated identification of bioactive compounds and drug target profiling. We successfully applied this computational technique to finding inhibitors of Helicobacter pylori protease HtrA with new molecular scaffolds, and to deorphanizing of a compound from a combinatorial oxadiazole library. Bioactive molecules were discovered with minimal experimental effort. The results demonstrate that visualization of ‘chemical space’ provides an intuitive approach to molecular design and virtual screening in drug discovery, even in the absence of a three-dimensional receptor structure. Visualization of chemical data can help understand the structure of compound distributions in chemical space and guide molecular design experiments. [1–5] Commonly applied visualization techniques in chemistry are principal component analysis (PCA) [6] and self-organizing maps (SOMs, Kohonen networks). [7–9] Both methods have proven their value for visualization of compound libraries and virtual screening. Still, they suffer from several drawbacks. For example, a SOM’s quality to separate data depends on the chosen map size, i.e. the number of ‘neurons’ (local clusters, Voronoi fields), and determining the actual quality of a computed SOM projection is nontrivial. A perceived disadvantage is that SOMs lack immediate interpretability due to nonlinear projection. While for low-dimensional data linear projection by PCA seems to be preferable, SOMs have shown to produce more robust projections of high-dimensional data. [10] Here, we present a method for visualizing and interpreting high-dimensional chemical data, which is complementary to SOM and PCA projection and overcomes some of their disadvantages and limitations. The projection is based on stochastic proximity embedding (SPE). [11] SPE embeds data in a low-dimensional space in such a way that pairwise distances between compounds are preserved. As a consequence, patterns in the original high-dimensional data distribution become accessible to visual inspection. Making such patterns visible supports our intuitive interpretation how a molecular representation might distinguish between sets of compounds (e.g., active vs inactive) and create some kind of order in data space. Once activity islands are identified in the visualization, the compounds that form such local clusters can be extracted and subjected to biochemical tests. [12–15]

Correction for Schneider et al., Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells

Volume 79, no. 6, p. [2362–2371][1], 2011. Page 2368: Figure 5 should appear as shown below. ![Figure][2] [1]: /lookup/doi/10.1128/IAI.01350-10 [2]: pending:yes