Host/pathogen interaction

Suche


Molecular function and signal transduction of the serine protease HtrA: a novel secreted effector protein of bacterial pathogens
Stable adhesion complexes are crucial for maintenance of the cell-to-cell integrity in healthy epithelia of humans and represent the first barrier for microbial pathogens. Alterations in these complexes are key events in the development and progression of many diseases including various cancers. One of the major proteins involved in maintaining epithelial adhesion is the tumor-suppressor and junctional transmembrane protein E-cadherin. E-cadherin also controls the transcription factor b-catenin and other signaling components, and is involved in cell morphogenesis, adhesion, recognition and communication. Inactivation of E-cadherin’s adhesive properties is often a key step in various infections, tumor progression and metastasis. We have recently identified a novel secreted effector protein, the serine protease HtrA, ofthe foodborne pathogen Campylobacter jejuni and the gastric pathogen and type-I carcinogen Helicobacter pylori. HtrA of these microbes can be secreted into the extracellular space where it can cleave-off the extracellular domain of E-cadherin on polarized gastric or intestinal epithelial cells. Since the htrA gene is conserved in many bacterial pathogens our findings could provide a potential novel mechanism how these microbes may destroy cellular junctions in mucosal epithelial cells, in order to get access to deeper tissues and cause disease by degrading E-cadherin and probably other cellular factors. This project is therefore designed to contribute to the understanding of mechanisms underlying basic HtrA functions in bacterial pathogenesis. In particular, we investigate the molecular mechanisms/requirements for HtrA proteolytic activity, pinpoint novel HtrA targets on host cells and to study downstream signaling events. In addition, we aim to identify the secretion pathway of HtrA across the two bacterial membranes because this could give hints how HtrA is transported. Our studies will take advantage of powerful technologies including fluorescence microscopy and live cell imaging as well as proteomics-based and cellular signal transduction approaches.

 Structure and Function of the H. pylori cag type IV secretion system
Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA/B, SabA, AlpA/B, OipA and HopZ) and the cag (cytotoxin-associated genes) pathogenicity island (PAI) encoding a type IV secretion system (T4SS). The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA can bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin β1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA/B during infection are still unknown. We are interested in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. Of particular interest is the T4SS encoded by the cagPAI. T4SSs are macromolecular assemblies used by numerous bacteria to transport molecules across their membranes including proteins and DNA. T4SS are generally composed of a set of 12 proteins (VirB1-11 and the VirD4 coupling factor) plus some accessory proteins. This together represents a dynamic membrane-anchored machinery powered by three encoded NTPases. Since T4SSs are widespread in many pathogenic bacteria where they are often used to deliver effectors into host cells, we investigate the unique and complex interaction of various components of the H. pylori T4SS as a model system. Its interaction with the integrin β1 receptor will help to better understand the molecular mechanisms of how protein translocation events work in H. pylori. We therefore use specific bacterial mutant strains, proteomics-based methods, protein-protein interaction studies such as Biacore and sophisticated microscopic technologies.

Identification and characterization of novel Helicobacter species in mammals

As Helicobacter pylori was the first bacterium cultivated from human gastric biopsy specimens in 1982, it has become apparent that Helicobacter subspecies exhibit a broad host spectrum and can be isolated from the gastrointestinal tracts of humans, non-human primates, cats, dogs, cheetahs, ferrets, rodents, cows, sheep, pigs, dolphins, and birds. Currently there are 32 validated Helicobacter species and several other described isolated candidates. Members of the genus Helicobacter are helical curved, spiral or fusiform, Gram-negative bacteria with or without helical periplasmic fibers. We are interested in the investigation and characterization of novel enterohepatic Helicobacter subspecies (EHS), which are an emerging group of microaerobic, motile pathogens carrying flagella with variable styles in number and locations. EHS are known to persistently colonize multiple animal species. They can be isolated from the lower intestine, hepatobiliary system, and diarrheic feces and are potentially associated with chronic inflammation and epithelial cell hyperproliferation leading to neoplasic disease. Helicobacter hepaticus, considered the prototype of all known EHS, has been shown to induce chronic active hepatitis and hepatocellular carcinoma in A⁄JCr and B6C3F1 mice strains as well as typhlocolitis in A⁄JCr inbred mice. This species is also used experimentally to induce cholesterol gallstones, inflammatory bowel disease (IBD), and in certain strains of mice induces colon cancer. Because lesions caused by EHS in mice often mimic those seen in humans with cholecystitis, their possible role in hepatobiliary disease in humans has been proposed. In addition, the possible zoonotic origin of important clinical manifestations in humans and the health status of mice housed in research facilities have recently attracted the attention of scientists. In contrast to H. pylori, almost nothing is known about potential virulence factors in EHS. To evaluate the prevalence of EHS infections in mouse strains harbored in specific-pathogen-free (SPF) facilities, we tested 40 mouse lines that were permanently living in nine colony rooms using a group-specific PCR, which detects all Helicobacter species currently known. When Helicobacter-negative and infected mice shared the same cage, transmission of the infection occurred within two weeks at very high frequency (100%). Furthermore, we found that mice from commercial breeding facilities may carry undetected Helicobacter infections. We also showed that infection with EHS may occur and spread frequently in mice under SPF conditions, and despite extensive safety conditions. Due to the lack of available methods, EHS infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. We therefore isolate and investigate live pathogens, and develop methods to rapidly identify novel EHS.