Functional characterisation of a novel type IV secretion system associated with the severe disease marker, dupA
Lead Research Organisation:
UNIVERSITY OF NOTTINGHAM
Department Name: Nottingham Digestive Diseases Centre
Abstract
Helicobacter pylori is a common bacterial infection of the human stomach, affecting approximately half of the world?s population. In most people the infection is harmless, however, in ~20% of infected individuals, H. pylori causes serious disease, including gastric/duodenal ulcers, and gastric cancer. The presence of several genes encoded within the H. pylori genome have been shown to be putative markers for the development of severe disease coincident with H. pylori infection. We have shown that these markers are part of a single large cluster of genes which encode proteins known to assemble into a specialised bacterial ?type IV? secretion system (T4SS). Bacterial T4SSs function to transport protein-DNA complexes into other bacterial cells in the exchange of genetic material, or in the case of bacteria which cause diseases in plants, function to transport and integrate bacterial DNA into the host plant cell chromosome. This latter process genetically transforms the plant cell and initiates the development of disease.
Due to previously established association of individual H. pylori T4SS genes with disease and the presence of certain components within the T4SS gene cluster, which we call tfs4, we hypothesise that tfs4 may similarly transport protein-DNA complexes into infected cells within the human stomach. We propose to determine whether tfs4 activity (on/off status) similarly associates with disease presentation in strains isolated from infected individuals and to fully investigate the function of tfs4.
In plant pathogens, a protein called VirD2 is responsible for the transport and integration of bacterial DNA into the host plant cell chromosome. H. pylori tfs4 encodes a similar VirD2 protein. We will determine what other proteins and specific DNA sequences tfs4 VirD2 binds to and establish whether it can be transported, in complex with bacterial DNA, via the novel H. pylori T4SS to different types of human gastric cells. This may reveal a novel, uncharacterised and extremely important underlying mechanism by which H. pylori is able to cause serious diseases, such as gastric cancer in some infected individuals. Our work will be important for determining which strains of H. pylori have the most potential for causing disease and may provide insight into how the tfs4 T4SS can be exploited in future for the treatment of gastric disorders. Our observations will be published in the scientific press, on our publicly accessible websites and potentially also communicated to the national media via our Press Office to capture and stimulate broad public attention.
Due to previously established association of individual H. pylori T4SS genes with disease and the presence of certain components within the T4SS gene cluster, which we call tfs4, we hypothesise that tfs4 may similarly transport protein-DNA complexes into infected cells within the human stomach. We propose to determine whether tfs4 activity (on/off status) similarly associates with disease presentation in strains isolated from infected individuals and to fully investigate the function of tfs4.
In plant pathogens, a protein called VirD2 is responsible for the transport and integration of bacterial DNA into the host plant cell chromosome. H. pylori tfs4 encodes a similar VirD2 protein. We will determine what other proteins and specific DNA sequences tfs4 VirD2 binds to and establish whether it can be transported, in complex with bacterial DNA, via the novel H. pylori T4SS to different types of human gastric cells. This may reveal a novel, uncharacterised and extremely important underlying mechanism by which H. pylori is able to cause serious diseases, such as gastric cancer in some infected individuals. Our work will be important for determining which strains of H. pylori have the most potential for causing disease and may provide insight into how the tfs4 T4SS can be exploited in future for the treatment of gastric disorders. Our observations will be published in the scientific press, on our publicly accessible websites and potentially also communicated to the national media via our Press Office to capture and stimulate broad public attention.
Technical Summary
H. pylori colonises the gastric mucosa of over half the world?s population causing gastritis, peptic ulceration and gastric adenocarcinoma. Recent studies identify several marker genes significantly associated with severe clinical outcome of infection encoded within plasticity regions of the H. pylori genome. Our preliminary analyses show that all these marker genes are genetically linked and encoded within a single novel cluster of type IV secretion system (T4SS) genes. This analysis indicates that the activity of the T4SS, termed here tfs4, underlies the development of severe disease associated with H. pylori infection in some human populations. We propose two main avenues of research to investigate tfs4.
First, we will determine the prevalence and activation status of tfs4 using PCR to screen for complete tfs4 clusters in an extensive collection of geographically diverse clinical strains (400+), then assess activation status using specific antibodies. Association of tfs4 presence/activity with known clinical presentation will be determined using appropriate statistical analysis.
Second, we will investigate the function of tfs4. Within the tfs4 gene cluster we identify homologues of Agrobacterium tumefaciens genes VirD2 and VirC1 which are critical to the ability of the pathogen to genetically transform host plant cells. In A. tumefaciens, VirD2 functions as an endonuclease to excise and covalently bind to specific transfer(T)-DNA sequences. In association with other Vir proteins it pilots the T-DNA through the T4SS into the host plant cell, where it localises to the nucleus and integrates T-DNA into the host chromosome. These diverse VirD2 activities are dependent upon several sequence motifs which are similarly conserved in the sequence of H. pylori tfs4 VirD2. We will therefore comprehensively investigate the protein-protein and protein-DNA interactions of tfs4 VirD2 in both complex and candidate yeast two hybrid screens, co-immunoprecipitation and column pulldown approaches. We will determine whether VirD2 is translocated to the host cell and confirm its cellular localisation using an established GSK-tag reporter and novel protein transfection approaches in combination with immunostaining and fluorescence microscopy. We will also determine the sequence of DNA to which VirD2 binds and its sequence context. Finally, we will provide preliminary confirmation of tfs4 activity in vivo using immunohistochemical analysis of existing gastric cell samples. Our collective results are anticipated to improve determination of pathogenic potential of H. pylori strains, inform clinical management of infected individuals and present an opportunity for biotechnological exploitation of tfs4 activity for the treatment of gastric diseases in the future.
First, we will determine the prevalence and activation status of tfs4 using PCR to screen for complete tfs4 clusters in an extensive collection of geographically diverse clinical strains (400+), then assess activation status using specific antibodies. Association of tfs4 presence/activity with known clinical presentation will be determined using appropriate statistical analysis.
Second, we will investigate the function of tfs4. Within the tfs4 gene cluster we identify homologues of Agrobacterium tumefaciens genes VirD2 and VirC1 which are critical to the ability of the pathogen to genetically transform host plant cells. In A. tumefaciens, VirD2 functions as an endonuclease to excise and covalently bind to specific transfer(T)-DNA sequences. In association with other Vir proteins it pilots the T-DNA through the T4SS into the host plant cell, where it localises to the nucleus and integrates T-DNA into the host chromosome. These diverse VirD2 activities are dependent upon several sequence motifs which are similarly conserved in the sequence of H. pylori tfs4 VirD2. We will therefore comprehensively investigate the protein-protein and protein-DNA interactions of tfs4 VirD2 in both complex and candidate yeast two hybrid screens, co-immunoprecipitation and column pulldown approaches. We will determine whether VirD2 is translocated to the host cell and confirm its cellular localisation using an established GSK-tag reporter and novel protein transfection approaches in combination with immunostaining and fluorescence microscopy. We will also determine the sequence of DNA to which VirD2 binds and its sequence context. Finally, we will provide preliminary confirmation of tfs4 activity in vivo using immunohistochemical analysis of existing gastric cell samples. Our collective results are anticipated to improve determination of pathogenic potential of H. pylori strains, inform clinical management of infected individuals and present an opportunity for biotechnological exploitation of tfs4 activity for the treatment of gastric diseases in the future.