Structural biology of Helicobacter pylori type IV secretion system

Background

Helicobacter pylori chronically infects the gastric mucosa of millions of people annually worldwide: it has been estimated that over 50% of the world population carries this infection. H. pylori has been associated with the development of several diseases, like chronic gastritis, gastric and duodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphoma [1–3].

The complete genome sequence of two different isolates of H. pylori (J99 and 26995) is known. The strains that contain a 37 kb foreign DNA region, called cag pathogenicity island (cag-PAI), cause the most severe form of virulence [4].

The cag-PAI encodes for a functional type IV secretion apparatus homologous to the VirB/D4 Type IV Secretion System (T4SS) of the plant pathogen Agrobacterium tumefaciens and other Gram-negative bacteria [5]. T4SSs are involved in conjugal DNA transfer, in the DNA delivery to (or uptake from) the environment, for instance the release of oncogenic DNA into infected plant cells by A. tumefaciens, or in the translocation of effector proteins [6, 7].

The T4SS encoded by the cag-PAI of H. pylori is responsible for the translocation into the host cell of the protein CagA, a major antigenic virulence factor encoded within the cag-PAI. Once secreted into the gastric epithelial cells, CagA induces cellular modifications, such as elongation and spreading of host cells [8].

The aim of this structural genomic project is to determine the three-dimensional structure of most of the proteins encoded by the cag-PAI, a task that will allow to elucidate the function and the organization of the entire T4SS of such a relevant pathogenic bacterium [9].

Results

Protein production for structural studies presents one of the most difficult and challenging tasks for heterologous expression in E. coli. Generally, the protein must be native, active, soluble, highly pure, and concentrated. We have identified protein insolubility/aggregation as the major bottlenecks in the process towards the determination of protein structures by X-ray diffraction. Each protein often needs separate handling and analysis to determine tag choice, growth and buffer conditions for optimal solubility. To speed up recombinant protein production, we have adopted a strategy of parallel expression of a protein from a variety of vectors containing different tags and/or fusion partners, and a variety of E. coli host strains.

To this point in time, we have cloned, expressed, and purified several proteins of the cag pathogenicity island of H. pylori. They all have been expressed in E. coli. We have already determined the structure of CagZ [10] and CagS, using the Se-Met method. We have also obtained crystals of an other protein, along with crystallization tests on other cag proteins.

Perspectives

We believe these studies will also furnish valuable information for vaccine production and provide insights into the mechanism of H. pylori pathogenesis.