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Research > Medical Microbiology and Hospital Epidemiology > WG PD Dr. W. Fischer > Research > 

Functional analysis of the H. pylori Cag type IV secretion apparatus

The cag pathogenicity island, and the type IV protein secretion system encoded on this island, are considered as major virulence determinants of H. pylori. Important functions of this secretion system are the induction of a proinflammatory response in gastric tissues, and direct translocation of the bacterial CagA protein into the cytoplasm of various host cells. Once inside the target cell, CagA becomes phosphorylated at several tyrosine residues and causes changes in cell morphology and gene expression.
The Cag type IV secretion system uses at least 15 different proteins to form a secretion apparatus spanning both bacterial membranes, and pilus-like appendages at the bacterial surface. We have recently defined a comprehensive interaction network including all components of the system, resulting in identification of distinct apparatus subcomplexes. We are analyzing the structure and function of these individual components and subcomplexes, using genetic, cell biological and biochemical techniques. As an example, we were able to show by localization and interaction studies that the CagI and CagL proteins form a functional complex in the periplasm and/or at the bacterial cell surface. Since these unique components mediate binding of the secretion apparatus to integrin receptors on target cells, formation of this complex presumably represents a crucial step in secretion apparatus assembly.

Signal recognition mechanisms in the Cag type IV secretion system

The precise, but dynamic sorting of proteins to their respective destinations is a process of fundamental importance for all living cells. However, the underlying molecular mechanisms, for example signal sequences that target proteins to Sec-independent bacterial secretion systems, are still only poorly understood. We have recently developed novel reporter assays to monitor type IV secretion in H. pylori, and we are using them to examine the signals that target CagA to the type IV secretion apparatus. Furthermore, we characterize the molecules that are involved in recognition of these signals, and examine accessory proteins such as type IV secretion chaperones. We have previously shown that the dedicated CagA translocation factors CagF, Cagβ and CagZ assemble in a cytoplasmic membrane-associated substrate recognition complex. A closer examination of the secretion chaperone CagF has revealed that CagF binds to several domains of the CagA substrate and thereby probably keeps CagA in a translocation-competent state.
A detailed knowledge about these molecular interactions and processes is not only important for understanding H. pylori pathogenicity and type IV secretion in general, but may also be useful for engineering this molecular machine as a transporter for foreign proteins.

Helicobacter pylori plasticity zones as mobile genomic islands

One of the most remarkable features of H. pylori is its unusual genetic diversity, resulting from high mutation and recombination rates as well as from efficient horizontal gene transfer mechanisms. Different isolates also exhibit considerable variability in their gene content, with many strain-specific genes clustered in distinct genomic regions termed plasticity zones (PZs). Whole genome sequencing of several H. pylori strains and comparative genome analysis has revealed that PZs are organized as mobile genome islands (or integrating conjugative elements) that can insert via site-specific recombination into the bacterial chromosome. Although the function of these islands is currently unknown, the presence of certain PZ genes has recently been associated with development of duodenal ulcer.
A thorough comparative analysis of PZs from more than 40 H. pylori strains has revealed that many of these regions encode complete type IV secretion systems. Integration of PZs may occur at numerous sites in the chromosome, but all currently known sites share a common sequence motif which is duplicated upon PZ insertion. We have demonstrated horizontal gene transfer of such a region from donor to recipient cells. It occurs via a non-classical, conjugation-like mechanism in which the recipient bacteria take advantage of the transport machinery that is also involved in natural transformation competence.