Outer membrane targeting of passenger proteins by the vacuolating cytotoxin autotransporter of Helicobacter pylori.

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by beta-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal beta-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter.

Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter.

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E. coli. The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact.

Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.

The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA(+)) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins.

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori.

The pattern of mutations induced by neocarzinostatin and methyl methanesulfonate in the ataxia telangiectasia-like Chinese hamster cell line V-E5.

The Chinese hamster cell line V-E5 is a mutant cell line isolated from V79 cells. The phenotypic characteristics of V-E5 strongly resemble those of cells from patients suffering from the genomic instability syndrome ataxia telangiectasia. In order to further characterize the mutant cell line and to get insight into the underlying genetic defect we compared the clastogenic and mutagenic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS) in V-E5 and V79 wild-type cells (V79-LE). V-E5 cells were 2-3 times more sensitive to the cytotoxic effect of NCS or MMS. The clastogenic action of NCS was characterized by the predominant induction of chromosome breaks and dicentrics in both cell lines, whereas MMS mainly induced chromatid-type aberrations. The frequency of mutations induced by NCS as well as MMS was slightly enhanced in V-E5 cells compared to V79 cells treated with the same dose. However, the mutant cell line was found to be hypomutable when considering the same survival level as in the parental cell line. Molecular analysis of mutants induced by NCS revealed a high frequency of total deletions of the hprt gene in both cell lines. In contrast, among MMS-induced mutations only 11% deletion mutations were found in V79-LE, whereas in V-E5 MMS-induced deletions were seen in 52% of the hprt-deficient mutants. These results are discussed with respect to a possible relation between genomic instability, cell cycle control and mutational spectra.

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells.

Adriamycin (AM), a widely used chemotherapeutic drug, induced a broad spectrum of gene mutations at the hprt locus of V79 cells. The frequency and distribution of AM-induced deletions was analyzed with multiplex polymerase chain reaction in two V79 cell lines, which differed considerably in their spontaneous deletion frequency. Among AM-induced mutants, deletions predominated in both cell lines. Apart from total deletions of the hprt gene, partial deletions were found which were distributed all over the hprt gene with breakpoints in nearly all introns. Under the same experimental conditions, chromosome aberrations were induced by AM which mainly represented chromatid-type aberrations. Neither the induction of gene mutations nor the induction of chromosome aberrations was enhanced by the repair inhibitor 3-aminobenzamide. These results are discussed in the context with our earlier findings on bleomycin-induced mutations and it is suggested that at least two mechanisms lead to the formation of gene deletions. One of them seems to be associated with a misrepair process of frank DNA double-strand breaks and related to chromosome aberrations while the other is not.