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Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the roTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydiai transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation. more...

Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with thetight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region. more...

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR~P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR~P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR~P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene. more...

Research concerning Escherichia coli adhesin autotransporter domain is presented. The involvement of this domain in diffuse adherence that is genetically fused to periplasmatic enzyme beta-lactamase is discussed. more...

Results on the characterization of adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter show that the autotransporter is localized to the membrane. The linker and the beta-barrel components of the autotransporter are essential for its export function and the latter represents the C-terminal domain of AIDA-I autotransporter. more...

A suitable tester plasmid was constructed to screen a plasmid library of Neisseria gonorrhoeae genomic DNA fragments to determine the presence of a site-specific recombinase in gonococci. Analysis of the plasmid library of Neisseria gonorrhoeae with a tester plasmid containing the Moraxella invertible region indicated the presence of a putative gonococcal recombinase. Furthermore, Neisseria gonorrhoeae encoded a protein that was capable of functionally replacing the Moraxella lacunata piv gene in DNA inversion assays. more...

The structure of the C-terminal domain of the Escherichia coli AIDA-I adhesin was analyzed to determine the efficiency of the autotransporter domain in the translocation of proteins from the outer membrane. Insertion of the autotransporter domain in the outer membrane of Escherichia coli induced resistance to exogenous and endogenous protease treatment which was mediated by the autotransporter function of AIDA-I. Furthermore, AIDA-I autotransporter-mediated secretion of passenger proteins had no significant influence on Escherichia coli growth rate. more...

The expression of Neisseria gonorrhoeae pilin in Pseudomonas aeruginosa was examined. It was shown that P. aeruginosa K/2PfS has the ability to express pili of N. gonorrhoeae MS11 when transformed with an expression plasmid carrying the foreign pilE1 pilin gene. P. aeruginosa produced only high levels of gonococcal MS11 pili with no indication of production of P. aeruginosa PAK/2PfS pili. more...

The review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. It discusses the future biotechnical and biomedical applications of the above applications. more...

Live attenuated Salmonella strains expressing antigens of pathogens are promising oral vaccine candidates. There is growing evidence that the topology of expression of the foreign antigens can have a dramatic impact on the immunogenicity. We examined the potential of the AIDA-I (Escherichia coli adhesin involved in diffuse adherence) autotransporter domain to display antigenic fragments of the urease A subunit of Helicobacter pylori for the induction of a protective immune response. In the murine H. pylori model, protection is mainly mediated by CD4(+) T cells, and we therefore used the AIDA-I expression system to successfully express both nearly full-length UreA and defined T-helper-cell epitopes on the surface of an attenuated Salmonella enterica serovar Typhimurium vaccine strain. Surface exposure of the large UreA fragment or of one UreA T-cell epitope mediated a significant reduction in the level of H. pylori in immunized mice after challenge infection, whereas conventional cytoplasmic expression of UreA in Salmonella had no effect. These results support the concept that surface display increases the immunogenicity of recombinant antigens expressed on oral live vaccine carriers and further demonstrate the feasibility of immunizing against H. pylori with Salmonella vaccine strains expressing CD4(+) T-cell epitopes. more...

Infection with cag+ but not cag-negative Helicobacter pylori leads to the formation of large homotypic aggregates of macrophage-like cells. Intracellular adhesion molecule 1 is up-regulated and recruited to the cell surface of infected cells and mediates the aggregation via lymphocyte function-associated molecule 1. This signaling may regulate cell-cell interactions and inflammatory responses. more...

Although Helicobacter pylori has generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number of H. pylori colonies recovered was lower than that for Salmonella enterica serovar Typhimurium X22, Escherichia coli expressing InvA, and Yersinia enterocolitica YO:9 grown at 25 degrees C but higher than that for Neisseria gonorrhoeae VP1 and Y. enterocolitica YO:9 grown at 37 degrees C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. Internalized H. pylori was bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry of H. pylori in a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry of H. pylori into AGS cells. Collectively, these results demonstrate that the entry of H. pylori into AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events. more...

The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering ("hummingbird") phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model. more...