Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as specific gastric cancers. H. pylori infects approximately 50% of the world's population, and infections can persist throughout the lifetime of the host. Motility and chemotaxis have been shown to be important in the infection process of H. pylori. We sought to address the specific roles of chemotaxis in infection of a mouse model system. We found that mutants lacking cheW, cheA, or cheY are all nonchemotactic and infect FVB/N mice with an attenuated phenotype after 2 weeks of infection. If infections proceeded for 6 months, however, this attenuation disappeared. Histological and culture analysis revealed that nonchemotactic mutants were found only in the corpus of the stomach, while the wild type occupied both the corpus and the antrum. Further analysis showed that nonchemotactic H. pylori isolates had an increased 50% infectious dose and were greatly outcompeted when coinfected with the wild type. If nonchemotactic mutants were allowed to establish an infection, subsequent infection with the wild type partially displaced the nonchemotactic mutants, indicating a role for chemotaxis in maintenance of infection. The data presented here support four roles for chemotaxis in H. pylori mouse infections: (i) establishing infection, (ii) achieving high-level infection, (iii) maintaining an infection when there are competing H. pylori present, and (iv) colonizing all regions of the stomach.Helicobacter pylori is a motile, chemotactic bacterium that colonizes the stomachs of ϳ50% of the world's population (13). Infection with H. pylori can persist throughout the lifetime of the host and can cause symptoms ranging from mild gastritis to gastric and duodenal ulcers to cancers, such as mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma (4, 39).Motility and chemotaxis, directed swimming, are survival factors for many bacterial species. Both motility and chemotaxis aid commensal and pathogenic infections, but little is known of the precise benefits of these processes. For example, the 50% lethal dose (LD 50 ) of Vibrio anguillarum nonchemotactic mutants for trout is increased 400-fold (28) (for reviews see references 22 and 30). In contrast, some nonchemotactic mutants of V. cholerae colonize infant mice better than does the wild type, in part at least because they occupy a larger portion of the gastrointestinal tract (11,20). In most of these cases, however, it is not known why motility mutants fare differently than the wild type. H. pylori is an excellent organism with which to ascertain motility's role in infection, because this bacterium is thought to lack a significant environmental niche outside of the human host and, thus, likely uses motility and chemotaxis within the host. In fact, disruption of genes involved in either motility or chemotaxis attenuates colonization of mice and piglets (16,17,19,24,29). Although these findings indicate that motility and chemotaxis play important roles during...

A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW-and responseregulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants. cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour. INTRODUCTIONMany micro-organisms move in a directed fashion in response to their environment. A common mechanism for movement is via rotary motor organelles called flagella. These motors are regulated by the chemotaxis signal transduction system, which transduces environmental cues into a swimming response. The core of this signal transduction system consists of chemoreceptors, a kinase (CheA), a receptor-kinase coupler (CheW) and a phosphorylatable response regulator (CheY) that controls flagellar rotation (Blair, 1995;Szurmant & Ordal, 2004).There are additional proteins that modulate the amount of phosphorylated CheY (CheY-P). Accessory proteins responsible for adaptation and other functions abound, such as methylation of the chemoreceptors by CheR. As more and more prokaryotic genomes have been sequenced, it is becoming clear that motile microbes each have the core signal transduction proteins with a somewhat unique set of these modulator proteins, although it is not yet apparent why particular microbes have particular sets (Blair, 1995;Szurmant & Ordal, 2004). One such microbe is Helicobacter pylori, a bacterium that uses flagellar motility and chemotaxis to fully colonize animal stomachs (Eaton et al., 1992(Eaton et al., , 1996 Foynes et al., 2000;Ottemann & Lowenthal, 2002;Terry et al., 2005). Understanding some of the less-usual attributes of this microbe's chemotactic signal transduction system was the goal of this study.H. pylori contains h...

Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralizationresistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described ␣4␤7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to ␣4␤7.A major goal in human immunodeficiency virus type 1 (HIV-1) vaccine research is the identification of immunogens able to elicit protective immunity from HIV-1 infection. Results from the recent RV144 clinical trial in Thailand (53) have provided evidence that immunization with vaccines containing the recombinant HIV-1 envelope glycoprotein gp120 (6, 7) can protect humans from HIV infection when incorporated in a prime/boost immunization regimen. Although the level of protection observed in the RV144 trial (31%) was modest, it represents a significant advance in HIV-1 vaccine research and has rekindled the efforts to identify improved subunit vaccine antigens that might achieve even higher levels of protection. In these studies, we have sought to understand the molecular determinants of neutralization sensitivity and resistance in HIV-1 envelope proteins for the purpose of developing improved vaccine antigens.In previous studies (47), we have described a novel method of mutational analysis of the HIV-1 envelope protein, termed swarm analysis, for identification of mutations that confer sensitivity and/or resistance to broadly neutralizing antibodies (bNAbs). This method makes use of the natural amino acid sequence virus variation that occurs in each HIV-infected individual to establish panels of closely related envelope proteins that differ from each other by a limited number of amino acid substitutions. We have previously used this method to identify a novel amino acid substitution in gp41 that conferred sensitivity to neutralization by monoclonal and polyclonal antibodies as we...

SummaryBacterial chemotaxis is a colonization factor for the ulcer-causing pathogen Helicobacter pylori. H. pylori contains genes encoding the chemotaxis signalling proteins CheW, CheA and CheY; CheW couples chemoreceptors to the CheA kinase and is essential for chemotaxis. While characterizing a cheW mutant, we isolated a spontaneous, chemotactic variant (Che + ). We determined that this phenotype was caused by a genetic change unlinked to the original cheW mutation. To locate the underlying Che + mutation, we compared total protein profiles of the nonchemotactic mutant (cheW) with those from the cheW Che + variant by two-dimensional differential in-gel electrophoresis. One protein was found only in the cheW Che + variant. This protein was identified by MS/MS as HP0170, a hypothetical protein with no known function. DNA sequencing verified that hp0170 was mutated in the cheW Che + suppressor, and deletion of this open reading frame in the cheW background nearly recapitulated the Che + suppressor phenotype. Using hidden Markov models, we found that HP0170 is a remote homologue of E. coli CheZ. CheZ interacts with phosphorylated CheY and stimulates its autodephosphorylation. CheZ was not predicted to be present in e-proteobacteria. We found that chemotaxis in the cheW Che + suppressor depended on both cheY and cheA. We hypothesize that a small amount of phosphorylated CheY is generated via CheA in the cheW mutant, and this amount is sufficient to affect flagellar rotation when HP0170 is removed. Our results suggest that HP0170 is a remote homologue of CheZ, and that CheZ homologues are found in a broader range of bacteria than previously supposed.

Secreted frizzled related proteins (Sfrps) are thought to bind and regulate Wnt activity through a cysteine rich domain that is highly similar to that of Frizzled receptors. To investigate possible roles for Sfrps in chick development, we have isolated partial cDNAs encoding Sfrp-1 and Sfrp-2 and have thoroughly characterized the expression patterns of both genes. Both sfrp-1 and sfrp-2 are expressed at all stages of development analyzed, ranging from Hamburger and Hamilton stage 4 through stage 32. Expression of both sfrp-1 and sfrp-2 is observed in mesodermal and ectodermal derivatives, while sfrp-1 is also found in endodermal lineages.