Helicobacter pylori has been shown to require flagella for infection of the stomach. To analyze whether flagella themselves or motility is needed by these pathogens, we constructed flagellated nonmotile mutants. This was accomplished by using both an insertion mutant and an in-frame deletion of the motB gene. In vitro, these mutants retain flagella (Fla ؉ ) but are nonmotile (Mot ؊ ). By using FVB/N mice, we found that these mutants had reduced ability to infect mice in comparison to that of their isogenic wild-type counterparts. When these mutants were coinfected with wild type, we were unable to detect any motB mutant. Finally, by analyzing the 50% infectious dose, we found that motility is needed for initial colonization of the stomach mucosa. These results support a model in which motility is used for the initial colonization of the stomach and also to attain full infection levels.Many bacterial pathogens swim, but little is known about how these microbes use this ability when inside animal hosts. Motility is often driven by flagella, complex extracellular structures that require large amounts of energy for operation. A bacterium presumably benefits if it makes flagella only when needed and regulates their activity so as not to swim at random.Flagella are often used by bacteria inside the animals they infect. Flagellar mutants of Helicobacter pylori (13), Campylobacter jejuni (32, 45), Campylobacter coli (37), and Vibrio anguillarum (35) are less virulent than their wild-type counterparts in animal models, indicating that flagella are crucial to the infection process of these pathogens. Additionally, human immunoglobulins are often directed against flagellar proteins in H. pylori (28), Pseudomonas aeruginosa (3, 44), C. jejuni (46), and C. coli (27), implying that flagella are expressed by these bacteria when inside the host.The functions that flagella play during infection, however, are not thoroughly understood. Flagella are best known for conferring motility, although recently they have been shown to play a variety of other roles. These include serving as an export apparatus for virulence factors (47) and sensing the viscosity of a medium (29). If flagella are required for infection, they could be used for any or all of these processes.H. pylori is a human pathogen that colonizes gastric tissue, causing symptoms ranging from mild gastritis to ulcers, and confers an increased risk of gastric cancer (10,34,43). Human colonization by this bacterium is very prevalent; it is estimated that over 50% of people worldwide are infected but only a subset develop disease. Several bacterial factors, including flagella, and various enzymes and toxins contribute to the full virulence of H. pylori (7,10,26,39,40).H. pylori carries a tuft of about five sheathed flagella located at one pole. Each flagellar filament is composed of two flagellins, FlaA and FlaB (22). The minor flagellin species, FlaB, localizes to the base of the flagellum, while the more abundant one, FlaA, lies in the outer regions (22). Elimination of fla...

Helicobacter pylori uses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, the H. pylori genome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three of Escherichia coli or Bacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, while fliY and fliN are partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of an H. pylori strain containing an R54C substitution in fliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions in H. pylori much as it does in other organisms.

Microbes have chemotactic signaling systems that enable them to detect and follow chemical gradients in their environments. The core of these sensory systems consists of chemoreceptor proteins coupled to the CheA kinase via the scaffold or coupler protein CheW. Some bacterial chemotaxis systems replace or augment CheW with a related protein CheV, which is less well understood. CheV consists of a CheW domain fused to a phosphorylatable receiver domain. Our review of the literature, as well as comparisons of CheV and CheW sequence and structure, suggest that CheV proteins conserve CheW residues that are crucial for coupling. Phosphorylation of the CheV receiver domain might adjust the efficiency of its coupling, and thus allow the system to modulate the response to chemical stimuli in an adaptation process.

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...