Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA⁺ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.     

异源表达的幽门螺杆菌cag4蛋白有溶菌糖基转移酶活性

摘要【目的】构建融合基因原核表达载体pET-28a- cag4,并表达重组融合蛋白cag4,分析重组融合蛋白的酶活性,为新型抗生素（或是抗菌药物）的研发提供作用靶位。【方法】本研究利用PCR技术从幽门螺杆菌NCTC11637中克隆了cag4基因;经T-A克隆,酶切鉴定,构建了原核表达载体pET-28a- cag4;经测序鉴定正确后,转化进入大肠埃希菌 BL21(DE3)进行异源表达。利用IPTG体外诱导后,经SDS-PAGE和Western Bolt鉴定目的蛋白表达后,采用Ni2＋-NTA柱在变性条件下纯化目的蛋白,并对重组蛋白进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析。【结果】在大肠埃希菌 BL21(DE3)中获得高效表达的重组蛋白; 经SDS-PAGE和Western Bolt鉴定表达后,采用Ni2＋-NTA柱在变性条件下纯化,并进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析,表明目的蛋白具有明显的肽聚糖水解活性; 通过监测浊度下降速率,比较其在不同pH条件下活性的变化,即?A/（min?mg protein）,结果表明,幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。【结论】幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。     

Helicobacter pylori cag Pathogenicity Island's Role in B7-H1 Induction and Immune Evasion

During Helicobacter pylori (H. pylori) infection CD4⁺ T cells in the gastric lamina propria are hyporesponsive and polarized by Th1/Th17 cell responses controlled by T_reg cells. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation, effector function, and induce T_reg cells in vitro. In this study, we investigated the underlying mechanisms and the functional relevance of B7-H1 induction by H. pylori infection to chronic infection. Using H. pylori wild type (WT), cag pathogenicity island (cag PAI^-) and cagA^- isogenic mutant strains we demonstrated that H. pylori requires its type 4 secretion system (T4SS) as well as its effector protein CagA and peptidoglycan (PG) fragments for B7-H1 upregulation on GEC. Our study also showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, which has a functional T4SS delivery system, but not with H. pylori SS1 strain lacking a functional T4SS, led to a strong upregulation of B7-H1 expression in the gastric mucosa, increased bacterial load, induction of T_reg cells in the stomach, increased IL-10 in the serum. Interestingly, B7-H1^-/- mice showed less T_reg cells and reduced bacterial loads after infection. These studies demonstrate how H. pylori T4SS components activate the p38 MAPK pathway, upregulate B7-H1 expression by GEC, and cause T_reg cell induction; thus, contribute to establishing a persistent infection characteristic of H. pylori.     

cag pathogenicity island of Helicobacter pylori in Korean children

Background. cag pathogenicity island is reported to be a major virulence factor of Helicobacter pylori. The aim of this study was to investigate the status of cag pathogenicity island genes and gastric histology in Korean children with H. pylori gastritis. Methods. Helicobacter pylori DNA was extracted from antral biopsy specimens from 25 children with H. pylori gastritis. Specific polymerase chain reaction assays were used for four genes of cag pathogenicity island. The features of gastritis were scored in accordance with the updated Sydney System. Results. cagA was present in 23 (92%) of 25 children, and cagE in 24 (96%). Twenty‐two (88%) children were cagT positive and 19 (76%) virD4 positive. All of the selected genes of the cag pathogenicity island were present in 17 (68%) children and completely deleted in one child. There were no differences in neutrophil activity and chronic inflammation between children infected with intact cag pathogenicity island strains and those with partially or totally deleted‐cag pathogenicity island strains. Conclusion. cag pathogenicity island is not a uniform, conserved entity in Korea. Completeness of cag pathogenicity island may not be the major factor to determine the severity of H. pylori gastritis in children.     

A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system

Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag-PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin-8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src-family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10-homologous protein. HP0527 is also clustered in the outer membrane. The VirB7-homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS-associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.     

Lipopolysaccharide Diversity Evolving in Helicobacter pylori Communities through Genetic Modifications in Fucosyltransferases

Helicobacter pylori persistently colonizes the gastric mucosa of half the human population. It is one of the most genetically diverse bacterial organisms and subvariants are continuously emerging within an H. pylori population. In this study we characterized a number of single-colony isolates from H. pylori communities in various environmental settings, namely persistent human gastric infection, in vitro bacterial subcultures on agar medium, and experimental in vivo infection in mice. The lipopolysaccharide (LPS) O-antigen chain revealed considerable phenotypic diversity between individual cells in the studied bacterial communities, as demonstrated by size variable O-antigen chains and different levels of Lewis glycosylation. Absence of high-molecular-weight O-antigen chains was notable in a number of experimentally passaged isolates in vitro and in vivo. This phenotype was not evident in bacteria obtained from a human gastric biopsy, where all cells expressed high-molecular-weight O-antigen chains, which thus may be the preferred phenotype for H. pylori colonizing human gastric mucosa. Genotypic variability was monitored in the two genes encoding α1,3-fucosyltransferases, futA and futB, that are involved in Lewis antigen expression. Genetic modifications that could be attributable to recombination events within and between the two genes were commonly detected and created a diversity, which together with phase variation, contributed to divergent LPS expression. Our data suggest that the surrounding environment imposes a selective pressure on H. pylori to express certain LPS phenotypes. Thus, the milieu in a host will select for bacterial variants with particular characteristics that facilitate adaptation and survival in the gastric mucosa of that individual, and will shape the bacterial community structure.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

幽门螺杆菌cag PAI编码的Ⅳ型分泌系统

幽门螺杆菌(Helicobacter pylori,H.pylori)是定植于人胃部特定的病原菌,感染呈全球分布,感染率高达50%以上。现已证实它是轻度胃炎,消化性溃疡及胃癌的主要病因。Ⅰ型H.pylori菌株含有一个约40kb的特殊基因片段,即cag致病岛(cytotoxin associated gene pathogenicity island,cag PAI),该片段只出现于致病相关菌株,基因呈高密度分布并编码一个分泌转运系统称为Ⅳ型分泌系统(type Ⅳ secretion system,TFSS),通过转运相关毒素而参与H.pylori诱导上皮细胞细胞内的酪氨酸磷酸化、细胞骨架重排、基垫结构形成、活化核转录因子NF-κB、诱导促炎细胞因子白细胞介素-8的表达等,故在H.pylori的致病中起着关键作用。近年来,研究者们致力于研究Ⅳ型分泌系统的功能,但是对于这个装置是如何转运蛋白进入宿主细胞的确切机制还是知之甚少,因此,对Ⅳ型分泌系统的研究将有助于进一步明确H.pylori致病机制,并为临床诊断和治疗提供新的靶点。     

Helicobacter pylori cag Pathogenicity Island (cagPAI) Involved in Bacterial Internalization and IL-8 Induced Responses via NOD1- and MyD88-Dependent Mechanisms in Human Biliary Epithelial Cells

Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5β1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5β1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.     

Diversity of restriction-modification gene homologues in Helicobacter pylori

The complete genome sequences of two Helicobacter pylori strains have recently become available. We have searched them for homologues of restriction-modification genes. One strain (26695) carried 52 such homologues, and the other (J99) carried 53. Their sequence alignments were arranged in the form of a phylogenetic tree and compared with the tree based on rRNA. The trees showed that the homologues are scattered among diverse groups of bacteria. They also revealed high polymorphism within the species--there are 42 pairs with high homology, 10 specific to 26695, and 11 specific to J99. Many of the restriction-modification homologues were characterized by a GC content lower than that of the average gene in the genome. Some of the restriction-modification homologues showed a different codon use bias from the average genes. These observations are interpreted in terms of horizontal transfer of the restriction-modification genes.     

幽门螺杆菌Ⅳ型分泌系统（T4SS）研究进展

Ⅳ型分泌系统（T4SS）广泛存在于革兰阴性菌中,细菌可通过该系统将生物大分子或毒力因子等运输至靶细胞中并发挥相应功能。目前在H. pylori中已发现了至少三种T4SS,其中研究较为透彻的是cag致病岛（cagPAI）编码的cagT4SS系统,此外可塑区编码的tfs3系统和comB系统也有相关的报道。H. pylori的T4SS作为其与致病相关的重要结构已受到很多学者关注,对该菌T4SS系统的研究有助于进一步明确H. pylori的致病机制,并为临床诊断和治疗相关胃十二指肠疾病提供新的靶点。本文将对H. pylori的T4SS相关研究进展作一简要综述。     

Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells

Helicobacter pylori is recognized as the main cause of gastritis and is associated with gastric carcinogenesis. Syndecan-4 represents the major source of heparan sulfate (HS) in the gastric cells. HS proteoglycans expressed on the cell surface constitute targets for H. pylori at the early stage of infection. The aim of this study was to determine whether H. pylori induction of syndecan-4 expression is affected by the virulence characteristics of the infecting strain, namely the cytotoxic-associated gene ( cag ) pathogenicity island (PAI). We observed that individuals infected with highly pathogenic H. pylori strains express syndecan-4 in the foveolar epithelium of the gastric mucosa. The association between the cag PAI status of the infecting strain and syndecan-4 expression was further demonstrated by infection of gastric epithelial cell lines with a panel of cag PAI⁺ and cag PAI⁻ H. pylori strains, showing that expression of syndecan-4 was significantly increased in response to infection with the highly pathogenic strains. Moreover, infection of gastric cells with cag A and cag E mutant strains further confirmed that syndecan-4 induction is dependent on an intact cag PAI. The present study shows that highly pathogenic H. pylori strains induce syndecan-4 expression, both in human gastric mucosa and in gastric cell lines, in a cag PAI-dependent manner.     

Functional characterization of Helicobacter pylori DnaB helicase

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5′ to 3′ helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.     

cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

Conservation of the cytotoxin-associated (cag A) gene of Helicobacter pylori and investigation of association with vacuolating-cytotoxin activity and gastroduodenal disease

Abstract Polymerase chain reaction (PCR) amplification and DNA hybridization analyses were used to test for the presence of the cytotoxin-associated ( cag A) gene in 108 strains of Helicobacter pylori . Fifty-two geographically diverse strains of known vacuolating cytotoxin activity, and 56 recent UK clinical isolates from patients with duodenal ulceration ( n =28) and from healthy individuals who were endoscopically normal ( n =28) were studied. Overall, cag A was detected by PCR in 74 (69%) strains and DNA hybridization provided evidence of gene homologues in a further eight strains. For 96% of the cytotoxin-producing strains and 46% of the non-cytotoxin producing strains, there was a close association either with presence or absence of cag A. At the genomic level, Southern blot DNA hybridization showed that cag A was probably present in a single copy in most of the H. pylori tested, and that Hae III restriction site variation within and around the gene provided additional markers of diversity for the species. As 40% of the cag A containing strains did notnproduce an active cytotoxin, and no significant association between cag A presence and DU-disease was observed, we concluded that the presence of the cag A gene in H. pylori could not be used as a single reliable predictor of higher risk patients.     

Functional Analysis of the Helicobacter pylori Flagellar Switch Proteins

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.Flagellar motility is important for gastric colonization by the ulcer-causing bacterium Helicobacter pylori and also for suborgan localization within the stomach (16-18, 33, 45). Flagellar motility is regulated by a set of signal transduction proteins, collectively referred to as the chemotaxis pathway, that control the migration of microbes in response to environmental cues. This pathway is well elucidated in organisms such as Escherichia coli, Salmonella enterica serovar Typhimurium (referred to hereinafter as S. Typhimurium), and Bacillus subtilis. Sequence analysis of the genomes of other flagellated bacteria, including H. pylori, has suggested that there is diversity in the set of chemotaxis proteins that a particular microbe contains. Here we analyze the diversity of H. pylori''s flagellar switch proteins, which control flagellar rotational direction.The molecular mechanisms underlying chemotactic signal transduction in E. coli and S. Typhimurium have been extensively studied (7, 50) The overall function of this pathway is to convert the perception of local environmental conditions into a swimming response that drives bacteria toward beneficial conditions and away from harmful ones. Such migration is accomplished by interspersing straight, or smooth, swimming with periods of random reorientations or tumbles. Smooth swimming occurs when the flagella rotate counterclockwise (CCW), while reorienting occurs when the flagella rotate clockwise (CW). The chemotaxis signal transduction system acts to appropriately alter flagellar rotation. The canonical chemotaxis pathway consists of a chemoreceptor bound to the coupling protein CheW, which is in turn bound to the histidine kinase CheA. If a beneficial/attractant ligand is not bound (or a repellant is bound) to the chemoreceptor, CheA autophosphorylates and passes a phosphate to the response regulator CheY. Phosphorylated CheY (CheY-P) interacts with a protein complex called the flagellar switch (discussed at more length below). This interaction causes a switch in the direction of flagellar rotation from CCW to CW, thus reorienting the cells, via an as-yet-unknown mechanism (reviewed in references 23 and 29).Bacterial flagella are complex, multiprotein organelles (reviewed in references 23, 25, and 29). Each flagellum is composed of several parts, including the filament, the hook, and the basal body (listed from outside the cell to inside the cytoplasm). The flagellar basal body spans from the outer membrane to the cytoplasm and is responsible for rotating the flagellum. This part of the flagellum is further made up of several subassemblies that are named for their locations. The innermost is called the switch or C ring, based on its location in the cytoplasm. The switch is comprised of three proteins in E. coli, FliM, FliN, and FliG (reviewed in references 23 and 29). Experimental evidence strongly suggests that these proteins, along with the stator proteins MotA and MotB, drive motor rotation, because one can obtain point mutations in these proteins that disrupt rotation but not flagellation. Null mutations, however, in fliM, fliN, or fliG also result in aflagellated cells, a phenotype that has been proposed to arise because these proteins are needed to complete the flagellar export apparatus (23).There is extensive structural information about each of the switch proteins and their arrangement in the flagellum (reviewed in references 23 and 29, with additional key references added below). There are 26 copies of FliG, 34 copies of FliM, and ∼136 copies of FliN, arranged in a circular structure at the base of each flagellum. FliM is positioned between FliG and FliN and interacts with both. FliM also binds CheY-P via sequences in the first 16 amino acids, and elsewhere (15), to play a key role in switching flagellar rotation direction. FliG, the switch protein closest to the cytoplasmic membrane, interacts with the stator protein MotA, the FliF membrane protein that forms the flagellar basal-body MS ring, and the membrane-bound respiratory protein fumarate reductase (11). FliG has the most direct role in creating flagellar rotation. FliN is the most cytoplasmic component of the switch, and its role is not fully understood. FliN may play a role in switching by possibly binding CheY-P directly (36) and an additional role in flagellar assembly, because it binds to the flagellar export protein FliH and localizes it, along with its interaction partners FliI and FliJ, to the flagellum (20, 28, 36). FliN contains significant sequence similarity to secretion proteins of type III secretion systems of Yersinia pestis and Shigella flexneri. The conserved domain comprises most of FliN and is called a SpoA or PFAM PF01052 domain. Other FliN homologs include YscL and Spa33 (25).The flagellar switch of another well-studied chemotactic microbe, B. subtilis, differs slightly in its protein makeup from that of E. coli. B. subtilis contains FliM and FliG, which function similarly to their E. coli counterparts, but instead of FliN it has a protein called FliY (6, 42). FliY of B. subtilis has two functional domains, one of which is homologous to E. coli FliN, while the other shares similarity with the B. subtilis chemotaxis protein CheC, which functions to dephosphorylate CheY-P. FliY is the most active known phosphatase of CheY-P in B. subtilis (40, 41).H. pylori contains homologs of many of the chemotaxis and flagellar genes found in other organisms (32, 48). Curiously, its genome encodes four predicted flagellar switch proteins, FliG, FliM, and both FliY and FliN, although FliY was not annotated in the original genome analysis. Previous work had determined that H. pylori strain SS1 lacking fliG was aflagellated (1), but the other switch proteins had not been analyzed. As noted above, FliN and FliY share a FliN domain and so could have functional redundancy. fliY and fliM appear to reside in an operon, suggesting that the two encoded proteins function together (see Fig. S1 in the supplemental material).Since having all four flagellar switch proteins in one microbe is unusual, we were curious as to whether all four serve “switch” functions. As noted above, fliM and fliG deletions typically result in an aflagellated phenotype in other organisms. Others had previously shown that fliG mutations have this phenotype in H. pylori (1), and we additionally show here that fliM null mutants are also almost completely aflagellate. In spite of a shared domain that might indicate functional redundancy, we show that fliN and fliY are each necessary for normal numbers of flagellated cells. Finally, we characterize a fliM point mutant that results in a lock-smooth swimming bias and demonstrate physical interaction between CheY-P and FliM, indicating that FliM responds to CheY signaling in H. pylori in a manner similar to that found in E. coli, S. Typhimurium, B. subtilis, and other studied organisms.