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.     

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA_C) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA_C. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA_C consists of at least three subdomains, a flexible linker, FlhA_CN, and FlhA_CC, and that FlhA_CN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA_C not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.The bacterial flagellum, which is responsible for motility, is a supramolecular complex of about 30 different proteins, and it consists of at least three substructures: the basal body, the hook, and the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. Many of the flagellar component proteins are translocated into the central channel of the growing flagellar structure and then to the distal end of the structure for self-assembly by the flagellar type III protein export apparatus (11, 16, 22). This export apparatus consists of six integral membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and three soluble proteins, FliH, FliI, and FliJ (18, 21). These protein components show significant sequence and functional similarities to those of the type III secretion systems of pathogenic bacteria, which directly inject virulence factors into their host cells (11, 16).FliI is an ATPase (4) and forms an FliH₂-FliI complex with its regulator, FliH, in the cytoplasm (20). FliI self-assembles into a homo-hexamer and hence exhibits full ATPase activity (1, 8, 17). FliH and FliI, together with FliJ and the export substrate, bind to the export core complex, which is composed of the six integral membrane proteins, to recruit export substrates from the cytoplasm to the core complex (14) and facilitate the initial entry of export substrates into the export gate (23). FliJ not only prevents premature aggregation of export substrates in the cytoplasm (13) but also plays an important role in the escort mechanism for cycling export chaperones during flagellar assembly (3). The export core complex is believed to be located in the central pore of the basal body MS ring (11, 16, 22). In fact, it has been found that FlhA, FliP, and FliR are associated with the MS ring (5, 9). The FliR-FlhB fusion protein is partially functional, suggesting that FliR and FlhB interact with each other within the MS ring (29). The export core complex utilizes a proton motive force across the cytoplasmic membrane as the energy source to drive the successive unfolding of export substrates and their translocation into the central channel of the growing flagellum (23, 27). Here we refer to the export core complex as the “export gate,” as we have previously (8, 16, 23, 24).FlhA is a 692-amino-acid protein consisting of two regions: a hydrophobic N-terminal transmembrane region with eight predicted α-helical transmembrane spans (FlhA_TM) and a hydrophilic C-terminal cytoplasmic region (FlhA_C) (12, 15). FlhA_TM is responsible for the association with the MS ring (9). FlhA_C interacts with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB (6, 12, 21, 24) and plays a role in the initial export process with these proteins (28). It has been shown that the V404M mutation in FlhA_C increases not only the probability of FliI binding to the export gate in the absence of FliH (14) but also the efficiency of substrate translocation through the export gate in the absence of FliH and FliI (23). Recently, it has been shown that FlhA_C is also required for substrate recognition (7). These observations suggest that an interaction between FlhA_C and FliI is coupled with substrate entry, although it is not clear how.In order to understand the mechanism of substrate entry into the export gate, we characterized a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, whose mutation blocks the flagellar protein export process at 42°C (28). We show here that this mutation severely inhibits translocation of flagellar proteins through the export gate after the FliH-FliI-FliJ complex binds to the FlhA-FlhB platform of the gate and that the impaired ability of the flhA(G368C) mutant to export flagellar proteins is restored almost to wild-type levels by intragenic second-site mutations that may alter the interactions between subdomains of FlhA_C for possible rearrangement for the export function.     

Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.     

幽门螺杆菌热休克蛋白70基因的克隆与表达

从幽门螺杆菌染色体ＤＮＡ,用ＰＣＲ方法扩增得到了热休克蛋白７０基因。序列分析表明,我国Ｈｐ临床分离株Ｙ２的热休克蛋白７０基因与经全基因组序列测定的两株幽门螺杆菌２６６９５和ｊ９９有高度同源性。将该基因克性到融合分泌表达载体ｐＭＡＬ－ｐ２中,转化大肠杆菌,在ＩＰＴＧ诱导下表达出与预期大小相符的１１３ｋＤ的融合表达蛋白。该蛋白质３０℃诱导表达５ｈ后,可达到细菌周质总蛋白质的１９．４％。用免疫印迹分析表     

Affinity of Helicobacter pylori to cholesterol and other steroids

Helicobacter pylori has a particular affinity to cholesterol. It is not known, however, whether other steroidal substances are bound as well. In order to characterize the specificity and nature of the H. pylori-steroid interaction, the affinity of H. pylori to cholesterol and several steroidal hormones was investigated. Seven strains of H. pylori (five reference strains, two wild strains) and one strain each of Staphylococcus epidermidis and Escherichia coli were cultured on a cholesterol-free medium. Cholesterol-free bacteria were incubated with cyclodextrin-mediated cholesterol and several cyclodextrin-mediated steroidal hormones (beta-estradiol, testosterone, progesterone, hydrocortisone, dexamethasone). The steroid contents of the bacteria were determined by gas liquid chromatography. High amounts of cholesterol were detected in all H. pylori strains, whilst steroidal hormones were not found. Neither S. epidermidis nor E. coli showed an appreciable amount of cholesterol in the chromatographic examinations. Bacterial pretreatment with proteinase K diminished cholesterol adsorption of H. pylori. These data indicate a specific affinity of H. pylori to cholesterol. This unique property might serve as a pathogenicity component enabling survival and colonization of H. pylori in the gastric environment.     

MacA,a Periplasmic Membrane Fusion Protein of the Macrolide Transporter MacAB-TolC,Binds Lipopolysaccharide Core Specifically and with High Affinity

The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject''s stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.Helicobacter pylori, a gram-negative bacterium, is remarkable for its ability to persist in the human stomach for decades. Colonization with H. pylori increases risk for peptic ulcer disease and gastric adenocarcinoma (53, 70) and elicits a vigorous immune response (15). The persistence of H. pylori occurs in a niche in the human body previously considered inhospitable to microbial colonization: the acidic stomach replete with proteolytic enzymes.H. pylori strains exhibit substantial genetic diversity, including extensive variation in the presence, arrangement, order, and identity of genes (2, 4-7, 25, 51, 74). Furthermore, analyses of multiple single-colony H. pylori isolates from separate stomach biopsy specimens of individual patients have demonstrated diversity, both within hosts (27, 65), and over time (36). The mechanisms that generate H. pylori genetic diversity may be among the factors that enable persistence in this environment (3, 28).While the natural ability of H. pylori for transformation and recombination may explain some of the intra- and interhost genetic variation observed in this bacterium (43), point mutations and interspecies recombination alone are not sufficient for explaining the extent of the variation in H. pylori (14, 32). The initial genomic sequencing of H. pylori strains 26695 and J99 (6, 72) revealed large amounts of repetitive DNA (1, 59). DNA repeats in bacteria are associated with mechanisms of plasticity, such as phase variation (49, 67); slipped-strand mispairing (41, 46); and increased rates of recombination, deletion, and insertion (17, 60, 62). Because many of the recombination repair and mismatch repair mechanisms common in bacteria are absent or modified in H. pylori (28-30, 56, 76), this organism may be particularly susceptible to the diversifying effects of repetitive DNA. In fact, loci in the H. pylori genome containing repetitive DNA have been shown to exhibit extensive inter- and intrahost variation (9, 10, 28, 37).We hypothesized that identification of repetitive DNA hotspots in H. pylori would allow the recognition of genes whose variation could aid in persistence. To examine this hypothesis, we conducted in silico analyses to identify open reading frames (ORFs) enriched for DNA repeats and then used a combination of sequence analyses and immunoassays to examine the patterns associated with the specific repetitive DNA observed. Our approach led to the realization that a previously identified H. pylori-specific gene family (19, 52) exhibits extensive genetic variation at multiple levels.     

Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (K_d = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.     

Interaction between FliJ and FlhA,Components of the Bacterial Flagellar Type III Export Apparatus

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH_X-FliI₆ ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues—Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83—of FliJ. A structural model of the FliI₆-FliJ ring complex suggests that they extend out of the FliI₆ ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.