Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection.

Toll-like receptors (TLRs) expressed by mucosal epithelium play an essential role in the defense against microbes by recognizing conserved bacterial molecules. For the first time TLR4, TLR5 and TLR9 have been microanatomically localized in patients with noninflamed gastric mucosa and Helicobacter pylori gastritis by immunohistochemistry. Because polarized expression of TLRs in apical and basolateral epithelial compartments is thought to modulate mucosal immunity, subcellular TLR distribution by gastric epithelium was investigated using confocal microscopy. TLR4, TLR5 and TLR9 were expressed by gastric epithelium in antrum and corpus of all patients with H. pylori gastritis (n = 14) and with noninflamed gastric mucosa (n = 5). TLR4 was expressed at the apical and the basolateral pole of the gastric epithelium as well in noninflamed gastric mucosa as in H. pylori gastritis. TLR5 and TLR9 expression in the noninflamed gastric mucosa was identical to that of TLR4 with localization at the apical and the basolateral epithelial pole. However, in H. pylori gastritis TLR5 and TLR9 expression on the gastric epithelium changed to an exclusive basolateral localization without detectable expression at the apical pole. In the human stomach, the gastric epithelium expressed TLR4, TLR5 and TLR9, which gives it the possibility to interact with H. pylori. Furthermore, gastric epithelial TLR4 expression is highly polarized in an apical and a basolateral compartment, whereas TLR5 and TLR9 polarization seems to be a process dynamically influenced by H. pylori infection. This polarized and dynamically regulated gastric epithelial expression of TLRs supports a sentinel role for these receptors in the mucosal immunity to H. pylori.

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+.

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.

Switching of flagellar motility in Helicobacter pylori by reversible length variation of a short homopolymeric sequence repeat in fliP, a gene encoding a basal body protein.

The genome of Helicobacter pylori contains numerous simple nucleotide repeats that have been proposed to have regulatory functions and to compensate for the conspicuous dearth of master regulatory pathways in this highly host-adapted bacterium. H. pylori strain 26695, whose genomic sequence was determined by The Institute for Genomic Research (TIGR), contains a repeat of nine cytidines in the fliP flagellar basal body gene that splits the open reading frame in two parts. In this work, we demonstrate that the 26695(C9) strain with a split fliP gene as sequenced by TIGR was nonflagellated and nonmotile. In contrast, earlier isolates of strain 26695 selected by positive motility testing as well as pig-passaged derivatives of 26695 were all flagellated and highly motile. All of these motile strains had a C(8) repeat and consequently a contiguous fliP reading frame. By screening approximately 50,000 colonies of 26695(C9) for motility in soft agar, a motile revertant with a C(8) repeat could be isolated, proving that the described switch is reversible. The fliP genes of 20 motile clinical H. pylori isolates from different geographic regions possessed intact fliP genes with repeats of eight cytidines or the sequence CCCCACCC in its place. Isogenic fliP mutants of a motile, C(8) repeat isolate of strain 26695 were constructed by allelic exchange mutagenesis and found to be defective in flagellum biogenesis. Mutants produced only small amounts of flagellins, while the transcription of flagellin genes appeared unchanged. These results strongly suggest a unique mechanism regulating motility in H. pylori which relies on slipped-strand mispairing-mediated mutagenesis of fliP.

Adherence of isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae to human and ferret gastric epithelial cells.

Isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae were screened for their ability to adhere to primary human and ferret gastric epithelial cells, respectively. We also evaluated the adherence of an H. pylori strain with a mutation in the flbA gene, a homologue of the flbF/lcrD family of genes known to be involved in the regulation of H. pylori flagellar biosynthesis. H. pylori and H. mustelae mutants deficient in production of FlaA or FlaB and mutants deficient in the production of both FlaA and FlaB showed no reduction in adherence to primary human or ferret gastric epithelial cells compared with the wild-type parental strains. However, adherence of the H. pylori flbA mutant to human gastric cells was significantly reduced compared to the adherence of the wild-type strain. These results show that flagella do not play a direct role in promoting adherence of H. pylori or H. mustelae to gastric epithelial cells. However, genes involved in the regulation of H. pylori flagellar biosynthesis may also regulate the production of an adhesin.

In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results.

We describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice. This method was used to study the in vivo local distribution of bacteria within the mucus of Helicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyzed, within less than 1 min, as a native preparation. To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 micron of mucus from the tissue-mucus interface), a luminal layer (the last 25 micron to the mucus-lumen interface), and the remaining central mucus layer. The volume of the analyzed segments in the sample was between 4 and 9 pl. The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer. The mean distance of H. felis to the epithelial surface was 16 microm. A total of 75% of all H. felis bacteria resided in the mucus zone between 5 and 20 micron from the tissue surface, with no bacteria closer than 5 micron to the epithelial surface. This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision.

Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis.

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.

Virulence factors of Helicobacter pylori: implications for vaccine development.

Helicobacter pylori is one of the most common infectious diseases in humans and causes gastritis, peptic ulcer disease and malignant tumours of the stomach. This review discusses how H. pylori can colonize the human stomach, an ecological niche that is protected against all other bacteria. Knowledge about the virulence factors of H. pylori has accumulated rapidly over the last decade. Together with the information contained in the complete H. pylori genome sequence, this knowledge is now being applied in the search for a vaccine against this global pathogen.

Green fluorescent protein as a novel marker and reporter system in Helicobacter sp.

In order to be able to study gene regulation in single, live Helicobacter pylori bacteria in vitro or in contact with host cells, we established the green fluorescent protein gene gfp from Aequorea victoria as a reporter gene for use with Helicobacter species. We describe here the construction of genomic transcriptional fusions of the promoterless gfp gene with the flaA and flaB promoters of H. pylori. We have also constructed a Mini-Tn3-km-gfp transposon to be used for shuttle transposon mutagenesis in H. pylori and H. mustelae. A marker strain with wild-type phenotype, carrying multiple plasmid-borne copies of gfp under the control of the H. pylori flaB promoter, was constructed for studies of bacterial distribution and transmission in animal models.

Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae.

Helicobacter mustelae, like Helicobacter pylori, possesses two flagellin proteins, FlaA and FlaB. Isogenic mutant strains of H. mustelae have been constructed by disruption of the flaA or flaB gene with a kanamycin resistance cassette or by introduction of both a kanamycin and a chloramphenicol resistance gene to produce a double mutant. To determine whether one or both flagellin proteins are necessary for colonization and persistence of infection with H. mustelae, 19 ferrets, specific pathogen free for H. mustelae, were given either the HMF1 flaA::km (weakly motile), ATCC 43772 flaB::km (moderately motile), or HMF1 flaA::cat flaB::km (non-motile) mutant strain, the wild-type parent strains, or sterile broth. Gastric tissue samples were obtained during sequential gastric biopsies beginning at 3 weeks postinoculation and ending at necropsy at 3 months postinoculation. H. mustelae infection status was determined by culture, histology, and serology. The wild-type parent strains of H. mustelae infected all ferrets at all time points. The double-mutant strain was unable to colonize; the flaA and flaB single-mutant strains were able to initially colonize at a low level and establish persistent infection with increasing numbers of organisms over time. The severity of gastritis produced by infection with these strains of H. mustelae correlated with the number of organisms present in the gastric mucosa. Flagellar motility is an important virulence factor for colonization and pathogenesis in the H. mustelae ferret model.

Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes.

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.

Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes.

Helicobacterpylori possesses two flagellin molecules, MA, the major species, and FlaB, which is expressed in minor amounts. This study sought to determine if one or both flagellin species are necessary for colonization or persistence by H. pylon. Thirty-six gnotobiotic piglets from six litters were given one of four isogenic strains of H. pylon orally. The bacterial strains used were strain N6, the wild type, which produced both FlaA and FlaB and was fully motile; N6flaB::km, which produced FlaA but not FlaB and was weakly motile; N6flaA::km, which expressed FlaB but not FlaA and was nonmotile; and N6flaA::cat/flaB::km, which produced neither flagellin and was nonmotile. Strain N6 colonized all piglets and persisted for 2, 4, and 10 days after inoculation. Both N6flaA::km and N6flaB::km colonized for 2 and 4 but not 10 days, and colonization was weak. N6flaA::cat/flaB:: km colonized for 2 days but did not persist for 4 or 10 days after inoculation. These findings demonstrate that both flagellin species are necessary for full colonization by H. pylon. Colonization for up to 4 days is possible in the absence of either flagellin species but not both.

Construction and characterization of an isogenic urease-negative mutant of Helicobacter mustelae.

Helicobacter mustelae infects the ferret stomach and provides an opportunity to study pathogenic determinants of a Helicobacter species in its natural host. We constructed an isogenic urease-negative mutant of H. mustelae which produced no detectable urease and showed a reduced acid tolerance. This mutant provides an opportunity to further evaluate the role of urease in the pathogenesis of Helicobacter infection.

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.

Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange.

Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.

Biochemical studies of Helicobacter mustelae fatty acid composition and flagella.

The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0, 3-OH-C14:0, C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.