Identification of a D-glycero-D-manno-heptosyltransferase gene from Helicobacter pylori

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.     

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.     

Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.     

Structural studies on lipopolysaccharides of serologically non-typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants.

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewis(x)+ (Le(x)) and Lewis(y)++ (++Le(y)) antigenic determinants in the O-specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non-typable with anti-Le(x) and anti-Le(y) antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H-NMR spectroscopy and electrospray ionization-mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O-specific polysaccharide chains. The O-specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly-N-acetyl-beta-lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Le(x)chain terminating with Le(x) or Le(y) units:[sequence: see text] where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0-2, 6-7 in strain AF1 and m = 0-2, 6-7 or approximately 40 in strain 007, the medium-size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti-Le(x) and anti-Le(y) may reflect the presence of a polymeric Le(x) chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a D-glycero-D-manno-heptose residue in the outer core varied in the two strains, and an extended DD-heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core L-glycero-D-manno-heptose region of both strains carried a 2-aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.     

Direct analysis of the extracellular proteome from two strains of Helicobacter pylori

Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.     

Characterization of the pilin ortholog of the Helicobacter pylori type IV cag pathogenicity apparatus, a surface-associated protein expressed during infection

The Helicobacter pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) involved in host interaction and pathogenicity. Previously, seven cag PAI proteins were identified as homologs of Agrobacterium tumefaciens Vir proteins, which form a paradigm T4SS. The T pilus composed of the processed VirB2 pilin is an external structural part of the A. tumefaciens T4SS. In H. pylori, cag-dependent assembly of pili has not been observed so far, nor has a pilin (VirB2) ortholog been characterized. We have here identified, using a motif-based search, an H. pylori cag island protein (HP0546) that possesses sequence and predicted structural similarities to VirB2-like pilins of other T4SSs. The HP0546 protein displays interstrain variability in its terminal domains. HP0546 was expressed as a FLAG-tagged fusion protein in Escherichia coli, A. tumefaciens, and H. pylori and was detected as either two or three bands of different molecular masses in the insoluble fraction, indicating protein processing. As reported previously, isogenic H. pylori mutants in the putative cag pilin gene had reduced abilities to induce cag PAI-dependent interleukin-8 secretion in gastric epithelial cells. Fractionation analysis of H. pylori, using a specific antiserum raised against an N-terminal HP0546 peptide, showed that the protein is partially surface exposed and that its surface localization depended upon an intact cag system. By immunoelectron microscopy, HP0546 was localized in surface appendages, with surface exposure of an N-terminal epitope. Pronounced strain-to-strain variability of this predicted surface-exposed part of HP0546 indicates a strong selective pressure for variation in vivo.     

Lipopolysaccharide structures of Helicobacter pylori genomic strains 26695 and J99, mouse model H. pylori Sydney strain, H. pylori P466 carrying sialyl Lewis X, and H. pylori UA915 expressing Lewis B classification of H. pylori lipopolysaccharides into glycotype families.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.     

Identification of genes associated with natural competence in Helicobacter pylori by transposon shuttle random mutagenesis.

To identify genes involved in DNA transformation, we generated 1500 insertion mutants of a Helicobacter pylori strain by transposon shuttle mutagenesis. All mutant strains were screened for their frequency of natural transformation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic loci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknockout of HP1326 revealed no impairment in natural transformation, while the other 5 mutants showed the same defective in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels compared with competent strains. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Therefore, our data indicate that HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation.     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Functional genomics of Helicobacter pylori: identification of a beta-1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide

A previously annotated open reading frame (ORF) (HP0826) from Helicobacter pylori was cloned and expressed in Escherichia coli cells and determined to be a beta-1,4-galactosyltransferase that used GlcNAc as an acceptor. Mutational analysis in H. pylori strains demonstrated that this enzyme plays a key role in the biosynthesis of the type 2 N-acetyl-lactosamine (LacNAc) polysaccharide O-chain backbone, by catalysing the addition of Gal to GlcNAc. To examine the potential role of this O-chain structure in bacterial colonization of the host stomach, the mutation was introduced into H. pylori strain SS1 which is known to be capable of colonizing the gastric mucosa of mice. Compared with the parental strain, mutated SS1 was less efficient at colonizing the murine stomach.     

The crystal structure of ADP-L-glycero-D-manno-heptose-6-epimerase (HP0859) from Helicobacter pylori

Helicobacter pylori, the human pathogen that affects about half of the world population and that is responsible for gastritis, gastric ulcer and adenocarcinoma and MALT lymphoma, owes much of the integrity of its outer membrane on lipopolysaccharides (LPSs). Together with their essential structural role, LPSs contribute to the bacterial adherence properties, as well as they are well characterized for the capability to modulate the immuno-response. In H. pylori the core oligosaccharide, one of the three main domains of LPSs, shows a peculiar structure in the branching organization of the repeating units, which displayed further variability when different strains have been compared. We present here the crystal structure of ADP-L-glycero-D-manno-heptose-6-epimerase (HP0859, rfaD), the last enzyme in the pathway that produces L-glycero-D-manno-heptose starting from sedoheptulose-7-phosphate, a crucial compound in the synthesis of the core oligosaccharide. In a recent study, a HP0859 knockout mutant has been characterized, demonstrating a severe loss of lipopolysaccharide structure and a significant reduction of adhesion levels in an infection model to AGS cells, if compared with the wild type strain, in good agreement with its enzymatic role. The crystal structure reveals that the enzyme is a homo-pentamer, and NAD is bound as a cofactor in a highly conserved pocket. The substrate-binding site of the enzyme is very similar to that of its orthologue in Escherichia coli, suggesting also a similar catalytic mechanism.     

Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori

Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3-5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.     

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.     

HP0333, a Member of the dprA Family, Is Involved in Natural Transformation in Helicobacter pylori

Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.     

Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells.

An LD-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS-PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis - mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE-->7)-L-alpha-D-Hepp-(1-->5)-alpha-Kdop-(2-->6)-Lipid A, where PE represents a phosphoethanolamine group, LD-Hep represents L-glycero-D-manno-heptose, and Kdo represents 3-deoxy-D-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.     

Serum-resistant mutants of Escherichia coli O111 contain increased lipopolysaccharide, lack an O antigen-containing capsule, and cover more of their lipid A core with O antigen.

Escherichia coli strains of group O111 were characterized with respect to sensitivity to complement killing, amount of lipopolysaccharide and O antigen-containing capsule, and distribution of O antigen. All wild-type E. coli O111 strains were resistant to complement killing in the absence of specific antibody. Presensitization of strains with antibody to whole cells (OK antibody), followed by incubation in 50% pooled normal human serum as a source of complement, subdivided wild-type strains into three types: completely resistant, partially resistant, and sensitive. Completely and partially resistant mutants were isolated by cycles of serum killing, starting with one sensitive strain. Completely resistant mutants had no O antigen-containing capsule, but had 50% more lipopolysaccharide than did the parent, and this lipopolysaccharide had 30% fewer lipid A core molecules devoid of O antigen. Partially resistant mutants still had O antigen-containing capsule, but contained 40% more lipopolysaccharide than did the parent; the extent of coverage of lipid A core with O antigen remained unchanged. No correlations were found between outer membrane protein composition and the degree of serum resistance. Since the terminal membrane attack complex (C5b-9) must stably insert into a hydrophobic membrane site to effect killing, we conclude that both increased lipid A core and increased coverage of lipid A core with O antigen preclude access of C5b-9 to lethal sites on the cell surface.     

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.     

Lewis epitopes on outer membrane vesicles of relevance to Helicobacter pylori pathogenesis

BACKGROUND: Helicobacter pylori extrudes protein- and lipopolysaccharide-enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host. MATERIALS AND METHODS: H. pylori strains were examined for outer membrane vesicle production using transmission electron microscopy and Lewis antigen expression probed using immunoelectron microscopy. Sera from patients were analyzed for cross-reacting anti-Lewis antibodies and, subsequently, absorbed using outer membrane vesicle preparations to remove the cross-reacting antibodies. RESULTS: The formation of outer membrane vesicles by H. pylori was observed in both in vitro and in vivo samples. Furthermore, vesicles were produced following culture in either liquid or solid medium by all strains examined. Moreover, we observed the presence of Lewis epitopes on outer membrane vesicles using immunoelectron microscopy and immunoblotting. Circulating anti-Lewis antibodies were found in the sera of gastric cancer patients but not in the sera of H. pylori-negative control subjects. Absorption of patient sera with outer membrane vesicles decreased the levels of anti-Lewis autoantibodies. CONCLUSIONS: Our results demonstrate the ability of H. pylori to generate outer membrane vesicles bearing serologically recognizable Lewis antigens on lipopolysaccharide molecules which may contribute to the chronic immune stimulation of the host. The ability of these vesicles to absorb anti-Lewis autoantibodies indicates that they may, in part, play a role in putative autoimmune aspects of H. pylori pathogenesis.     

Multiple in vivo passages enhance the ability of a clinical Helicobacter pylori isolate to colonize the stomach of Mongolian gerbils and to induce gastritis

The Mongolian gerbil is an excellent animal model for Helicobacter pylori-induced gastritis in humans. In this study, initially low colonization rates of the H. pylori strains ATCC 43504, SS1, or HP87 inoculated into gerbils caused difficulties in establishing this model. In order to increase the colonization ability and pathogenicity, the clinical HP87 isolate was selected for adaptation to the gerbil stomach by multiple in vivo passages through gerbils. Development of gastritis was examined histologically at 4-52 weeks after infection. The proportion of gerbils which tested positive for H. pylori by culture at four weeks after inoculation gradually increased from 11.1% of gerbils inoculated with HP87 without prior in vivo passage (P0) to 100% of gerbils inoculated with HP87 with seven in vivo passages (P7). In addition, adaptation of HP87 resulted in more severe histopathological changes. Gerbils infected with adapted HP87 (P7) exhibited severe infiltration by monomorphonuclear and polymorphonuclear leukocytes in the mucosa, submucosa, and subserosa of the gastric antrum, as well as epithelial changes consisting of hyperplasia, erosion, and ulceration. Histopathological changes increased in severity from four to 52 weeks after infection. Adaptation of HP87 during its passages through gerbils could be due to genetic changes in bacterial colonization factors. Identification of these changes might be useful to understand the underlying mechanism of gastric adaptation and pathogenesis of H. pylori.