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.     

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.     

Failure of surface ring mutant strains of Helicobacter mustelae to persistently infect the ferret stomach

Helicobacter mustelae, the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genus Helicobacter, including H. pylori. The unique ring structures are composed of a protein named Hsr. To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free for H. mustelae were inoculated with an Hsr-deficient mutant strain or the wild-type H. mustelae strain. Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain. However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced. This decline contrasted the robust antral colonization by the wild-type strain. The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets. Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutant H. mustelae strain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain. Scant colonization by the Hsr-negative H. mustelae strain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response.     

A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach.

The urease of Helicobacter pylori has been proposed to be one of its pathogenic factors. A kanamycin resistance determinant was inserted in a cloned urease gene, and transformation-mediated allelic exchange mutagenesis was carried out to introduce the disrupted gene into the corresponding wild-type chromosomal region of a clinical isolate of H. pylori, CPY3401. The resulting mutant, HPT73, had the null activity of urease. Nude mouse stomachs were challenged with these two isogenic strains to examine the role of urease in pathogenesis. Gastritis was found in the CPY3401-challenged stomachs, from which bacteria indistinguishable from CPY3401 were recovered. There was no gastritis in the HPT73-challenged stomachs, and we could not recover H. pylori from them. These results indicated that H. pylori urease is essential for colonizing the nude mouse stomach.     

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.     

Presence of active aliphatic amidases in Helicobacter species able to colonize the stomach

Ammonia production is of great importance for the gastric pathogen Helicobacter pylori as a nitrogen source, as a compound protecting against gastric acidity, and as a cytotoxic molecule. In addition to urease, H. pylori possesses two aliphatic amidases responsible for ammonia production: AmiE, a classical amidase, and AmiF, a new type of formamidase. Both enzymes are part of a regulatory network consisting of nitrogen metabolism enzymes, including urease and arginase. We examined the role of the H. pylori amidases in vivo by testing the gastric colonization of mice with H. pylori SS1 strains carrying mutations in amiE and/or amiF and in coinfection experiments with wild-type and double mutant strains. A new cassette conferring resistance to gentamicin was used in addition to the kanamycin cassette to construct the double mutation in strain SS1. Our data indicate that the amidases are not essential for colonization of mice. The search for amiE and amiF genes in 53 H. pylori strains from different geographic origins indicated the presence of both genes in all these genomes. We tested for the presence of the amiE and amiF genes and for amidase and formamidase activities in eleven Helicobacter species. Among the gastric species, H. acinonychis possessed both amiE and amiF, H. felis carried only amiF, and H. mustelae was devoid of amidases. H. muridarum, which can colonize both mouse intestine and stomach, was the only enterohepatic species to contain amiE. Phylogenetic trees based upon the sequences of H. pylori amiE and amiF genes and their respective homologs from other organisms as well as the amidase gene distribution among Helicobacter species are strongly suggestive of amidase acquisition by horizontal gene transfer. Since amidases are found only in Helicobacter species able to colonize the stomach, their acquisition might be related to selective pressure in this particular gastric environment.     

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro.

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Comparison of isolates of Helicobacter pylori and Helicobacter mustelae.

On the basis of analysis of protein profiles, isolates of Helicobacter pylori and Helicobacter mustelae were less than 40% similar. Cytotoxin produced by H. pylori was not detected in isolates of H. mustelae. Both bacterial species agglutinated human erythrocytes. These results substantiate a taxonomic difference between H. pylori and H. mustelae.     

Purification and characterization of Helicobacter mustelae urease.

Helicobacter mustelae is a urease-rich bacterium associated with gastritis in ferrets. The ureases of H. mustelae and Helicobacter pylori, a bacterium implicated in human gastritis, share many characteristics. Helicobacter sp. ureases appear to be unique among bacterial enzymes in exhibiting submillimolar Km values and in being composed of two subunits.     

Inability to express fimbriae results in impaired ability of Haemophilus influenzae b to colonize the nasopharynx.

We cloned into the structural fimbrial subunit gene from a fimbriated Haemophilus influenzae b a 1.5-kb kanamycin resistance gene. The resultant strain (RKAW5) was tested by Southern analysis, hemagglutination, and electron-micrographic examination to confirm gene inactivation. In comparison with the parent, RKAW5 exhibited a significant decrease in adherence to human buccal epithelial cells and in nasal colonization of yearling rhesus monkeys.     

Ability of an avirulent mutant of Vibrio cholerae to colonize in the infant mouse upper bowel.

Vibrio cholerae strain 3083 (biotype El Tor, serotype Ogawa) and Texas Star-SR (SR), a mutant derived from 3083 that produces the B (binding) but not the A (toxic) subunit of choleragen, were compared in their abilities to: (i) associate with the infant mouse upper bowel; (ii) survive and multiple there; and (iii) induce diarrhea. Vibrios labeled with 35SO4 were used to determine association with the upper bowel and ability to multiply. The parental strain associated significantly better than SR, although viable mutant cells were found in the infant mouse intestine 16 to 18 h after challenge. Addition of exogenous toxin enhanced the rate at which labeled SR (but not 3083) was cleared, further suggesting that SR associates less well with the upper bowel. Both SR and 3083 multiplied in the upper bowel but, due perhaps to slight net killing during the first 3 h and its more rapid rate of clearance, SR achieved a population size only 10% that of 3083 by 8 h postchallenge. Strain 3083 elicited diarrhea in infant mice but SR did not, even after 10 successive passages through the infant mouse intestine. Strain SR was slightly temperature sensitive at 37 and 40 degrees C. Its potential use as a live vaccine is discussed.     

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.     

Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric Helicobacter.

Helicobacter mustelae has been isolated from stomachs of ferrets with chronic gastritis and ulcers. When H. mustelae is inoculated orally into H. mustelae-negative ferrets, the animals become colonized and develop gastritis, a significant immune response, and a transient hypochlorhydria. All of these features mimic Helicobacter pylori-induced gastric disease in humans. Because the epidemiology of H. pylori infection is poorly understood and its route of transmission is unknown, the feces of weanling and adult ferrets were cultured for the presence of H. mustelae. H. mustelae was isolated from the feces of 11 of 36 ferrets by using standard helicobacter isolation techniques. H. mustelae was identified by biochemical tests, ultrastructural morphology, reactivity with specific DNA probes, and 16S rRNA sequencing. H. mustelae was not recovered from 20-week-old ferrets which had been H. mustelae positive as weanlings, nor was H. mustelae recovered from 1-year-old ferrets. Isolation of H. mustelae from feces may correspond to periods of transient hypochlorhydria, or H. mustelae may be shed in feces intermittently. The H. mustelae-colonized ferret provides an ideal model for studying the pathogenesis and transmission of H. pylori-induced gastric disease.     

Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.     

Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal acid glycoprotein

An isogenic mutant of Streptococcus pyogenes Manfredo that lacks the ability to make streptococcal acid glycoprotein (SAGP) has been constructed by inserting a deletion in the sagp gene using the method of allelic exchange. An assay of cell extracts (CE) prepared from the wild-type and mutant Manfredo strains for the enzyme arginine deiminase (AD) showed that significant activity was present in wild-type CE but none could be detected in mutant CE. These findings confirm our earlier conclusion that SAGP has AD activity (B. A. Degnan, J. M. Palmer, T. Robson, C. E. D. Jones, M. Fischer, M. Glanville, G. D. Mellor, A. G. Diamond, M. A. Kehoe, and J. A. Goodacre, Infect. Immun. 66:3050-3058, 1998). Wild-type CE but not mutant CE potently inhibited human peripheral blood mononuclear cell proliferation in response to phytohemagglutinin, and this inhibition was overcome by the addition of L-arginine to proliferation assay mixtures. Invasion assays showed that the isogenic mutant organisms lacking SAGP, and thus AD activity, were between three and five times less able to enter epithelial cells (Hep-2C and A549) than were the wild-type streptococci. Both wild-type and mutant S. pyogenes bacteria were extremely sensitive to low pH. However, L-arginine (1 mM or above) significantly increased the viability of the wild type but not the isogenic mutant organisms under acidic conditions. The difference in acid susceptibility between wild-type and mutant bacteria may explain the reduced capacity of the isogenic mutant bacteria to invade and survive intracellularly.     

Sequence and antigenic variability of the Helicobacter mustelae surface ring protein Hsr

We have identified an array of more than 500 repetitive sequences flanking the hsr gene, which encodes the major surface protein of the ferret pathogen Helicobacter mustelae. The repeats show identity exclusively to the amino-terminal half of Hsr. Analysis of Hsr from three strains indicated variability of exposed epitopes. Characterization of an hsr mutant showed that Hsr is not an adhesin.     

Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae.

Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni2+ and Zn2+. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.     

An ultrastructural study of Helicobacter mustelae and evidence of a specific association with gastric mucosa.

The ultrastructure of Helicobacter mustelae, a natural inhabitant of the ferret stomach, has been studied and compared with the human gastroduodenal pathogen H. pylori. H. mustelae is a short, slightly curved rod, 2 microns x 0.5 micron, with four or more sheathed flagella. The most common flagellar configuration is a single flagellum at one terminus, bipolar arrangement at the other end and a lateral flagellum. Dense inclusion bodies were observed below the flagellar insertion sites. It is suggested that this configuration is a specialised adaptation to motility in a viscous environment. On examination of the ferret gastric mucosa, similarities to H. pylori were observed such as adherence to gastric tissue and the formation of adhesion pedestals. However, unlike H. pylori, significant numbers of bacteria were intracellular. Furthermore, a much greater proportion of H. mustelae were attached to the mucosa with few bacteria lying free in the mucus, as is seen with H. pylori. It is concluded that the ferret is an important model for the study of adherence of bacteria to gastric mucosa and their possible role in peptic ulceration.     

Loss of virulence associated with absence of flagellum in an isogenic mutant of Pseudomonas aeruginosa in the burned-mouse model.

To test the importance of flagella as a virulence factor, virulent strain Pseudomonas aeruginosa M-2 was mutagenized, and a Fla mutant was isolated. An M-2 Fla spontaneous motile revertant was isolated from M-2 Fla. The three strains were biochemically and morphologically identical. Assay of Fla in the burned-mouse model showed a severe loss of virulence. The Fla revertant was fully virulent. The importance of P. aeruginosa motility (flagella) for virulence in burns infections is indicated.     

Construction of an isogenic leukotoxin deletion mutant ofPasteurella haemolyticaserotype 1: characterization and virulence

Allelic replacement was used to generate two isogeniclktAdeletion mutants ofPasteurella haemolyticaserotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed thatlktAsequences were absent in the twoP. haemolyticadeletion mutants. Culture supernatants and whole cell lysates from the wild typeP. haemolytica, D153 parent strain, but not thelktAdeletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of thelktAdeletion mutant,lktA77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in thelktAmutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with thelktAmutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.