Non-motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production

Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N-terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non-motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.     

Structural,genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno -n o n-ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.     

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori

The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.     

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension.

In the accompanying paper, we report that Drosophila gastrulae genetically depleted for the 5-HT(2Dro) serotonin receptor or for serotonin show abnormal germband extension. In wild-type gastrulae, peaks of both the 5-HT(2Dro) receptor and serotonin coincide precisely with the onset of germband extension. Here, we assessed the genetic requirement for this peak of serotonin. We report the characterisation of the serotonin content of individual Drosophila embryos, progeny from flies heterozygous for mutations in genes that are involved in the serotonin synthesis pathway and include the GTP-cyclohydrolase, tryptophan hydroxylase and DOPA decarboxylase loci. The peak of serotonin synthesis at the beginning of germband extension appears strictly dependent upon the maternal deposition of biopterins, products of GTP-cyclohydrolase and cofactors of tryptophan hydroxylase and upon the zygotic synthesis of both tryptophan hydroxylase and DOPA decarboxylase enzymes. Mutant embryos with an impairment in this peak of serotonin synthesis die with a cuticular organisation which is also observed in embryos deficient for the 5-HT(2Dro) receptor. This characteristic cuticular phenotype is thus the hallmark of desynchronisation of the morphogenetic movements during gastrulation. Together, these findings provide additional support for the notion that serotonin, acting through the 5-HT(2Dro) receptor, is necessary for proper gastrulation.     

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

Comparative ultrastructural studies of spermiogenesis and spermatozoa in some species of Polyplacophora (Mollusca)

Summary

Comparative data on the ultrastructure of spermiogenesis and spermatozoa of the Polyplacophora Acanthochitona crinita, Chaetopleura angulata and Callochiton septemvalvis are presented in this study. In contrast to what has been described for this and other classes of Mollusca, no acrosome is present in the spermatozoa of these Polyplacophora. The nucleus is extended by a long, thin apical point. In A. crinita and C. angulata the mitochondria are situated at the basal and lateral regions of the nucleus. They do not present a typical middle piece. These species present a pericentriolar process. In C. septemvalvis the mitochondria are situated at the base of the nucleus, surrounding the centrioles, which are orthogonally positioned in all species. The ultrastructural development during spermiogenesis is similar. In middle spermatids of A. crinita, the chromatin is arranged in fine filaments. In C. septemvalvis and C. angulata the chromatin filaments are thicker, forming coarse bands. In late spermatids elongation of the nucleus continues, it becomes rather electron-dense and the chromatin filaments are more condensed. Finally, the nucleus has a uniformly electron-dense appearance, with no signs of filamentous organization. Considering the ultrastructural modifications observed, the Polyplacophora spermatozoa could be included in a modified type.     

Comparative and functional studies of Drosophila species invasion by the gypsy endogenous retrovirus.

Gypsy is an endogenous retrovirus of Drosophila melanogaster. Phylogenetic studies suggest that occasional horizontal transfer events of gypsy occur between Drosophila species. gypsy possesses infective properties associated with the products of the envelope gene that might be at the origin of these interspecies transfers. We report here the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster, suggesting that potentially infective gypsy copies able to spread between sexually isolated species can occur. The ability of gypsy to invade the genome of a new species is conditioned by its capacity to be expressed in the naive genome. The genetic basis for the regulation of gypsy activity in D. melanogaster is now well known, and it has been assigned to an X-linked gene called flamenco. We established an experimental simulation of the invasion of the D. melanogaster genome by gypsy elements derived from other Drosophila species, which demonstrates that these non- D. melanogaster gypsy elements escape the repression exerted by the D. melanogaster flamenco gene.     

Colonization and tissue tropism of Helicobacter pylori and a novel urease-negative Helicobacter species in ICR mice are independent of route of exposure

Background. In humans, Helicobacter pylori is known to colonize the stomach and to induce persistent gastritis; selected reports also suggest it causes extragastric disease, including hepatitis. H. pylori and a novel urease-negative Helicobacter sp. induce gastritis and typhlocolitis, respectively, when inoculated orally into mice. Experimental typhlocolitis and hepatitis have been caused by intraperitoneal (IP) injection of H. hepaticus, H. bilis, and the novel Helicobacter spp. However, the route by which IP-inoculated organisms localize to specific areas of the gastrointestinal system is unknown. Materials and Methods. To determine whether Helicobacter spp. can be isolated from blood, can preferentially colonize specific tissues, and can cause pathological changes, we inoculated 6-week-old outbred mice orally or intraperitoneally with H. pylori or a novel Helicobacter sp. Results. When these mice were inoculated by the IP route, H. pylori was cultured from lungs, spleen, liver, cecum, and stomach on day 1 after inoculation, from liver and stomach mucosa on day 3 after inoculation, and from the stomach on day 30 after inoculation, suggesting preferential colonization of the stomach. After inoculation by the IP route, the novel intestinal Helicobacter sp. was cultured from the blood, lungs, spleen, liver, kidneys, cecum, and feces but not from stomach mucosa on day 1 after inoculation. By day 30 after inoculation, the novel Helicobacter sp. was cultured from cecum and feces only, suggesting that it had preferentially colonized the lower bowel. By the IP route, the novel Helicobacter sp. induced hepatitis that persisted for 30 days after inoculation. Though mice inoculated intraperitoneally with H. pylori developed an acute hepatitis, the liver lesion began to resolve 30 days after inoculation. Mice inoculated orally with either H. pylori or the novel Helicobacter sp. did not have hepatitis on day 30 after inoculation but developed 100% colonization of stomach and cecum, respectively. Conclusion. The isolation of H. pylori and the novel Helicobacter sp. from multiple tissues infers that a transient helicobacter bacteremia occurs when Helicobacter spp. are injected intraperitoneally, but organisms are cleared rapidly from nontarget tissues and preferentially colonize specific regions of the gastrointestinal tract.     

Urease-positive, acid-sensitive mutants of Helicobacter pylori: urease-independent acid resistance involved in growth at low pH

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.     

Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants.

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer.     

Epidemiology of Helicobacter pylori: transmission, translocation and extragastric reservoirs.

Although H. pylori infection is endemic and despite more than 10 years of research, the mode and route of transmission remain elusive. This may, in part, be due to the inherent problems of detecting H. pylori noninvasively. The prevalence of infection varies between countries and is closely related to Growth Domestic Product. An age-cohort effect and data from longitudinal studies suggest that the incidence of infection is much higher in children than adults. In developing countries the prevalence of infection is often more than 80% in young adults, in contrast to less than 10% for similar age groups in developed countries. The observations of mosaicism (in the VacA gene) and a panmycytic population structure imply exchange of genetic material either in or outside of the host, which is supported by the increasing recognition of polyclonal infection and suggests that secondary infection occurs after primary acquisition. In addition, in children persistent primary infection may sometimes occur only after previous (repeated) exposure and/or transient colonisation of the gastric mucosa. H. pylori and other gastric Helicobacter spp are always noninvasive, but other human nongastric Helicobacter spp have sometimes been isolated from the systemic circulation in immunocompromised patients. For nonhuman hosts, intestinal Helicobacter spp are thought to translocate more frequently from the colon to the liver. Within the human host, the oral cavity is the principal extragastric reservoir, although case reports suggest that H. pylori may sometimes be found beyond the 2nd part of the duodenum. The hypothesis that H. pylori is a zoonosis or transmitted as coccoid forms by a vector (pets, houseflies) is not supported by recent research showing that H. pylori is entirely unable to support an aerobic or anaerobic metabolism and that coccoid forms are non-viable. H. pylori is primarily acquired in infancy, most probably via the oroorogastric route, from other family members or close contacts encountered after weaning or socialisation. Further studies to support or refute this hypothesis are required.     

Nucleotide sequence of the Wolinella succinogenes flagellin, which contains in the antigenic domain two conserved regions also present in Campylobacter spp. and Helicobacter pylori.

Wolinella succinogenes possesses one polar flagellum, which shows a characteristic surface pattern of parallel lines along the axis of the filament in electron microscopic images. We determined the gene sequence of the Wolinella flagellin, which is, as in most other bacteria, the only structural component of the filament. Sequence comparison with other members of the Proteobacteria revealed two highly conserved regions in the central part of the flagellin molecule among Campylobacter spp. and Helicobacter pylori, an area that had previously been described as highly variable. Similar surface patterns are found in related polarly flagellated bacteria, but not in Escherichia coli and Bacillus subtilis, which also lack these conserved regions.     

Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.