Progressive dysbiosis of human orodigestive microbiota along the sequence of gastroesophageal reflux, Barrett's esophagus and esophageal adenocarcinoma.

The incidence of esophageal adenocarcinoma (EA) has drastically increased in the United States since 1970s for unclear reasons. We hypothesized that the widespread usage of antibiotics has increased the procarcinogenic potential of the orodigestive microbiota along the sequence of gastroesophageal reflux (GR), Barrett's esophagus (BE) and EA phenotypes. This case control study included normal controls (NC) and three disease phenotypes GR, BE and EA. Microbiota in the mouth, esophagus, and stomach, and rectum were analyzed using 16S rRNA gene sequencing. Overall, we discovered 44 significant pairwise differences in abundance of microbial taxa between the four phenotypes, with 12 differences in the mouth, 21 in the esophagus, two in the stomach, and nine in the rectum. Along the GR→BE→EA sequence, oral and esophageal microbiota were more diversified, the dominant genus Streptococcus was progressively depleted while six other genera Atopobium, Actinomyces, Veillonella, Ralstonia, Burkholderia and Lautropia progressively enriched. In NC, Streptococcus appeared to control populations of other genera in the foregut via numerous negative and positive connections, while in disease states, the rich network was markedly simplified. Inferred gene functional content showed a progressive enrichment through the stages of EA development in genes encoding antibiotic resistance, ligands of Toll-like and NOD-like receptors, nitrate-nitrite-nitric oxide pathway and acetaldehyde metabolism. The orodigestive microbiota is in a progressive dysbiotic state along the GR-BE-EA sequence. The increasing dysbiosis and antibiotic and procarcinogenic genes in the disease states warrants further study to define their roles in EA pathogenesis.

Periodontal pathogens are a risk factor of oral cavity squamous cell carcinoma, independent of tobacco and alcohol and human papillomavirus.

Over the past decade, there has been a change in the epidemiology of oral cavity squamous cell cancer (OC-SCC). Many new cases of OC-SCC lack the recognized risk factors of smoking, alcohol and human papilloma virus. The aim of this study was to determine if the oral microbiome may be associated with OC-SCC in nonsmoking HPV negative patients. We compared the oral microbiome of HPV-negative nonsmoker OC-SCC(n = 18), premalignant lesions(PML) (n = 8) and normal control patients (n = 12). Their oral microbiome was sampled by oral wash and defined by 16S rRNA gene sequencing. We report that the periodontal pathogens Fusobacterium, Prevotella, Alloprevotella were enriched while commensal Streptococcus depleted in OC-SCC. Based on the four genera plus a marker genus Veillonella for PML, we classified the oral microbiome into two types. Gene/pathway analysis revealed a progressive increase of genes encoding HSP90 and ligands for TLRs 1, 2 and 4 along the controls→PML → OC-SCC progression sequence. Our findings suggest an association between periodontal pathogens and OC-SCC in non smoking HPV negative patients.

Conserved Noncoding Elements in the Most Distant Genera of Cephalochordates: The Goldilocks Principle.

Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120-160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes in several vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over >500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates.

Low coverage sequencing of three echinoderm genomes: the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis.

BACKGROUND: There are five major extant groups of Echinodermata: Crinoidea (feather stars and sea lillies), Ophiuroidea (brittle stars and basket stars), Asteroidea (sea stars), Echinoidea (sea urchins, sea biscuits, and sand dollars), and Holothuroidea (sea cucumbers). These animals are known for their pentaradial symmetry as adults, unique water vascular system, mutable collagenous tissues, and endoskeletons of high magnesium calcite. To our knowledge, the only echinoderm species with a genome sequence available to date is Strongylocentrotus pupuratus (Echinoidea). The availability of additional echinoderm genome sequences is crucial for understanding the biology of these animals. FINDINGS: Here we present assembled draft genomes of the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis from Illumina sequence data with coverages of 12.5x, 22.5x, and 21.4x, respectively. CONCLUSIONS: These data provide a resource for mining gene superfamilies, identifying non-coding RNAs, confirming gene losses, and designing experimental constructs. They will be important comparative resources for future genomic studies in echinoderms.

Timing and Scope of Genomic Expansion within Annelida: Evidence from Homeoboxes in the Genome of the Earthworm Eisenia fetida.

Annelida represents a large and morphologically diverse group of bilaterian organisms. The recently published polychaete and leech genome sequences revealed an equally dynamic range of diversity at the genomic level. The availability of more annelid genomes will allow for the identification of evolutionary genomic events that helped shape the annelid lineage and better understand the diversity within the group. We sequenced and assembled the genome of the common earthworm, Eisenia fetida. As a first pass at understanding the diversity within the group, we classified 363 earthworm homeoboxes and compared them with those of the leech Helobdella robusta and the polychaete Capitella teleta. We inferred many gene expansions occurring in the lineage connecting the most recent common ancestor (MRCA) of Capitella and Eisenia to the Eisenia/Helobdella MRCA. Likewise, the lineage leading from the Eisenia/Helobdella MRCA to the leech H. robusta has experienced substantial gains and losses. However, the lineage leading from Eisenia/Helobdella MRCA to E. fetida is characterized by extraordinary levels of homeobox gain. The evolutionary dynamics observed in the homeoboxes of these lineages are very likely to be generalizable to all genes. These genome expansions and losses have likely contributed to the remarkable biology exhibited in this group. These results provide a new perspective from which to understand the diversity within these lineages, show the utility of sub-draft genome assemblies for understanding genomic evolution, and provide a critical resource from which the biology of these animals can be studied.

Groundwater ecosystem resilience to organic contaminations: microbial and geochemical dynamics throughout the 5-year life cycle of a surrogate ethanol blend fuel plume.

The capacity of groundwater ecosystem to recover from contamination by organic chemicals is a vital concern for environmental scientists. A pilot-scale aquifer system was used to investigate the long-term dynamics of contaminants, groundwater geochemistry, and microbial community structure (by 16S rRNA gene pyrosequencing and quantitative real-time PCR) throughout the 5-year life cycle of a surrogate ethanol blend fuel plume (10% ethanol + 50 mg/L benzene + 50 mg/L toluene). Two-year continuous ethanol-blended release significantly changed the groundwater geochemistry (resulted in anaerobic, low pH, and organotrophic conditions) and increased bacterial and archaeal populations by 82- and 314-fold respectively. Various anaerobic heterotrophs (fermenters, acetogens, methanogens, and hydrocarbon degraders) were enriched. Two years after the release was shut off, all contaminants and their degradation byproducts disappeared and groundwater geochemistry completely restored to the pre-release states (aerobic, neutral pH, and oligotrophic). Bacterial and archaeal populations declined by 18- and 45-fold respectively (relative to the time of shut off). Microbial community structure reverted towards the pre-release states and alpha diversity indices rebounded, suggesting the resilience of microbial community to ethanol blend releases. We also found shifts from O2-sensitive methanogens (e.g., Methanobacterium) to methanogens that are not so sensitive to O2 (e.g., Methanosarcina and Methanocella), which is likely to contribute to the persistence of methanogens and methane generation following the source removal. Overall, the rapid disappearance of contaminants and their metabolites, rebound of geochemical footprints, and resilience of microbial community unequivocally document the natural capacity of groundwater ecosystem to attenuate and recover from a large volume of catastrophic spill of ethanol-based biofuel.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Joint assembly and genetic mapping of the Atlantic horseshoe crab genome reveals ancient whole genome duplication.

BACKGROUND: Horseshoe crabs are marine arthropods with a fossil record extending back approximately 450 million years. They exhibit remarkable morphological stability over their long evolutionary history, retaining a number of ancestral arthropod traits, and are often cited as examples of "living fossils." As arthropods, they belong to the Ecdysozoa, an ancient super-phylum whose sequenced genomes (including insects and nematodes) have thus far shown more divergence from the ancestral pattern of eumetazoan genome organization than cnidarians, deuterostomes and lophotrochozoans. However, much of ecdysozoan diversity remains unrepresented in comparative genomic analyses. RESULTS: Here we apply a new strategy of combined de novo assembly and genetic mapping to examine the chromosome-scale genome organization of the Atlantic horseshoe crab, Limulus polyphemus. We constructed a genetic linkage map of this 2.7 Gbp genome by sequencing the nuclear DNA of 34 wild-collected, full-sibling embryos and their parents at a mean redundancy of 1.1x per sample. The map includes 84,307 sequence markers grouped into 1,876 distinct genetic intervals and 5,775 candidate conserved protein coding genes. CONCLUSIONS: Comparison with other metazoan genomes shows that the L. polyphemus genome preserves ancestral bilaterian linkage groups, and that a common ancestor of modern horseshoe crabs underwent one or more ancient whole genome duplications 300 million years ago, followed by extensive chromosome fusion. These results provide a counter-example to the often noted correlation between whole genome duplication and evolutionary radiations. The new, low-cost genetic mapping method for obtaining a chromosome-scale view of non-model organism genomes that we demonstrate here does not require laboratory culture, and is potentially applicable to a broad range of other species.

Human papillomavirus community in healthy persons, defined by metagenomics analysis of human microbiome project shotgun sequencing data sets.

UNLABELLED: Human papillomavirus (HPV) causes a number of neoplastic diseases in humans. Here, we show a complex normal HPV community in a cohort of 103 healthy human subjects, by metagenomics analysis of the shotgun sequencing data generated from the NIH Human Microbiome Project. The overall HPV prevalence was 68.9% and was highest in the skin (61.3%), followed by the vagina (41.5%), mouth (30%), and gut (17.3%). Of the 109 HPV types as well as additional unclassified types detected, most were undetectable by the widely used commercial kits targeting the vaginal/cervical HPV types. These HPVs likely represent true HPV infections rather than transitory exposure because of strong organ tropism and persistence of the same HPV types in repeat samples. Coexistence of multiple HPV types was found in 48.1% of the HPV-positive samples. Networking between HPV types, cooccurrence or exclusion, was detected in vaginal and skin samples. Large contigs assembled from short HPV reads were obtained from several samples, confirming their genuine HPV origin. This first large-scale survey of HPV using a shotgun sequencing approach yielded a comprehensive map of HPV infections among different body sites of healthy human subjects. IMPORTANCE: This nonbiased survey indicates that the HPV community in healthy humans is much more complex than previously defined by widely used kits that are target selective for only a few high- and low-risk HPV types for cervical cancer. The importance of nononcogenic viruses in a mixed HPV infection could be for stimulating or inhibiting a coexisting oncogenic virus via viral interference or immune cross-reaction. Knowledge gained from this study will be helpful to guide the designing of epidemiological and clinical studies in the future to determine the impact of nononcogenic HPV types on the outcome of HPV infections.

Adaptive microbial population shifts in response to a continuous ethanol blend release increases biodegradation potential.

The fate of fuel releases largely depends on the poorly-understood response in microbial community structure and function. Here, we evaluate the impacts to the microbial community resulting from a pilot-scale continuous release (10 months) of a 10% v:v ethanol solution mixed with benzene and toluene (50 mg/L each). Microbial population shifts were characterized by pyrosequencing-based 16S rRNA analysis and by quantitative PCR targeting Bacteria, Archaea, and functional genes for methanogenesis (mcrA), acetogenesis (fhs) and aerobic degradation of aromatic hydrocarbons (PHE), which could occur in hypoxic micro-environments. The release stimulated microbial growth, increased species richness and diversity, and selected for genotypes involved in fermentative degradation (the relative abundance of mcrA and fhs increased 18- and 6-fold, respectively). The growth of putative hydrocarbon degraders and commensal anaerobes, and increases in microbial diversity and in degradation rates suggest an adaptive response that increases the potential for natural attenuation of ethanol blend releases.

Diversity of 5S rRNA genes within individual prokaryotic genomes.

We examined intragenomic variation of paralogous 5S rRNA genes to evaluate the concept of ribosomal constraints. In a dataset containing 1161 genomes from 779 unique species, 96 species exhibited > 3% diversity. Twenty-seven species with > 10% diversity contained a total of 421 mismatches between all pairs of the most dissimilar copies of 5S rRNA genes. The large majority (401 of 421) of the diversified positions were conserved at the secondary structure level. The high diversity was associated with partial rRNA operon, split operon, or spacer length-related divergence. In total, these findings indicated that there are tight ribosomal constraints on paralogous 5S rRNA genes in a genome despite of the high degree of diversity at the primary structure level.

Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome.

AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database. CONCLUSION: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Diversity of 16S rRNA genes within individual prokaryotic genomes.

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

Helicobacter pylori activation of PARP-1: usurping a versatile regulator of host cellular health.

Chronic infection of the human stomach by Helicobacter pylori is an important risk factor for gastric cancer. H. pylori produces a cache of virulence factors that promote colonization and persistence, which, in turn, contributes to a robust inflammatory response at the host-pathogen interface. Recently, we reported that H. pylori activates the abundant nuclear regulator poly(ADP-ribose) polymerase (PARP)-1, resulting in the production of the catabolite poly(ADP-ribose) (PAR). PARP-1 is emerging as a key player in establishing homeostasis at the host-pathogen interface. In this article, we summarize the discovery of H. pylori-dependent PARP-1 activation, and discuss potential roles for PARP-1 in H. pylori-mediated gastric disease. In light of the remarkable successes that have reported for treating inflammatory disorders and cancers with PARP-1 inhibitors, we discuss the prospects of targeting PARP-1 for treatment of H. pylori-associated gastric disease.

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori.

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.

Diversity of 23S rRNA genes within individual prokaryotic genomes.

BACKGROUND: The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. METHODOLOGY/PRINCIPAL FINDINGS: Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%-4.04%). Significant (1.17%-4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. CONCLUSIONS/SIGNIFICANCE: These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.

Inflammation and intestinal metaplasia of the distal esophagus are associated with alterations in the microbiome.

BACKGROUND & AIMS: Gastroesophageal reflux causes inflammation and intestinal metaplasia and its downstream sequelum adenocarcinoma in the distal esophagus. The incidence of esophageal adenocarcinoma has increased approximately 6-fold in the United States since the 1970s, accompanied with a significant increase in the prevalence of gastroesophageal reflux disease (GERD). Despite extensive epidemiologic study, the cause for GERD and the unexpected increases remain unexplainable. Microbes are among the environmental factors that may contribute to the etiology of GERD, but very little research has been done on the esophageal microbiome, particularly in its relation to GERD. This is the first comprehensive reported correlation between a change in the esophageal microbiome and esophageal diseases. METHODS: Biopsy samples of the distal esophagus were collected from 34 patients. Host phenotypes were histologically defined as normal, esophagitis, or Barrett's esophagus (intestinal metaplasia). Microbiomes from the biopsy samples were analyzed by bacterial 16S ribosomal RNA gene survey and classified into types using unsupervised cluster analysis and phenotype-guided analyses. Independence between host phenotypes and microbiome types were analyzed by Fisher exact test. RESULTS: Esophageal microbiomes can be classified into 2 types. The type I microbiome was dominated by the genus Streptococcus and concentrated in the phenotypically normal esophagus. Conversely, the type II microbiome contained a greater proportion of gram-negative anaerobes/microaerophiles and primarily correlated with esophagitis (odds ratio, 15.4) and Barrett's esophagus (odds ratio, 16.5). CONCLUSIONS: In the human distal esophagus, inflammation and intestinal metaplasia are associated with global alteration of the microbiome. These findings raise the issue of a possible role for dysbiosis in the pathogenesis of reflux-related disorders.

Modification of a mammalian cell protein in the presence of [32P-adenylate]NAD: evidence for ADP ribosylation activity associated with Helicobacter pylori.

Culture filtrates from Helicobacter pylori promote the transfer of the radiolabel from [32P-adenylate]NAD to one or more heat-labile factors within extracts prepared from several mammalian cell lines, with the predominate radiolabeled species exhibiting an apparent molecular mass of greater than 130 kDa. Our results suggest that several H. pylori strains release a factor that ADP-ribosylates a mammalian target protein.