Grincamycins P-T: Rearranged Angucyclines from the Marine Sediment-Derived Streptomyces sp. CNZ-748 Inhibit Cell Lines of the Rare Cancer Pseudomyxoma Peritonei.

While marine natural products have been investigated for anticancer drug discovery, they are barely screened against rare cancers. Thus, in our effort to discover potential drug leads against the rare cancer pseudomyxoma peritonei (PMP), which currently lacks effective drug treatments, we screened extracts of marine actinomycete bacteria against the PMP cell line ABX023-1. This effort led to the isolation of nine rearranged angucyclines from Streptomyces sp. CNZ-748, including five new analogues, namely, grincamycins P-T (1-5). The chemical structures of these compounds were unambiguously established based on spectroscopic and chemical analyses. Particularly, grincamycin R (3) possesses an S-containing α-l-methylthio-aculose residue, which was discovered in nature for the first time. All of the isolated compounds were evaluated against four PMP cell lines and some exhibited low micromolar inhibitory activities. To identify a candidate biosynthetic gene cluster (BGC) encoding the grincamycins, we sequenced the genome of the producing strain, Streptomyces sp. CNZ-748, and compared the BGCs detected with those linked to the production of angucyclines with different aglycon structures.

Parapseudoflavitalea muciniphila gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from a human peritoneal tumour and reclassification of Pseudobacter ginsenosidimutans as Pseudoflavitalea ginsenosidimutans comb. nov.

A Gram-stain-negative, microaerophilic, non-motile, rod-shaped bacterium strain designated PMP191FT, was isolated from a human peritoneal tumour. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the organism formed a lineage within the family Chitinophagaceae that was distinct from members of the genus Pseudoflavitalea (95.1-95.2 % sequence similarity) and Pseudobacter ginsenosidimutans (94.4 % sequence similarity). The average nucleotide identity values between strain PMP191FT and Pseudoflavitalea rhizosphaerae T16R-265T and Pseudobacter ginsenosidimutans Gsoil 221T was 68.9 and 62.3% respectively. The only respiratory quinone of strain PMP191FT was MK-7 and the major fatty acids were iso-C15 : 0, iso-C15 : 1 G and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids consisted of phosphatidylethanolamine and some unidentified amino and glycolipids. The G+C content of strain PMP191FT calculated from the genome sequence was 43.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strain PMP191FT represents a novel species and genus for which the name Parapseudoflavitalea muciniphila gen. nov., sp. nov. is proposed. The type strain is PMP191FT (=DSM 104999T=ATCC BAA-2857T = CCUG 72691T). The phylogenetic analyses also revealed that Pseudobacter ginsenosidimutans shared over 98 % sequence similarly to members of the genus Pseudoflavitalea. However, the average nucleotide identity value between Pseudoflavitalea rhizosphaerae T16R-265T, the type species of the genus and Pseudobacter ginsenosidimutans Gsoil 221T was 86.8 %. Therefore, we also propose that Pseudobacter ginsenosidimutans be reclassified as Pseudoflavitalea ginsenosidimutans comb. nov.

Role of the HefC efflux pump in Helicobacter pylori cholesterol-dependent resistance to ceragenins and bile salts.

The human gastric pathogen Helicobacter pylori modifies host cholesterol via glycosylation and incorporates the glycosylated cholesterol into its membrane; however, the benefits of cholesterol to H. pylori are largely unknown. We speculated that cholesterol in the H. pylori membrane might alter the susceptibility of these organisms to membrane-disrupting antibacterial compounds. To test this hypothesis, H. pylori strains were cultured in Ham's F-12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 10 bile salts and four ceragenins, which are novel bile salt derivatives that mimic membrane-disrupting activity of antimicrobial peptides. H. pylori cultured with cholesterol was substantially more resistant to seven of the bile salts and three ceragenins than H. pylori cultured without cholesterol. In most cases, these cholesterol-dependent differences ranged from 2 to 7 orders of magnitude; this magnitude depended on concentration of the agent. Cholesterol is modified by glycosylation using Cgt, a cholesteryl glycosyltransferase. Surprisingly, a cgt knockout strain still maintained cholesterol-dependent resistance to bile salts and ceragenins, indicating that cholesterol modification was not involved in resistance. We then tested whether three putative, paralogous inner membrane efflux pumps, HefC, HefF, or HefI, played a role. While HefF and HefI appeared unimportant, HefC was shown to play a critical role in the resistance to bile salts and ceragenins by multiple methods in multiple strain backgrounds. Thus, both cholesterol and the putative bile salt efflux pump HefC play important roles in H. pylori resistance to bile salts and ceragenins.

Helicobacter pylori AlpA and AlpB bind host laminin and influence gastric inflammation in gerbils.

Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37.

The human gastric pathogen Helicobacter pylori steals host cholesterol, modifies it by glycosylation, and incorporates the glycosylated cholesterol onto its surface via a cholesterol glucosyltransferase, encoded by cgt. The impact of cholesterol on H. pylori antimicrobial resistance is unknown. H. pylori strain 26695 was cultured in Ham's F12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 12 antibiotics, six antifungals, and seven antimicrobial peptides (including LL-37 cathelicidin and human alpha and beta defensins). Of 25 agents tested, cholesterol-grown H. pylori cells were substantially more resistant (over 100-fold) to nine agents than were H. pylori cells grown without cholesterol. These nine agents included eight antibiotics and LL-37. H. pylori was susceptible to the antifungal drug pimaricin regardless of cholesterol presence in the culture medium. A cgt mutant retained cholesterol-dependent resistance to most antimicrobials but displayed increased susceptibility to colistin, suggesting an involvement of lipid A. Mutation of lpxE, encoding lipid A1-phosphatase, led to loss of cholesterol-dependent resistance to polymyxin B and colistin but not other antimicrobials tested. The cgt mutant was severely attenuated in gerbils, indicating that glycosylation is essential in vivo. These findings suggest that cholesterol plays a vital role in virulence and contributes to the intrinsic antibiotic resistance of H. pylori.

Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media.

Helicobacter pylori chronically infects the gastric mucosa, where it can be found free in mucus, attached to cells, and intracellularly. H. pylori requires iron for growth, but the sources of iron used in vivo are unclear. In previous studies, the inability to culture H. pylori without serum made it difficult to determine which host iron sources might be used by H. pylori. Using iron-deficient, chemically defined medium, we determined that H. pylori can bind and extract iron from hemoglobin, transferrin, and lactoferrin. H. pylori can use both bovine and human versions of both lactoferrin and transferrin, contrary to previous reports. Unlike other pathogens, H. pylori preferentially binds the iron-free forms of transferrin and lactoferrin, which limits its ability to extract iron from normal serum, which is not iron saturated. This novel strategy may have evolved to permit limited growth in host tissue during persistent colonization while excessive injury or iron depletion is prevented.

Immune and microRNA responses to Helicobacter muridarum infection and indole-3-carbinol during colitis.

BACKGROUND: Indole-3-carbinol (I3C) and other aryl hydrocarbon receptor agonists are known to modulate the immune system and ameliorate various inflammatory and autoimmune diseases in animal models, including colitis induced by dextran sulfate sodium (DSS). MicroRNAs (miRNAs) are also gaining traction as potential therapeutic agents or diagnostic elements. Enterohepatic Helicobacter (EHH) species are associated with an increased risk of inflammatory bowel disease, but little is known about how these species affect the immune system or response to treatment. AIM: To determine whether infection with an EHH species alters the response to I3C and how the immune and miRNA responses of an EHH species compare with responses to DSS and inflammatory bowel disease. METHODS: We infected C57BL/6 mice with Helicobacter muridarum (H. muridarum), with and without DSS and I3C treatment. Pathological responses were evaluated by histological examination, symptom scores, and cytokine responses. MiRNAs analysis was performed on mesenteric lymph nodes to further evaluate the regional immune response. RESULTS: H. muridarum infection alone caused colonic inflammation and upregulated proinflammatory, macrophage-associated cytokines in the colon similar to changes seen in DSS-treated mice. Further upregulation occurred upon treatment with DSS. H. muridarum infection caused broad changes in mesenteric lymph node miRNA expression, but colitis-associated miRNAs were regulated similarly in H. muridarum-infected and uninfected, DSS-treated mice. In spite of causing colitis exacerbation, H. muridarum infection did not prevent disease amelioration by I3C. I3C normalized both macrophage- and T cell-associated cytokines. CONCLUSION: Thus, I3C may be useful for inflammatory bowel disease patients regardless of EHH infection. The miRNA changes associated with I3C treatment are likely the result of, rather than the cause of immune response changes.

The Cancer Microbiome: Distinguishing Direct and Indirect Effects Requires a Systemic View.

The collection of microbes that live in and on the human body - the human microbiome - can impact on cancer initiation, progression, and response to therapy, including cancer immunotherapy. The mechanisms by which microbiomes impact on cancers can yield new diagnostics and treatments, but much remains unknown. The interactions between microbes, diet, host factors, drugs, and cell-cell interactions within the cancer itself likely involve intricate feedbacks, and no single component can explain all the behavior of the system. Understanding the role of host-associated microbial communities in cancer systems will require a multidisciplinary approach combining microbial ecology, immunology, cancer cell biology, and computational biology - a systems biology approach.

Pre- and post-operative antibiotics in conjunction with cytoreductive surgery and heated intraperitoneal chemotherapy (HIPEC) should be considered for pseudomyxoma peritonei (PMP) treatment.

Pseudomyxoma peritonei (PMP) is a subtype of peritoneal carcinomatosis that is traditionally treated by cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC). A growing body of evidence suggests that microbes are associated with various tumor types and have been found in organs and cavities that were once considered sterile. Prior and ongoing research from our consortium of PMP researchers strongly suggests that bacteria are associated with PMP tumors. While the significance of this association is unclear, in our opinion, further research is warranted to understand whether these bacteria contribute to the development, maintenance and/or progression of PMP. Elucidation of a possible causal role for bacteria in PMP could suggest a benefit for supplementation of antibiotics to current treatment protocols.

Both diet and Helicobacter pylori infection contribute to atherosclerosis in pre- and postmenopausal cynomolgus monkeys.

A number of viruses and bacterial species have been implicated as contributors to atherosclerosis, potentially providing novel pathways for prevention. Epidemiological studies examining the association between Helicobacter pylori and cardiovascular disease have yielded variable results and no studies have been conducted in nonhuman primates. In this investigation, we examined the relationship between H. pylori infection and atherosclerosis development in socially housed, pre- and postmenopausal cynomolgus macaques consuming human-like diets. Ninety-four premenopausal cynomolgus monkeys (Macaca fascicularis) were fed for 36 months an atherogenic diet deriving its protein from either casein lactalbumin(CL) or high isoflavone soy (SOY). Animals were then ovariectomized and fed either the same or the alternate diet for an additional 36 months. Iliac artery biopsies were obtained at the time of ovariectomy and iliac and coronary artery sections were examined at the end of the study. Evidence of H. pylori infection was found in 64% of the monkeys and 46% of animals had live H. pylori within coronary atheromas as determined by mRNA-specific in situ hybridization. There was a significant linear relationship between the densities of gastric and atheroma organisms. Helicobactor pylori infection correlated with increased intimal plaque area and thickness at both the premenopausal and postmenopausal time points and regardless of diet (p< 0.01), although animals consuming the SOY diet throughout had the least amount of atherosclerosis. Additionally, plasma lipid profiles, intimal collagen accumulation, ICAM-1, and plaque macrophage densities were adversely affected by H. pylori infection among animals consuming the CL diet, while the SOY diet had the opposite effect. Plaque measurements were more highly associated with the densities of cagA-positive H. pylori within coronary atheromas than with the densities of gastric organisms, whereas plasma lipid changes were associated with H. pylori infection, but not cagA status. This study provides strong evidence that live H. pylori infects atheromas, exacerbates atherosclerotic plaque development, and alters plasma lipid profiles independently of diet or hormonal status. Finally, socially subordinate animals relative to their dominant counterparts had a greater prevalence of H. pylori, suggesting a stress effect. The results indicate that early H. pylori eradication could prevent or delay development of cardiovascular disease.

Adherence of Helicobacter pylori to abiotic surfaces is influenced by serum.

Helicobacter pylori bacteria cultured in a chemically defined medium without serum readily adhere to a variety of abiotic surfaces. Growth produces microcolonies that spread to cover the entire surface, along with a planktonic subpopulation. Serum inhibits adherence. Initial attachment is protein mediated, but other molecules are responsible for more permanent attachment.

Clinical Risk Score for Prediction of Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae in Bloodstream Isolates.

OBJECTIVE To develop a risk score to predict probability of bloodstream infections (BSIs) due to extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBLE). DESIGN Retrospective case-control study. SETTING Two large community hospitals. PATIENTS Hospitalized adults with Enterobacteriaceae BSI between January 1, 2010, and June 30, 2015. METHODS Multivariate logistic regression was used to identify independent risk factors for ESBLE BSI. Point allocation in extended-spectrum ß-lactamase prediction score (ESBL-PS) was based on regression coefficients. RESULTS Among 910 patients with Enterobacteriaceae BSI, 42 (4.6%) had ESBLE bloodstream isolates. Most ESBLE BSIs were community onset (33 of 42; 79%), and 25 (60%) were due to Escherichia coli. Independent risk factors for ESBLE BSI and point allocation in ESBL-PS included outpatient procedures within 1 month (adjusted odds ratio [aOR], 8.7; 95% confidence interval [CI], 3.1-22.9; 1 point), prior infections or colonization with ESBLE within 12 months (aOR, 26.8; 95% CI, 7.0-108.2; 4 points), and number of prior courses of ß-lactams and/or fluoroquinolones used within 3 months of BSI: 1 course (aOR, 6.3; 95% CI, 2.7-14.7; 1 point), ≥2 courses (aOR, 22.0; 95% CI, 8.6-57.1; 3 points). The area under the receiver operating characteristic curve for the ESBL-PS model was 0.86. Patients with ESBL-PSs of 0, 1, 3, and 4 had estimated probabilities of ESBLE BSI of 0.7%, 5%, 24%, and 44%, respectively. Using ESBL-PS ≥3 to indicate high risk provided a negative predictive value of 97%. CONCLUSIONS ESBL-PS estimated patient-specific risk of ESBLE BSI with high discrimination. Incorporation of ESBL-PS with acute severity of illness may improve adequacy of empirical antimicrobial therapy and reduce carbapenem utilization. Infect Control Hosp Epidemiol 2017;38:266-272.

Altered gut microbiome in a mouse model of Gulf War Illness causes neuroinflammation and intestinal injury via leaky gut and TLR4 activation.

Many of the symptoms of Gulf War Illness (GWI) that include neurological abnormalities, neuroinflammation, chronic fatigue and gastrointestinal disturbances have been traced to Gulf War chemical exposure. Though the association and subsequent evidences are strong, the mechanisms that connect exposure to intestinal and neurological abnormalities remain unclear. Using an established rodent model of Gulf War Illness, we show that chemical exposure caused significant dysbiosis in the gut that included increased abundance of phylum Firmicutes and Tenericutes, and decreased abundance of Bacteroidetes. Several gram negative bacterial genera were enriched in the GWI-model that included Allobaculum sp. Altered microbiome caused significant decrease in tight junction protein Occludin with a concomitant increase in Claudin-2, a signature of a leaky gut. Resultant leaching of gut caused portal endotoxemia that led to upregulation of toll like receptor 4 (TLR4) activation in the small intestine and the brain. TLR4 knock out mice and mice that had gut decontamination showed significant decrease in tyrosine nitration and inflammatory mediators IL1ß and MCP-1 in both the small intestine and frontal cortex. These events signified that gut dysbiosis with simultaneous leaky gut and systemic endotoxemia-induced TLR4 activation contributes to GW chemical-induced neuroinflammation and gastrointestinal disturbances.

A Novel Member of Chitinophagaceae Isolated from a Human Peritoneal Tumor.

Peritoneal tumors from a rare malignancy, pseudomyxoma peritonei, frequently contain bacteria. Evidence suggests that tumor-associated bacteria contribute to pseudomyxoma peritonei development and/or progression. One unique isolate (PMP191F) was characterized via whole-genome sequencing using the Illumina MiSeq platform. PMP191F shows similarities to the Chitinophaga, Niastella, and Flavitalea genera.

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.

Beyond the stomach: an updated view of Helicobacter pylori pathogenesis, diagnosis, and treatment.

Helicobacter pylori (H. pylori) is an extremely common, yet underappreciated, pathogen that is able to alter host physiology and subvert the host immune response, allowing it to persist for the life of the host. H. pylori is the primary cause of peptic ulcers and gastric cancer. In the United States, the annual cost associated with peptic ulcer disease is estimated to be $6 billion and gastric cancer kills over 700000 people per year globally. The prevalence of H. pylori infection remains high (> 50%) in much of the world, although the infection rates are dropping in some developed nations. The drop in H. pylori prevalence could be a double-edged sword, reducing the incidence of gastric diseases while increasing the risk of allergies and esophageal diseases. The list of diseases potentially caused by H. pylori continues to grow; however, mechanistic explanations of how H. pylori could contribute to extragastric diseases lag far behind clinical studies. A number of host factors and H. pylori virulence factors act in concert to determine which individuals are at the highest risk of disease. These include bacterial cytotoxins and polymorphisms in host genes responsible for directing the immune response. This review discusses the latest advances in H. pylori pathogenesis, diagnosis, and treatment. Up-to-date information on correlations between H. pylori and extragastric diseases is also provided.

Antibiotic treatment decreases microbial burden associated with pseudomyxoma peritonei and affects ß-catenin distribution.

PURPOSE: Pseudomyxoma peritonei is an understudied cancer in which an appendiceal neoplasm invades the peritoneum and forms tumor foci on abdominal organs. Previous studies have shown that bacteria reside within pseudomyxoma peritonei tumors and mucin. Thus, we sought to analyze the effect of antibiotics on bacterial density and ß-catenin expression within pseudomyxoma peritonei samples. EXPERIMENTAL DESIGN: The study included 48 patients: 19 with disseminated peritoneal adenomucinosis (DPAM) and 29 with peritoneal mucinous carcinomatosis (PMCA). Fourteen patients were given antibiotics (30 mg lansoprazole, 1 g amoxicillin, and 500 mg clarithromycin) twice a day for 14 days. One week after completion of therapy, surgery was conducted and specimens were harvested for pathology, bacterial culture, ISH, and immunohistochemistry. RESULTS: ISH showed the presence of bacteria in 83% of the patient samples, with a higher Helicobacter pylori density observed in PMCA versus DPAM. PMCA patients treated with antibiotics had a significantly lower bacterial density and decreased ß-catenin levels in the cytoplasm, the cell nuclei, and mucin-associated cells. Although not significant, similar trends were observed in DPAM patients. Cell membrane ß-catenin was significantly increased in both DPAM and PMCA patients receiving antibiotics. CONCLUSIONS: Bacteria play an important role in pseudomyxoma peritonei. Antibiotic treatment improved the histopathology of tissue, particularly in PMCA patients. In PMCA, antibiotics decreased bacterial density and were associated with a significant ß-catenin decrease in the cytoplasm, cell nuclei, and mucin along with a small membrane increase. These results suggest that antibiotics offer potential protection against cell detachment, cellular invasion, and metastasis.

A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei.

BACKGROUND: Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. METHODS: To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. MAIN RESULTS: We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. CONCLUSIONS: Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.

Helicobacter pylori arginase mutant colonizes arginase II knockout mice.

AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H. pylori). METHODS: H. pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase II, since this enzyme is expressed in the stomach, and H. pylori has previously been shown to induce the expression of murine gastric arginase II. To test this hypothesis, wild-type and arginase (rocF) mutant H. pylori strain SS1 were inoculated into arginase II knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase II knockout mice. Moreover, feeding arginase II knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase I activity in several tissues, did not block the ability of the rocF mutant H. pylori to colonize. In contrast, BEC poorly inhibited H. pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. pylori urease is neither bacterial arginase nor host arginase II; instead, either residual host arginase I or agmatinase is probably responsible.