Isolation of Outer Membrane Vesicles from Helicobacter pylori.

Outer membrane vesicles (OMV) shed by pathogenic bacteria have multifunctional roles in disease initiation and progression. Further, their efficacy as novel vaccines has underscored their importance as potential therapeutics. Consequently, to advance allied research related to their immunogenicity and pathogenicity it is important to separate these vesicular structures from parental cells and demonstrate them to be free from cellular debris and other non-vesicle-related constituents such as protein aggregates. To do so represents a key step in initiating OMV-related studies and the techniques and strategies adopted by the H. pylori community to achieve this will be the focus of this chapter.The key methods used typically to obtain a heterogeneous mixture of OMV (size range: ~20-300 nm in diameter) include growth of bacteria in broth culture followed by differential centrifugation, filtration, and concentration to separate OMV from the intact organisms. Additional measures may be adopted to further size-fractionate the population of OMV including gel filtration or density gradient ultra-centrifugation in order to facilitate differentiation between the activities of small versus large OMV, as recent studies have demonstrated differential modes of entry into host cells as well as size-dependent differences in the OMV proteome (Turner et al., Front Immunol 9:1466, 2018). The OMV from H. pylori harbor many of the virulence factors associated with gastric disease including the CagA oncoprotein, the cytotoxin VacA, and the HtrA protease (Olofsson et al., mBio 5:e00979-14, 2014; Mullaney et al., Proteomics Clin Appl 3:785-96, 2009) and their close association with areas of cell-cell contact and efficient endocytosis supports a role for these complexes in gastric disease (Turkina et al., FEMS Microbiol Lett 362:fnv076, 2015).

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear. RESULTS: We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS. CONCLUSIONS: These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

Etiological Role and Repeated Infections of Sapovirus among Children Aged Less than 2 Years in a Cohort Study in a Peri-urban Community of Peru.

Human sapovirus has been shown to be one of the most important etiologies in pediatric patients with acute diarrhea. However, very limited data are available about the causative roles and epidemiology of sapovirus in community settings. A nested matched case-control study within a birth cohort study of acute diarrhea in a peri-urban community in Peru from 2007 to 2010 was conducted to investigate the attributable fraction (AF) and genetic diversity of sapovirus. By quantitative reverse transcription-real-time PCR (qPCR) sapovirus was detected in 12.4% (37/299) of diarrheal and 5.7% (17/300) of nondiarrheal stools (P = 0.004). The sapovirus AF (7.1%) was higher in the second year (13.2%) than in the first year (1.4%) of life of children. Ten known genotypes and one novel cluster (n = 5) within four genogroups (GI, GII, GIV, and GV) were identified by phylogenetic analysis of a partial VP1 gene. Further sequence analysis of the full VP1 gene revealed a possible novel genotype, tentatively named GII.8. Notably, symptomatic reinfections with different genotypes within the same (n = 3) or different (n = 5) genogroups were observed in eight children. Sapovirus exhibited a high attributable burden for acute gastroenteritis, especially in the second year of life, of children in a Peruvian community. Further large-scale studies are needed to understand better the global burden, genetic diversity, and repeated infections of sapovirus.

Bismuth coordination networks containing deferiprone: synthesis, characterisation, stability and antibacterial activity.

A series of bismuth-dicarboxylate-deferiprone coordination networks have been prepared and structurally characterised. The new compounds have been demonstrated to release the iron overload drug deferiprone on treatment with PBS and have also been shown to have antibacterial activity against H. pylori.

First detected Helicobacter pylori infection in infancy modifies the association between diarrheal disease and childhood growth in Peru.

BACKGROUND: In endemic settings, Helicobacter pylori infection can occur shortly after birth and may be associated with a reduction in childhood growth. MATERIALS AND METHODS: This study investigated what factors promote earlier age of first H. pylori infection and evaluated the role of H. pylori infection in infancy (6-11 months) versus early childhood (12-23 months) on height. We included 183 children near birth from a peri-urban shanty town outside of Lima, Peru. Field-workers collected data on socioeconomic status (SES), daily diarrheal and breast-feeding history, antibiotic use, anthropometrics, and H. pylori status via carbon 13-labeled urea breath test up to 24 months after birth. We used a proportional hazards model to assess risk factors for earlier age at first detected infection and linear mixed-effects models to evaluate the association of first detected H. pylori infection during infancy on attained height. RESULTS: One hundred and forty (77%) were infected before 12 months of age. Lower SES was associated with earlier age at first detected H. pylori infection (low vs middle-to-high SES Hazard ratio (HR) 1.59, 95% CI 1.16, 2.19; p = .004), and greater exclusive breast-feeding was associated with reduced likelihood (HR 0.63, 95% CI 0.40, 0.98, p = .04). H. pylori infection in infancy was not independently associated with growth deficits (p = .58). However, children who had their first detected H. pylori infection in infancy (6-11 months) versus early childhood (12-23 months) and who had an average number of diarrhea episodes per year (3.4) were significantly shorter at 24 months (-0.37 cm, 95% CI, -0.60, -0.15 cm; p = .001). DISCUSSION: Lower SES was associated with a higher risk of first detected H. pylori infection during infancy, which in turn augmented the adverse association of diarrheal disease on linear growth.

Surface layer proteins isolated from Clostridium difficile induce clearance responses in macrophages.

Clostridium difficile is the leading cause of hospital-acquired diarrhoea worldwide, and if the bacterium is not cleared effectively it can pose a risk of recurrent infections and complications such as colitis, sepsis and death. In this study we demonstrate that surface layer proteins from the one of the most frequently acquired strains of C. difficile, activate mechanisms in murine macrophage in vitro that are associated with clearance of bacterial infection. Surface layer proteins (SLPs) isolated from C. difficile induced the production of pro-inflammatory cytokines and chemokines and increased macrophage migration and phagocytotic activity in vitro. Furthermore, we also observed up-regulation of a number of cell surface markers on the macrophage, which are important in pathogen recognition and antigen presentation. The effects of SLPs on macrophages were reversed in the presence of a p38 inhibitor, indicating the potential importance of this signalling protein in how SLP activates the immune system. In conclusion this study shows that surface layer proteins from a common strain of C. difficile can activate a clearance response in macrophage and suggests that these proteins are important in clearance of C. difficile infection. Understanding how the immune system clears C. difficile infection could offer important insights for new treatment strategies.

Iron status and Helicobacter pylori infection in symptomatic children: an international multi-centered study.

OBJECTIVE: Iron deficiency (ID) and iron deficiency anaemia (IDA) are global major public health problems, particularly in developing countries. Whilst an association between H. pylori infection and ID/IDA has been proposed in the literature, currently there is no consensus. We studied the effects of H. pylori infection on ID/IDA in a cohort of children undergoing upper gastrointestinal endoscopy for upper abdominal pain in two developing and one developed country. METHODS: In total 311 children (mean age 10.7±3.2 years) from Latin America--Belo Horizonte/Brazil (n = 125), Santiago/Chile (n = 105)--and London/UK (n = 81), were studied. Gastric and duodenal biopsies were obtained for evaluation of histology and H. pylori status and blood samples for parameters of ID/IDA. RESULTS: The prevalence of H. pylori infection was 27.7% being significantly higher (p<0.001) in Latin America (35%) than in UK (7%). Multiple linear regression models revealed H. pylori infection as a significant predictor of low ferritin and haemoglobin concentrations in children from Latin-America. A negative correlation was observed between MCV (r = -0.26; p = 0.01) and MCH (r = -0.27; p = 0.01) values and the degree of antral chronic inflammation, and between MCH and the degree of corpus chronic (r = -0.29, p = 0.008) and active (r = -0.27, p = 0.002) inflammation. CONCLUSIONS: This study demonstrates that H. pylori infection in children influences the serum ferritin and haemoglobin concentrations, markers of early depletion of iron stores and anaemia respectively.

A short chain NAD(H)-dependent alcohol dehydrogenase (HpSCADH) from Helicobacter pylori: a role in growth under neutral and acidic conditions.

Toxic aldehydes produced by alcohol dehydrogenases have been implicated in the pathogenesis of Helicobacter pylori-related damage to the gastric mucosa. Despite this, the enzymes that might be responsible for producing such aldehydes have not been fully described. It was, therefore, of considerable interest to characterize the alcohol oxidizing enzymes in this pathogen. Previous work in this laboratory characterized two such H. pylori enzymes that had broad specificity for a range of aromatic alcohol substrates. However, an enzyme with specificity for aliphatic alcohols is likely to be required in order that H. pylori can metabolize the wide range of substrates encountered in the gastric mucosa. In this study we describe HpSCADH, an alcohol dehydrogenase from H. pylori 26695 with broad specificity for aliphatic alcohols. HpSCADH was classified in the cD1e subfamily of classical short chain alcohol dehydrogenases. The enzyme was a monomer of approximately 29kDa with a preference for NAD(+) as cofactor. Pyrazole was found to be a competitive inhibitor of HpSCADH. The physiological role of this enzyme was explored by construction of an HpSCADH isogenic mutant. At pH 7.0 the mutant showed reduced growth which became more pronounced when the pH was lowered to 5.0. When pyrazole was added to wild type H. pylori cells it caused growth profiles to be reduced to match those of the isogenic mutant suggesting that HpSCADH inhibition alone was responsible for growth impairment. Taken together, the data relating to the alcohol metabolizing enzymes of this pathogen indicate that they play an important role in H. pylori growth and adaptation to acidic environments. The therapeutic potential of targeting H. pylori alcohol dehydrogenases is discussed.

A nitrophenyl-based prodrug type for colorectal targeting of prednisolone, budesonide and celecoxib.

Celecoxib is a COX-2 inhibitor drug that can be used to reduce the risk of colorectal adenocarcinoma. Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to the use of both drug types is that they undergo absorption from the intestinal tract with serious side effects. The prodrug systems introduced here involve forming a nitro-substituted acylsulfonamide group in the case of celecoxib and a nitro-substituted 21-ester for the glucocorticoids. Drug release is triggered by the nitro reductase action of the colonic microflora, liberating a cyclization competent species. The release of the active parent drugs was evaluated in vitro using Clostridium perfringens and epithelial transport through Caco-2 monolayer evaluation was carried out to estimate the absorption properties of the prodrugs compared to the parental drugs.

Helicobacter pylori-associated hypochlorhydria in children, and development of iron deficiency.

AIMS: Acute Helicobacter pylori infection is associated with transient hypochlorhydria. In H pylori-associated atrophy, hypochlorhydria has a role in iron deficiency (ID) through changes in the physiology of iron-complex absorption. The aims were to evaluate the association between H pylori-associated hypochlorhydria and ID in children. METHODS: Symptomatic children (n=123) were prospectively enrolled. Blood, gastric juice and gastric biopsies were taken, respectively, for haematological analyses, pH assessment and H pylori determination, and duodenal biopsies for exclusion of coeliac disease. Stool samples were collected for parasitology/microbiology. Thirteen children were excluded following parasitology and duodenal histopathology, and five due to impaired blood analysis. RESULTS: Ten children were hypochlorhydric (pH>4) and 33 were H pylori positive. In H pylori-positive children with pH>4 (n=6) serum iron and transferrin saturation levels % were significantly lower (p<0.01) than H pylori-positive children with pH≤4. No differences in ferritin, or total iron binding capacity, were observed. In H pylori-negative children with pH>4, iron and transferrin saturation were not significantly different from children with pH≤4. CONCLUSIONS: Low serum iron and transferrin in childhood H pylori infection is associated with hypochlorhydria. In uninfected children, hypochlorhydria was not associated with altered serum iron parameters, indicating a combination of H pylori infection and/or inflammation, and hypochlorhydria has a role in the aetiology of ID. Although H pylori-associated hypochlorhydria is transient during acute gastritis, this alters iron homeostasis with clinical impact in developing countries with a high H pylori prevalence.

Structure requirements for anaerobe processing of azo compounds: implications for prodrug design.

This Letter generalizes the metabolism of the azo class of compounds by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analogue did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoreduction across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compound. Azo compounds with an electron donating group on the azo-containing aromatic ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compounds containing only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compound. This incomplete reduction is relevant to the design of azo-based prodrugs and the toxicology of azo-based dyes.

Azo-reductase activated budesodine prodrugs for colon targeting.

Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis.

Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection.

A double prodrug system for colon targeting of benzenesulfonamide COX-2 inhibitors.

The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.

Investigation into drug release from colon-specific azoreductase-activated steroid prodrugs using in-vitro models.

OBJECTIVES: The aim of this study was to investigate drug release from a double steroid prodrug, OPN501, which incorporates a phenylpropionate linker, and its phenylacetate analogue. The prodrugs, which were designed to deliver prednisolone to the colon for the treatment of inflammatory bowel disease, are based on a novel design that requires sequential azoreductase activity and cyclization of an amino ester to trigger drug release. We sought to explain the divergent effects of the two compounds in anti-inflammatory models and to justify the selection of OPN-501 for clinical development. METHODS: The compounds were incubated in mouse colonic contents (10%) fermented in brain heart infusion under anaerobic conditions. The disappearance of the prodrugs and release of prednisolone was monitored by HPLC. We then developed a method for assessment of prodrug activation using suspensions of Clostridium perfringens, an anaerobe from the human colon. The cyclization of the compounds was studied in various media, assessing the influence of pH and bulk solvent polarity on cyclization rate using HPLC and NMR. KEY FINDINGS: The prodrugs were activated via multiple pathways releasing prednisolone in mouse colonic ferment. The compounds released prednisolone by reduction-cyclization in C perfringens suspension. The active OPN-501 generated a stoichiometric amount of prednisolone following azoreductase activation, whereas its analogue did not. The pH rate profile for the cyclization of the amino intermediates of the two compounds revealed significant differences in rate at pH values relevant to the inflamed colon, which explain in part the different amounts of drug produced. CONCLUSIONS: The steroid prodrug OPN-501 has optimal drug release characteristics for colon targeting because of a kinetic advantage of a six-membered ring formation in the aminolysis reactions of anilides. The results are relevant to the development of OPN-501 but also to cyclization strategies in prodrug design especially for colon targeting.

Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.

Helicobacter pylori causes chronic gastritis, peptic ulcers, and gastric carcinoma. Gastric epithelial cells provide the first point of contact between H. pylori and the host. TLRs present on these cells recognize various microbial products, resulting in the initiation of innate immunity. Although previous reports investigated TLR signaling in response to intact H. pylori, the specific contribution of H. pylori LPS with regard to functional genomics and cell-signaling events has not been defined. This study set out to define downstream signaling components and altered gene expression triggered by H. pylori LPS and to investigate the role of the signaling protein tribbles 3 (TRIB3) during the TLR-mediated response to H. pylori LPS. Cotransfections using small interfering RNA and dominant-negative constructs demonstrated that H. pylori LPS functions as a classic TLR2 ligand by signaling through pathways involving the key TLR signaling components MyD88 adaptor-like, MyD88, IRAK1, IRAK4, TNFR-associated factor 6, IκB kinase ß, and IκBα. Microarray analysis, real-time PCR, and ELISA revealed the induction of a discrete pattern of chemokines as a direct effect of LPS:TLR2 signaling. H. pylori infection was associated with decreased expression of TRIB3 in human gastric epithelial cell lines and tissue samples. Additionally, H. pylori decreased expression of C/EBP homologous protein and activating transcription factor 4, the transcription factors involved in the induction of TRIB3 expression. Furthermore, knockdown of TRIB3 and C/EBP homologous protein enhanced TLR2-mediated NF-κB activation and chemokine induction in response to H. pylori LPS. Thus, modulation of TRIB3 by H. pylori and/or its products may be an important mechanism during H. pylori-associated pathogenesis.

Proteomics of bacterial pathogenicity: therapeutic implications.

Identification of the molecular mechanisms of host-pathogen interaction is becoming a key focus of proteomics. Analysis of these interactions holds promise for significant developments in the identification of new therapeutic strategies to combat infectious diseases, a process that will also benefit parallel improvements in molecular diagnostics, biomarker identification and drug discovery. This review highlights recent advances in functional proteomics initiatives in infectious disease with emphasis on studies undertaken within physiologically relevant parameters that enable identification of the infectious proteome rather than that of the vegetative state. Deciphering the molecular details of what constitutes physiologically relevant host-pathogen interactions remains an underdeveloped aspect of research into infectious disease. The magnitude of this deficit will be largely influenced by the ease with which model systems can be established to investigate such interactions. As the selective pressures exerted by the host on an infecting pathogen are numerous, the adequacy of certain model systems should be considered carefully.

A new mechanism of gastric epithelial injury induced by acid exposure: the role of Egr-1 and ERK signaling pathways.

The molecular mechanisms by which gastric acid causes epithelial injury in the stomach and initiates an inflammatory reaction are poorly understood. We aimed in the present study to investigate the role of the early growth response gene Egr-1 and ERK in gastric epithelial cells following acid exposure, and the signaling pathways involved. Western blotting was used to assess Egr-1 protein levels in AGS cells. A quantitative measurement of acid-induced Egr-1 and ERK translocation was performed using a high content analysis approach. Egr-1 functionality was assessed by transient transfection with Egr-1 antisense oligonucleotide. Exposure of AGS cells to acidic conditions induced Egr-1 protein expression in a pH- and time-dependent manner. Egr-1 expression was markedly increased as the pH was reduced from pH 7.4 to 6.4. High content analysis of Egr-1 activation showed acid-induced Egr-1 nuclear translocation; a maximum observed at 1-2 h followed by a decline to basal levels beyond 4 h. Acidic pH also activated ERK1/2 phosphorylation, whereas ERK1/2 inhibitors PD98059 and U0216 blocked both acid-induced Egr-1 and ERK translocation and expression. Moreover, acid exposure up-regulated VEGF expression, which was inhibited by the Egr-1 antisense oligonucleotide. Our results also demonstrate that exposure to acid induces Egr-1 via MEK-ERK1/2 pathway. These data suggest that Egr-1 activation might play a crucial role in gastric mucosal inflammation and associated epithelial injury.

Design, synthesis, and pharmacological effects of a cyclization-activated steroid prodrug for colon targeting in inflammatory bowel disease.

Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to their use is that they undergo absorption from the GIT before reaching the colon causing severe systemic side effects. We report here on a novel prodrug approach to targeting corticosteroids to the colon. The design involves attaching a 21-ester group that suppresses absorption during transit to the colon. The prodrug is designed to be primed by colonic microflora liberating an amino ester that cyclizes releasing the steroid. One of the prodrugs 5b was as efficacious as prednisolone in the murine DSS model but did not cause thymic atrophy, a marker for systemic steroid effects.