ORCID Profile
0000-0001-6817-6211
Current Organisations
Monash University
,
Hudson Institute of Medical Research
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Main Group Metal Chemistry | Inorganic Chemistry | Microbiology | Bacteriology | Infectious Agents | Organometallic Chemistry | Host-Parasite Interactions | Functional Materials | Bioinorganic Chemistry | Innate Immunity | Biochemistry and Cell Biology not elsewhere classified |
Expanding Knowledge in the Chemical Sciences | Infectious Diseases | Human Biological Preventatives (e.g. Vaccines) | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences
Publisher: Microbiology Society
Date: 1991
Abstract: Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of c ylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true c ylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.
Publisher: Hindawi Limited
Date: 03-2010
DOI: 10.1111/J.1462-5822.2009.01404.X
Abstract: Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.
Publisher: Microbiology Society
Date: 09-1988
Abstract: The urease enzyme of C ylobacter pylori was studied and compared with that of a related spiral-shaped bacterium, St1, isolated from the rodent ileum. Both bacteria possessed constitutive urease enzymes with activities up to 20-70 times that of Proteus vulgaris. This activity was retained on SDS-polyacrylamide gels. A major catalytic subunit of mol. wt 300,000 was located for all (six) strains of C. pylori subjected to SDS-PAGE whereas St1 had two active forms of mol. wts 140,000 and 150,000. Western-blot analysis indicated the presence of anti-urease antibodies in the sera of patients with C. pylori-associated gastritis. The response to C. pylori urease was not strain-specific but no cross-reactivity was detected between the C. pylori enzyme and that of St1. The very high urease activity of these bacteria is likely to be important in colonisation of the host. Possession of glutamate dehydrogenase activity by both organisms suggests that one role of the urease may be to assimilate the available urea nitrogen. Modification of the local environment to facilitate long-term colonisation is another possible function. Protection from acid is unlikely to be a primary role as the natural habitat of the organism St1 is the non-acid-secreting tissue of the small intestine.
Publisher: Wiley
Date: 03-1999
DOI: 10.1046/J.1365-2958.1999.01271.X
Abstract: Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.
Publisher: Hindawi Limited
Date: 21-11-2013
DOI: 10.1111/CMI.12055
Abstract: The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin-8 (IL-8) independently of CagA translocation and peptidoglycan-sensing nucleotide-binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL-8 secretion, requiring activation of α5 β1 integrin and the arginine-glycine-aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine-glycine-alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL-8. Comparison of IL-8 induction between H. pylori ΔvirD4 strains bearing wild-type or mutant cagL indicates that CagL-dependent IL-8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF-κB and inducing IL-8 without restoring CagA translocation. The CagA translocation-independent, CagL-dependent IL-8 induction involved host signalling via integrin α5 β1 , Src kinase, the mitogen-activated protein kinase (MAPK) pathway and NF-κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro-inflammatory responses independently of CagA translocation or NOD1 signalling.
Publisher: Public Library of Science (PLoS)
Date: 04-2010
Publisher: Wiley
Date: 09-02-2015
DOI: 10.1111/HEL.12196
Abstract: Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes.
Publisher: Frontiers Media SA
Date: 02-07-2018
Publisher: Elsevier BV
Date: 11-1998
DOI: 10.1016/S0167-4838(98)00207-6
Abstract: The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.
Publisher: Hindawi Limited
Date: 05-2010
DOI: 10.1111/J.1462-5822.2009.01421.X
Abstract: The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI(+)H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human beta-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI(+) bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAI-dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI(+)H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human beta-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI(+)H. pylori infection.
Publisher: Wiley
Date: 19-02-2018
DOI: 10.1002/IJC.31298
Publisher: Wiley
Date: 2000
DOI: 10.1002/1096-9896(2000)9999:9999<::AID-PATH619>3.0.CO;2-H
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-62703-005-2_20
Abstract: The human pathogen Helicobacter pylori causes inflammation in the stomach of infected hosts, leading in some cases to the development of gastric cancer. Several mouse models have been developed to study Helicobacter-induced carcinogenesis with similarities to gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (MALToma) in humans. These models require chronic infection of animals with mouse-colonizing isolates of H. pylori or with related gastric Helicobacter spp., such as the canine/feline species Helicobacter felis. Furthermore, consistent with the known influence of host and environmental factors in human gastric cancer, it is possible to manipulate the type and severity of gastric lesions in mouse Helicobacter infection models through the use of different mouse genetic backgrounds and/or by the administration of known cocarcinogens, such as alkylating agents (e.g., N-nitroso-N-methylurea), or even elevated quantities of dietary salt. Here, we describe protocols for the inoculation of mice with gastric Helicobacter spp. and the administration of these cocarcinogens. Furthermore, we will describe the various methodologies used to study gastric inflammation and carcinogenesis in Helicobacter-infected animals.
Publisher: Springer Science and Business Media LLC
Date: 15-05-2015
DOI: 10.1038/NRI3837
Abstract: Gram-negative bacteria shed extracellular outer membrane vesicles (OMVs) during their normal growth both in vitro and in vivo. OMVs are spherical, bilayered membrane nanostructures that contain many components found within the parent bacterium. Until recently, OMVs were dismissed as a by-product of bacterial growth however, findings within the past decade have revealed that both pathogenic and commensal bacteria can use OMVs to manipulate the host immune response. In this Review, we describe the mechanisms through which OMVs induce host pathology or immune tolerance, and we discuss the development of OMVs as innovative nanotechnologies.
Publisher: Frontiers Media SA
Date: 19-03-2018
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C3DT52544A
Abstract: Nine different β-thioxoketones of general formula R(1)C(=O)CH2C(=S)R(2) (R(1) = C6H5, R(2) = C6H5L1 R(1) = C6H5, R(2) = p-CF3C6H4L2 R(1) = p-MeOC6H4, R(2) = C6H5L3 R(1) = p-MeOC6H4, R(2) = p-CF3C6H4L4 R(1) = C5H4N, R(2) = C6H5L5 R(1) = p-IC6H4, R(2) = C6H5L6 R(1) = C6H5, R(2) = p-IC6H4L7 R(1) = C6H5, R(2) = C10H7L8 and R(1) = CH3, R(2) = C6H5L9) and their tris-substituted bismuth(III) complexes having the general formula [Bi{R(1)C(=O)CHC(=S)R(2)}3] were synthesised and fully characterised. The solid state structure of [Bi{C5H4NC(=O)CHC(=S)C6H5}3] B5 was determined by crystallography and revealed that the three β-thioxoketonato ligands are bound to bismuth(III) centre in a bidentate fashion through O and S atoms. The bismuth(III) complexes and the corresponding thioxoketones were assessed for their activity against H. pylori. All of the bismuth(III) complexes were highly active against H. pylori having a MIC of greater than or equal to 3.125 μg mL(-1), while the free acids were essentially not toxic to the bacteria. The anti-leishmanial activity of all the bismuth(III) β-thioxoketonates and the corresponding free acids were assessed against L. major promastigotes. The toxicity towards human fibroblast cells was also assessed. All of the free β-thioxoketones were selectively toxic to the L. major promastigotes displaying some potential as anti-leishmanial agents. Among these [C6H5C(=O)CH2C(=S)C6H5] L1 and [C5H4NC(=O)CH2C(=S)C6H5] L5 showed comparable activity to that of Amphotericin B, killing about 80% of the L. major promastigotes at a concentration of 25 μM (6.0 μg mL(-1)). The bismuth(III) β-thioxoketonate complexes were toxic to both the L. major promastigotes and fibroblast cells at high concentrations, but gave no improvement in anti-leishmanial activity over the free β-thioxoketones.
Publisher: Elsevier BV
Date: 07-2020
Publisher: Elsevier BV
Date: 1995
Publisher: Public Library of Science (PLoS)
Date: 29-04-2009
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S0016-5085(97)70094-5
Abstract: Orogastric immunization of mice with Helicobacter antigens, together with a mucosal adjuvant (cholera toxin), has been shown to confer immunity in the Helicobacter felis infection model. The aim of the study was to investigate the humoral immune responses associated with immunity and to compare these with responses in H. felis-infected mice. Enzyme-linked immunoassays were used to characterize the antibody-secreting cells and antibodies present at mucosal and systemic sites in mice. Animals were immunized orally with either whole-cell H. felis sonicates or Helicobacter pylon urease or heat-shock proteins. Infection of mice with H. felis preferentially induced the recruitment of plasma cells committed to immunoglobulin (Ig) A synthesis in salivary gland and gastric tissues. Antigen-specific IgA was the major antibody class detected in mucosal secretions recovered from these tissues. In contrast, immunization of mice against H. felis infection induced the proliferation of large numbers of IgG-secreting cells, as well as the synthesis of local IgG antibodies, in the gastric mucosa of the animals. Protection against H. felis infection occurred in the absence of gastric IgA responses in sonicate-immunized mice. It is proposed that locally synthesized specific IgG antibodies contribute to immunity against gastric Helicobacter infection.
Publisher: American Society for Microbiology
Date: 04-1992
DOI: 10.1128/JB.174.8.2466-2473.1992
Abstract: Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into C ylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.
Publisher: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.JSBMB.2016.07.005
Abstract: Des-acyl ghrelin is the unacylated form of the well-characterized appetite-stimulating hormone ghrelin. It affects a number of physiological processes, including increasing adipose lipid accumulation and inhibiting adipose tissue inflammation. Breast adipose tissue inflammation in obesity is associated with an increase in the expression of the estrogen biosynthetic enzyme, aromatase, and is hypothesized to create a hormonal milieu conducive to tumor growth. We previously reported that des-acyl ghrelin inhibits the expression and activity of aromatase in isolated human adipose stromal cells (ASCs), the main site of aromatase expression in the adipose tissue. The current study aimed to examine the effect of des-acyl ghrelin on the capacity of mouse macrophages (RAW264.7 cells) and human adipose tissue macrophages (ATMs) to stimulate aromatase expression in primary human breast ASCs. RAW264.7 cells were treated with 0, 10 and 100pM des-acyl ghrelin following activation with phorbol 12-myristate 13-acetate, and cells and conditioned media were collected after 6 and 24h. The effect of des-acyl ghrelin on macrophage polarization was examined by assessing mRNA expression of pro-inflammatory M1-specific marker Cd11c and anti-inflammatory M2-specific marker Cd206, as well as expression of Tnf and Ptgs2, known mediators of the macrophage-dependent stimulation of aromatase. TNF protein in conditioned media was assessed by ELISA. The effect of RAW264.7 and ATM-conditioned media on aromatase expression in ASCs was assessed after 6h. Results demonstrate des-acyl ghrelin significantly increases the expression of Cd206 and suppresses the expression of Cd11c, Tnf and Ptgs2 in activated RAW264.7 cells. Treatment of RAW264.7 and ATMs with des-acyl ghrelin also significantly reduces the capacity of these cells to stimulate aromatase transcript expression in human breast ASCs. Overall, these findings suggest that in addition to direct effects on aromatase in ASCs, des-acyl ghrelin also has the capacity to inhibit the macrophage-dependent induction of aromatase, and provides a novel mechanism for potential effects of des-acyl ghrelin to break the linkage between obesity and breast cancer.
Publisher: Wiley
Date: 12-1994
DOI: 10.1111/J.1365-2958.1994.TB01331.X
Abstract: All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-7568-6_10
Abstract: The gastrointestinal epithelium provides the first line of defense against invading pathogens, among which Helicobacter pylori is linked to numerous gastric pathologies, including chronic gastritis and cancer. Primary gastric epithelial cells represent a useful model for the investigation of the underlying molecular and cellular mechanisms involved in these H. pylori associated diseases. In this chapter, we describe a method for the isolation of primary gastric epithelial cells from mice and detection of epithelial cell adhesion molecule (EpCAM) expression in the isolated cells.
Publisher: Informa UK Limited
Date: 1991
Publisher: American Society for Microbiology
Date: 31-08-2010
Abstract: Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori , we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B 6 biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B 6 biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B 6 is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori . Thus, for the first time, we identify vitamin B 6 biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens. IMPORTANCE Approximately half of the world’s population is infected with H. pylori , yet how H. pylori bacteria establish chronic infections in human hosts remains elusive. From gene array studies, we identified two genes as representing potentially novel colonization factors for H. pylori . These genes encoded enzymes involved in the synthesis of vitamin B 6 , an important molecule for many metabolic reactions in living organisms. Little is currently known regarding vitamin B 6 biosynthesis in human pathogens. We showed that mutant H. pylori bacteria lacking an enzyme involved in de novo vitamin B 6 biosynthesis, PdxA, were unable to synthesize motility appendages (flagella) and were unable to establish chronic colonization in mice. Thus, this work identifies vitamin B 6 biosynthesis enzymes as novel virulence factors for bacterial pathogens. Interestingly, a number of human pathogens, but not their mammalian hosts, possess these genes, which suggests that Pdx enzymes may represent ideal candidates for new therapeutic targets.
Publisher: Springer Science and Business Media LLC
Date: 21-11-2016
Publisher: Public Library of Science (PLoS)
Date: 15-10-2013
Publisher: Cold Spring Harbor Laboratory
Date: 24-03-2019
DOI: 10.1101/587212
Abstract: The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been extensively studied, those involved in IL-18 processing, particularly in non-myeloid cells, are still poorly understood. Here, we have identified the cytosolic sensor NOD1 as a key regulator of IL-18 processing in epithelial cells responding to Helicobacter pylori infection. Importantly, NOD1 processing of IL-18 occurs independently of the canonical inflammasome proteins, NLRP3 and ASC. Instead, NOD1 interacts directly with caspase-1 via homotypic binding of caspase-activation recruitment domains. We show that IL-18 is important in maintaining tissue homeostasis and protecting against pre-neoplastic changes due to gastric H. pylori infection. These findings reveal an unanticipated role for NOD1 in a new type of inflammasome that regulates epithelial cell production of bioactive IL-18 with tissue protective functions.
Publisher: Wiley
Date: 04-2002
Publisher: Springer Berlin Heidelberg
Date: 1994
Publisher: Proceedings of the National Academy of Sciences
Date: 03-07-1995
Abstract: Helicobacter pylori is an important etiologic agent of gastroduodenal disease. In common with other organisms, H. pylori bacteria express heat shock proteins that share homologies with the GroES-GroEL class of proteins from Escherichia coli. We have assessed the heat shock proteins of H. pylori as potential protective antigens in a murine model of gastric Helicobacter infection. Orogastric immunization of mice with recombinant H. pylori GroES- and GroEL-like proteins protected 80% (n = 20) and 70% (n = 10) of animals, respectively, from a challenge dose of 10(4) Helicobacter felis bacteria (compared to control mice, P = 0.0042 and P = 0.0904, respectively). All mice (n = 19) that were immunized with a dual antigen preparation, consisting of H. pylori GroES-like protein and the B subunit of H. pylori urease, were protected against infection. This represented a level of protection equivalent to that provided by a sonicated Helicobacter extract (P = 0.955). Antibodies directed against the recombinant H. pylori antigens were predominantly of the IgG1 class, suggesting that a type 2 T-helper cell response was involved in protection. This work reports a protein belonging to the GroES class of heat shock proteins that was shown to induce protective immunity. In conclusion, GroES-like and urease B-subunit proteins have been identified as potential components of a future H. pylori subunit vaccine.
Publisher: MyJove Corporation
Date: 18-10-2018
DOI: 10.3791/56985
Publisher: Springer Science and Business Media LLC
Date: 26-06-2023
DOI: 10.1038/S41467-023-39487-1
Abstract: The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori . Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori -induced pathology.
Publisher: Informa UK Limited
Date: 2019
Publisher: American Society for Microbiology
Date: 07-1992
DOI: 10.1128/JB.174.13.4212-4217.1992
Abstract: Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.
Publisher: American Society for Microbiology
Date: 11-2010
DOI: 10.1128/IAI.00439-10
Abstract: Infection with Helicobacter pylori cag pathogenicity island ( cag PAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cag PAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α 5 β 1 integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α 5 β 1 integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS ( P 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α 5 β 1 integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α 5 β 1 integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori . These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.
Publisher: Wiley
Date: 19-04-2023
DOI: 10.1111/IMCB.12641
Abstract: Helicobacter pylori ( H. pylori ) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori –induced gastric pathology remain ill‐defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenesis of Helicobacter ‐induced gastric disease. Here, we reveal that AIM2 messenger RNA and protein expression levels are elevated in H. pylori –positive versus H. pylori –negative human gastric biopsies. Similarly, chronic Helicobacter felis infection in wild‐type mice augmented Aim2 gene expression levels compared with uninfected controls. Notably, gastric inflammation and hyperplasia were less severe in H. felis –infected Aim2 −/− versus wild‐type mice, evidenced by reductions in gastric immune cell infiltrates, mucosal thickness and proinflammatory cytokine and chemokine release. In addition, H. felis –driven proliferation and apoptosis in both gastric epithelial and immune cells were largely attenuated in Aim2 −/− stomachs. These observations in Aim2 −/− mouse stomachs correlated with decreased levels of inflammasome activity (caspase‐1 cleavage) and the mature inflammasome effector cytokine, interleukin‐1β. Taken together, this work uncovers a pathogenic role for the AIM2 inflammasome in Helicobacter ‐induced gastric disease, and furthers our understanding of the host immune response to a common pathogen and the complex and varying roles of AIM2 at different stages of cancerous and precancerous gastric disease.
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.MICINF.2017.06.005
Abstract: Persistent stomach infection with Helicobacter pylori causes chronic mucosal inflammation (gastritis), which is widely recognized as an essential precursor to gastric cancer. The IL-1 interleukin family cytokines IL-1β and IL-18 have emerged as central mediators of mucosal inflammation. Here, we review the regulation and functions of these cytokines in H. pylori-induced inflammation and carcinogenesis.
Publisher: Wiley
Date: 29-03-2019
DOI: 10.1111/IMCB.12244
Abstract: The NOD-like receptor (NLR) family plays an important role in innate immunity. Class II transactivator and NOD-like receptor caspase activation and recruitment domain CARD containing 5 (NLRC5) are unusual members of the NLR family that instead of recognizing pathogen-associated or damage-associated molecular patterns, form enhanceosomes with adaptor molecules and modulate major histocompatibility complex (MHC) class II and MHC class I expression, respectively. While NLRC5 has been shown to play a role during intracellular pathogen infection and tumor cell immune evasion, its role in regulating antigen-specific CD8
Publisher: The American Association of Immunologists
Date: 15-12-2009
Abstract: Helicobacter pylori rapidly activates MAPKs and transcription factors, NF-κB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largely dependent on the cag pathogenicity island (cagPAI)-encoded Type IV Secretion System. H. pylori was shown to translocate peptidoglycan through the Type IV Secretion System, which is recognized by the pathogen recognition molecule, NOD1, thus resulting in NF-κB activation. The mechanisms of H. pylori-induced MAPK and AP-1 activation, however, are less well defined and therefore, we assessed the contribution of NOD1 to their activation. For this, we used gastric epithelial cell lines, stably expressing siRNA to either NOD1 or a control gene. In siNOD1-expressing cells stimulated with cagPAI+ H. pylori, we observed significant reductions in p38 and ERK phosphorylation (p & 0.05), whereas the levels of Jnk phosphorylation remained unchanged. Consistent with a previous report, however, we were able to demonstrate NOD1-dependent Jnk phosphorylation by the invasive pathogen Shigella flexneri, highlighting pathogen-specific host responses to infection. We also show that NOD1 was essential for H. pylori induction of not only NF-κB, but also AP-1 activation, implying that NOD1 induces robust proinflammatory responses, in an attempt to rapidly control infection. Pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to H. pylori, further emphasizing the importance of MAPKs in innate immune responses to the pathogen. Thus, for the first time we have shown the important role for NOD1 in MAPK and AP-1 activation in response to cagPAI+ H. pylori.
Publisher: Wiley
Date: 27-03-2013
Abstract: The synthesis and characterisation of nine new tris-substituted bismuth(III) aminoarenesulfonates of the general formula [Bi(O3S-R(N))3] (R(N) = o-aminophenyl 1, m-aminophenyl 2, 6-amino-3-methoxyphenyl 3, p-aminophenyl 4, 2-pyridyl 5, o-aminonaphthyl 6, 5-aminonaphthyl 7, 4-amino-3-hydroxynaphthyl 8 and 5-isoquinolinyl 9) is described. Two synthetic strategies, using Ag2O and [Bi(OtBu)3], were explored and compared. The possibility to access heteroleptic bismuth(III) complexes with the new silver(I) metathesis reaction is demonstrated with the synthesis of the heteroleptic bismuth(III) aminoarenesulfonate complexes [PhBi(O3S-P2)2(dmso)] 10, [Ph2Bi(O3S-P2)]∞ 11 and [PhBi(O3S-P2)2]∞ 12, of which the solid state structures 10 and 12 are presented (2P-SO3(-) = 2-pyridinesulfonate). These complexes offer remarkable in-vitro activity against three standard laboratory strains of Helicobacter pylori (H. pylori) as demonstrated by their exceptionally low minimum inhibitory concentration (MIC) values of 0.049 μg mL(-1) for the strains 251 and B128, which places most MIC values in the nano-molar region. These results demonstrate the importance of the amino functionality in addition to the sulfonate group on the bactericidal properties against H. pylori.
Publisher: Wiley
Date: 25-04-2023
DOI: 10.1002/JEX2.84
Abstract: Contaminants within cell culture media often co‐isolate with eukaryotic extracellular vesicles (EVs) thus affecting their biological properties. It has yet to be investigated if this is also true for bacterial EVs (BEVs), especially for organisms grown in complex culture media containing animal‐derived products. To address this question, we isolated BEVs from the fastidious bacterium Helicobacter pylori grown in either standard Brain Heart Infusion (BHI) medium or BHI depleted of animal‐derived products (D‐BHI). We show that BEVs prepared from bacteria grown in D‐BHI medium have similar morphologies, size ranges and yields to those prepared from standard medium. Similarly, no differences were found in the ability of H. pylori BEVs to induce IL‐8 responses in epithelial cells. However, H. pylori BEVs prepared from D‐BHI medium were of higher purity than those prepared from standard medium. Importantly, proteomic analyses detected 3.4‐fold more H. pylori proteins and 10‐fold fewer bovine‐derived proteins in BEV s les prepared from D‐BHI rather than the standard method. Fifty‐seven H. pylori proteins were uniquely detected in BEV s les prepared from D‐BHI. In conclusion, we have described an improved method for BEV isolation. Furthermore, we demonstrate how animal‐derived products in bacteriological culture media may adversely affect proteomic analyses of BEVs.
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.JMB.2010.06.065
Abstract: Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.
Publisher: Wiley
Date: 04-2013
DOI: 10.1111/MMI.12205
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.CHOM.2014.04.001
Abstract: The intracellular innate immune receptor NOD1 detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. To date, the intracellular compartment in which PG is detected by NOD1 and whether NOD1 directly interacts with PG are two questions that remain to be resolved. To address this, we used outer membrane vesicles (OMVs) from pathogenic bacteria as a physiological mechanism to deliver PG into the host cell cytosol. We report that OMVs induced autophagosome formation and inflammatory IL-8 responses in epithelial cells in a NOD1- and RIP2-dependent manner. PG contained within OMVs colocalized with both NOD1 and RIP2 in EEA1-positive early endosomes. Further, we provide evidence for direct interactions between NOD1 and PG. Collectively, these findings demonstrate that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection.
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/C0DT00629G
Abstract: Three bis-phenylbismuth sulfonates [Ph(2)Bi(O(3)SR)](∞) (R = p-tolyl 1, mesityl 2 or S-(+)-10-c horyl 3) have been synthesised and characterised. Their tendency for ligand redistribution in solution, and activity against the bacterium Helicobacter pylori have been investigated. The structures of 2 and 3 have been authenticated by X-ray diffraction crystallography. They are structurally very similar with polymeric helical chain structures composed of four coordinate Bi atoms which bridge between two sulfonate O atoms with near linear O-Bi-O bond angles. The two phenyl rings are cis to one another and trans to the stereochemically active lone pair. Upon dissolution of the pure compounds 1, 2 and 3, a ligand redistribution reaction occurs in which the mono-phenylbismuth bis-sulfonates, the bismuth tris-sulfonates and triphenylbismuth are formed. Two further complexes of general formula [PhBi(O(3)SR)(2)](∞) (where R = p-tolyl 4, and mesityl 5) were thus obtained and their crystal structures determined. The presence of the single sulfonato ligand in compounds 1, 2 and 3 resulted in a dramatic increase in bacteriocidial activity towards H. pylori (MIC values of ≥6.25 μg mL(-1)) relative to BiPh(3) (>64 μg mL(-1)) and the sulfonic acids, which were essentially inactive.
Publisher: American Physiological Society
Date: 07-2017
DOI: 10.1152/PHYSREV.00026.2016
Abstract: Cell surface innate immune receptors can directly detect a variety of extracellular pathogens to which cytoplasmic innate immune sensors are rarely exposed. Instead, within the cytoplasm, the environment is rife with cellular machinery and signaling pathways that are indirectly perturbed by pathogenic microbes to activate intracellular sensors, such as pyrin, NLRP1, NLRP3, or NLRC4. Therefore, subtle changes in key intracellular processes such as phosphorylation, ubiquitination, and other pathways leading to posttranslational protein modification are key determinants of innate immune recognition in the cytoplasm. This concept is critical to establish the “guard hypothesis” whereby otherwise homeostatic pathways that keep innate immune sensors at bay are released in response to alterations in their posttranslational modification status. Originally identified in plants, evidence that a similar guardlike mechanism exists in humans has recently been identified, whereby a mutation that prevents phosphorylation of the innate immune sensor pyrin triggers a dominantly inherited autoinflammatory disease. It is also noteworthy that even when a cytoplasmic innate immune sensor has a direct ligand, such as bacterial peptidoglycan (NOD1 or NOD2), RNA (RIG-I or MDA5), or DNA (cGAS or IFI16), it can still be influenced by posttranslational modification to dramatically alter its response. Therefore, due to their existence in the cytoplasmic milieu, posttranslational modification is a key determinant of intracellular innate immune receptor functionality.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4CC07329K
Abstract: Bismuth( iii ) benzohydroxamates [Bi 2 (HBA) 3 ], [Bi( H -BHA) 3 ], [Bi(HBA)( H -HBA)] and [Bi 34 O 22 (BHA) 22 ( H -BHA) 14 (DMSO) 6 ], all show exceptional toxicity towards Helicobacter pylori (MIC 0.08–3.24 μM).
Publisher: Hindawi Limited
Date: 02-03-2021
DOI: 10.1111/CMI.13320
Abstract: Bacterial pathogens can subvert host responses by producing effector proteins that directly target the nucleus of eukaryotic cells in animals and plants. Nuclear-targeting proteins are categorised as either: "nucleomodulins," which have epigenetic-modulating activities or "cyclomodulins," which specifically interfere with the host cell cycle. Bacteria can deliver these effector proteins to eukaryotic cells via a range of strategies. Despite an increasing number of reports describing the effects of bacterial effector proteins on nuclear processes in host cells, the intracellular pathways used by these proteins to traffic to the nucleus have yet to be fully elucidated. This review will describe current knowledge about how nucleomodulins and cyclomodulins enter eukaryotic cells, exploit endocytic pathways and translocate to the nucleus. We will also discuss the secretion of nuclear-targeting proteins or their release in bacterial membrane vesicles and the trafficking pathways employed by each of these forms. Besides their importance for bacterial pathogenesis, some nuclear-targeting proteins have been implicated in the development of chronic diseases and even cancer. A greater understanding of nuclear-targeting proteins and their actions will provide new insights into the pathogenesis of infectious diseases, as well as contribute to advances in the development of novel therapies against bacterial infections and possibly cancer.
Publisher: Wiley
Date: 27-06-2011
DOI: 10.1002/PATH.2933
Abstract: Chronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by their ability to produce interleukin (IL)-17A (Th17), is associated with a host of inflammatory responses, including gastritis. However, the role of these Th17 cells in the pathogenesis of gastric cancer is less clear. To formally address this, we employed gp130(F/F) mice, which spontaneously develop gastric inflammation-associated tumours akin to human intestinal-type gastric cancer. At the molecular level, these tumours demonstrate hyper-activation of the latent transcription factor signal transducer and activator of transcription (STAT)3 via the IL-6 cytokine family member, IL-11. In gp130(F/F) mice, the generation of Th17 cells, as well as the gastric expression of IL-17a and other Th17-related factors (Rorγt, IL-23), were augmented compared to wild-type gp130(+/+) mice. Consistent with a role for IL-6 and STAT3 in regulating IL-17A, increased Th17 generation and gastric expression of Th17-related factors in gp130(F/F) mice were reduced to wild-type levels in gp130(F/F) :Stat3(-/+) mice displaying normalized STAT3 activity, and also in gp130(F/F) :IL-6(-/-) mice. Importantly, genetic ablation of IL-17A in gp130(F/F) :IL-17a(-/-) mice did not suppress the initiation and growth of gastric tumours. Furthermore, IL-17A and RORC gene expression was strongly increased in human gastric biopsies from patients with gastritis, but not gastric cancer. Collectively, our data suggest that increased expression of Th17-related factors does not correlate with the molecular pathogenesis of gastric tumourigenesis.
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2DT31360J
Abstract: Four new tris-substituted bismuth(III) sulfonates of general formula [Bi(O(3)SR)(3)] (R = phenyl 1, p-tolyl 2, 2,4,6-mesityl 3 and S-(+)-10-c horyl 4) have been synthesised and characterised. Their synthesis by solvent-free (SF) and solvent-mediated (SM) methods has been explored and their activity against Helicobacter pylori has been investigated. The compounds 1-4 display a remarkable in vitro activity against three laboratory strains of H. pylori (B128, 26,695 and 251) with minimum inhibitory concentration (MIC) values as low as 0.049 μg mL(-1) for the strains B128 and 26,695, and 0.781 μg mL(-1) for the clinical isolate 251. This places most MIC values in the nano-molar region and demonstrates the strong influence of the sulfonate group on the bactericidal properties. The novel solid state structure [Bi(8)(O(3)SMes)(20)(SO(4))(2)(H(2)O)(6)]·(C(7)H(8))(7)5·(C(7)H(8))(7), derived from the SM reaction under reflux conditions, is presented and the incorporation of the two inorganic sulfate anions in the centre of the wheel-like bismuth sulfonate cluster explained.
Publisher: Oxford University Press (OUP)
Date: 06-1999
DOI: 10.1093/JAC/43.6.753
Abstract: It was recently demonstrated that inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase, is associated with the development of resistance to metronidazole by Helicobacter pylori. In order to further evaluate the contribution of rdxA to metronidazole resistance, the sequence of the rdxA gene was determined for a series of metronidazole-sensitive and -resistant isolates derived from a single, metronidazole-sensitive strain using an H. pylori mouse model. These strains were cultured from the stomachs of mice experimentally infected with H. pylori strain SS1 and then treated orally with metronidazole. The sequence of the rdxA gene of all 10 sequenced metronidazole-sensitive and two (7%) of the 27 metronidazole-resistant isolates was identical to that of the parental strain. In contrast, the rdxA gene of the other 25 metronidazole-resistant isolates contained between one and three frameshift or missense mutations. This suggests that while the development of metronidazole resistance in H. pylori is frequently associated with mutational inactivation of the rdxA gene, other mechanisms of resistance are likely to exist in this bacterium.
Publisher: Hindawi Limited
Date: 22-07-2010
DOI: 10.1002/HUMU.21321
Abstract: Human Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16(+)CD14(+) differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8-dependent microbicidal response of infected macrophages.
Publisher: Springer Science and Business Media LLC
Date: 17-10-2004
DOI: 10.1038/NI1131
Abstract: Epithelial cells can respond to conserved bacterial products that are internalized after either bacterial invasion or liposome treatment of cells. We report here that the noninvasive Gram-negative pathogen Helicobacter pylori was recognized by epithelial cells via Nod1, an intracellular pathogen-recognition molecule with specificity for Gram-negative peptidoglycan. Nod1 detection of H. pylori depended on the delivery of peptidoglycan to host cells by a bacterial type IV secretion system, encoded by the H. pylori cag pathogenicity island. Consistent with involvement of Nod1 in host defense, Nod1-deficient mice were more susceptible to infection by cag pathogenicity island-positive H. pylori than were wild-type mice. We propose that sensing of H. pylori by Nod1 represents a model for host recognition of noninvasive pathogens.
Publisher: American Society for Microbiology
Date: 02-2008
DOI: 10.1128/IAI.01107-07
Abstract: Nuclear factor κB (NF-κB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-κB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-κB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-κB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-κB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island ( cag PAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-κB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear β-galactosidase activity, which is indicative of specific NF-κB activation. The numbers of β-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cag PAI, compared to naive animals ( P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-κB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori . This work demonstrates for the first time the induction of NF-κB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-κB signaling during chronic infection.
Publisher: Wiley
Date: 27-11-2019
DOI: 10.1113/JP278640
Abstract: Nucleotide binding oligomerization domain (Nod)‐like receptors regulate cognition, anxiety and hypothalamic–pituitary–adrenal axis activation. Nod‐like receptors regulate central and peripheral serotonergic biology. Nod‐like receptors are important for maintenance of gastrointestinal physiology. Intestinal epithelial cell expression of Nod1 receptors regulate behaviour. Gut–brain axis signalling is critical for maintaining health and homeostasis. Stressful life events can impact gut–brain signalling, leading to altered mood, cognition and intestinal dysfunction. In the present study, we identified nucleotide binding oligomerization domain (Nod)‐like receptors (NLR), Nod1 and Nod2, as novel regulators for gut–brain signalling. NLR are innate immune pattern recognition receptors expressed in the gut and brain, and are important in the regulation of gastrointestinal physiology. We found that mice deficient in both Nod1 and Nod2 (NodDKO) demonstrate signs of stress‐induced anxiety, cognitive impairment and depression in the context of a hyperactive hypothalamic–pituitary–adrenal axis. These deficits were coupled with impairments in the serotonergic pathway in the brain, decreased hippoc al cell proliferation and immature neurons, as well as reduced neural activation. In addition, NodDKO mice had increased gastrointestinal permeability and altered serotonin signalling in the gut following exposure to acute stress. Administration of the selective serotonin reuptake inhibitor, fluoxetine, abrogated behavioural impairments and restored serotonin signalling. We also identified that intestinal epithelial cell‐specific deletion of Nod1 (VilCre + Nod1 f/f ), but not Nod2, increased susceptibility to stress‐induced anxiety‐like behaviour and cognitive impairment following exposure to stress. Together, these data suggest that intestinal epithelial NLR are novel modulators of gut–brain communication and may serve as potential novel therapeutic targets for the treatment of gut–brain disorders.
Publisher: Wiley
Date: 03-01-2012
DOI: 10.1111/J.1523-5378.2011.00914.X
Abstract: Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch-to-batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid-rich supplement called AlbuMAX II(®) (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture. The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β-cyclodextrin or AlbuMAX II(®) (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF-κB luciferase assays and IL-8 ELISA. We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II(®) (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II(®) (Gibco BRL) than media containing β-cyclodextrin. Furthermore, bacteria grown in AlbuMAX II(®) (Gibco BRL) induced proinflammatory responses in AGS cells. AlbuMAX II(®) (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II(®) (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.
Publisher: Research Square Platform LLC
Date: 09-09-2020
DOI: 10.21203/RS.3.RS-49701/V1
Abstract: The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been well defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here, we report that the host defence molecule NOD1 regulates IL-18 processing in epithelial cells to the mucosal pathogens, Helicobacter pylori and Pseudomonas aeruginosa . We show that IL-18 is important in protecting against pre-neoplastic changes induced by gastric H. pylori infection in vivo . NOD1 mediates IL-18 processing via homotypic CARD-CARD interactions with caspase-1, and independently of canonical inflammasome proteins (NLRP3, ASC). These findings reveal an unanticipated role for NOD1 in the formation of bioactive IL-18, thereby underlining the differences in inflammasome functions between haematopoietic and non-haematopoietic cells.
Publisher: Wiley
Date: 07-1993
DOI: 10.1111/J.1365-2958.1993.TB01693.X
Abstract: Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.
Publisher: Elsevier BV
Date: 1997
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4DT02505A
Abstract: Bi( iii ) complexes, [BiL 3 ] and [Bi 2 L 3 ], derived from α-amino acids (LH) have been synthesised and characterised. Hydrolysis and oxido-cluster formation in water impacts significantly on their activity towards H. pylori .
Publisher: Elsevier BV
Date: 2004
Publisher: Elsevier BV
Date: 06-2020
Publisher: Wiley
Date: 17-02-2023
DOI: 10.1002/PATH.6053
Abstract: Mucosa‐associated lymphoid tissue (MALT) lymphoma is a B‐cell tumour that develops over many decades in the stomachs of in iduals with chronic Helicobacter pylori infection. We developed a new mouse model of human gastric MALT lymphoma in which mice with a myeloid‐specific deletion of the innate immune molecule, Nlrc5, develop precursor B‐cell lesions to MALT lymphoma at only 3 months post‐ Helicobacter infection versus 9–24 months in existing models. The gastric B‐cell lesions in the Nlrc5 knockout mice had the histopathological features of the human disease, notably lymphoepithelial‐like lesions, centrocyte‐like cells, and were infiltrated by dendritic cells (DCs), macrophages, and T‐cells (CD4 + , CD8 + and Foxp3 + ). Mouse and human gastric tissues contained immune cells expressing immune checkpoint receptor programmed death 1 (PD‐1) and its ligand PD‐L1, indicating an immunosuppressive tissue microenvironment. We next determined whether CD40L, overexpressed in a range of B‐cell malignancies, may be a potential drug target for the treatment of gastric MALT lymphoma. Importantly, we showed that the administration of anti‐CD40L antibody either coincident with or after establishment of Helicobacter infection prevented gastric B‐cell lesions in mice, when compared with the control antibody treatment. Mice administered the CD40L antibody also had significantly reduced numbers of gastric DCs, CD8 + and Foxp3 + T‐cells, as well as decreased gastric expression of B‐cell lymphoma genes. These findings validate the potential of CD40L as a therapeutic target in the treatment of human gastric B‐cell MALT lymphoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Publisher: Springer Science and Business Media LLC
Date: 08-2017
DOI: 10.1038/S41598-017-07288-4
Abstract: Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3CC40645H
Abstract: Reaction of Bi(O(t)Bu)3 with aspirin (acetylsalicylic acid = aspH) in dry toluene results in the bismuth(III) complex, [Bi(O2C(C6H4)OAc)3]∞ 1 (O2C(C6H4)OAc = asp), minimum inhibitory concentration (MIC) against Helicobacter pylori ≥ 6.25 μg mL(-1), while the inclusion of a stoichiometric equivalent of KO(t)Bu leads to crystals of the bismuthate salt [KBi(O2C(C6H4)OAc)4]∞ 2.
Publisher: CSIRO Publishing
Date: 2012
DOI: 10.1071/CH12042
Abstract: Two new substituted thiobenzoic acids, m-nitrothiobenzoic and m-sulfothiobenzoic acid, and six (four new) homo- and heteroleptic bismuth(iii) compounds derived from thiobenzoic acid and substituted thiobenzoic acid have been synthesised and fully characterised using both solvent free and solvent mediated methods Bi(SC(=O)C6H5)3 (3), PhBi(SC(=O)C6H5)2 (4), Ph2Bi(SC(=O)C6H5) (5), Bi(SC(=O)C6H4-m-NO2)3 (6), PhBi(SC(=O)C6H4-m-NO2)2 (7), and PhBi(SC(=O)C6H4-m-SO3) (8). The solid-state structures of the previously reported Bi(SC(=O)C6H5)3 (3) and PhBi(SC(=O)]C6H5)2 (4) complexes have now been confirmed by X-ray crystallography. In the solid-state complex 3 forms a column-like polymeric structure resembling stacked bowls through pyramidal intermolecular Bi–S3 bonds of distance 3.359 Å, providing a Bi(iii) centre with a nine coordinate environment. Complex 4 forms discrete tetrameric units cemented by long intermolecular Bi–S (3.774 Å), Bi–O(= C) (3.030, 3.071 Å) and Bi–C bonds (3.627 Å). The complexes were assessed for their activity against three strains of Helicobacter pylori and all show a minimum inhibitory concentration of 6.25 µg mL–1, indicating that the high level of bactericidal activity is insensitive to the degree of substitution at the Bi(iii) centre.
Publisher: Hindawi Limited
Date: 15-02-2018
DOI: 10.1111/CMI.12826
Abstract: Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL-33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL-33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide-Binding Oligomerisation Domain-Containing Protein 1 (NOD1) and its adaptor protein receptor-interacting serine-threonine Kinase 2, to promote production of both full-length and processed IL-33 in gastric epithelial cells. Furthermore, IL-33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1
Publisher: Elsevier BV
Date: 05-2005
DOI: 10.1016/J.MOLIMM.2004.12.001
Abstract: Toll-like receptor (TLR) molecules play a frontline role in the defence of the host against infection by microbial pathogens. These molecules, together with the recently described Nod family proteins, have been shown to trigger innate immune responses in host cells via the recognition of highly conserved microbial structures. TLR4, which is the best-characterised of these "pathogen-recognition molecules" (PRMs), was the first to be shown to recognise a specific microbial component: the lipopolysaccharide (LPS) from Gram-negative bacteria. The molecular specificities of the remaining PRMs have, in nearly all cases, now also been elucidated. Host cells belonging to the myeloid cell lineage are known to be particularly responsive to these microbial constituents. Conversely, other cell types such as epithelial cells, were generally thought to be hypo-responsive to stimulation by such molecules. New evidence suggests that these cells are in fact likely to play a fundamental role in host defence against pathogenic micro-organisms. Indeed, epithelial cells afford an initial barrier against the host microflora, and appear to be able to differentiate between pathogenic and commensal micro-organisms. This review article will discuss current knowledge regarding innate immune responses in epithelial and myeloid cells to the model non-invasive pathogen, Helicobacter pylori, which is a major cause of upper gastrointestinal tract disease in humans.
Publisher: Wiley
Date: 14-08-2018
DOI: 10.1111/IMCB.12190
Abstract: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both in vivo and in vitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1β and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P. aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H. pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P. aeruginosa OMVs in human monocytes. In contrast, free P. aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.
Publisher: Wiley
Date: 21-09-2012
DOI: 10.1111/MMI.12019
Abstract: The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram-negative pathogen Salmonella typhimurium and the Gram-positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.
Publisher: Frontiers Media SA
Date: 06-06-2017
Publisher: Elsevier BV
Date: 2013
Publisher: Hindawi Limited
Date: 05-2002
DOI: 10.1046/J.1462-5822.2002.00189.X
Abstract: Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)-8 secretion in gastric epithelial cells, via the activation of NF- kappa B, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI-mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n = 12) was cag PAI genotyped and tested in co-culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF- kappa B activation and IL-8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI(-) genotype and did not induce pro-inflammatory responses in these cells. Mouse-to-mouse passage of the two cag PAI(+) -colonizing strains yielded host-adapted variants that infected mice with bacterial loads 100-fold higher than those of the respective parental strains (P= 0.001). These mouse-adapted variants were affected in their capacity to induce pro-inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.
Publisher: American Chemical Society (ACS)
Date: 04-11-2011
DOI: 10.1021/OM2008869
Publisher: Springer Science and Business Media LLC
Date: 06-02-2009
Abstract: Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a Δ espADB mutant and a Δ escF T3SS mutant. We observed that flagellin was consistently and strongly present in the secretome of wild type EPEC and the Δ espADB mutant but present only weakly in the secretome of the Δ escF mutant. Given the ancestral relationship between the flagella export apparatus and virulence associated T3SSs, we investigated whether FliC could utilise the LEE-encoded T3SS for export. In the absence of a functional flagella export apparatus, we showed that FliC could be secreted by the LEE-encoded T3SS and stimulate (Toll-like receptor 5) TLR5 signalling but could not confer motility. Since the secretion of FliC during A/E lesion formation would presumably be disadvantageous for the pathogen, we propose that virulence associated T3SSs and flagella T3SSs have evolved through a system of chaperones and complex regulatory pathways to be functional at different times to ensure that FliC secretion does not occur during T3SS effector translocation.
Publisher: American Society for Microbiology
Date: 04-1999
DOI: 10.1128/AAC.43.4.777
Abstract: The Helicobacter pylori SS1 mouse model was used to characterize the development of resistance in H. pylori after treatment with metronidazole monotherapy and to examine the effect of prior exposure to metronidazole on the efficacy of a metronidazole-containing eradication regimen. Mice colonized with the metronidazole-sensitive H. pylori SS1 strain were treated for 7 days with either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole once a day or three times per day (TID). In a separate experiment, H. pylori -infected mice were administered either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole TID for 7 days, followed 1 month later by either peptone trypsin broth or the mouse equivalent of 20 mg of omeprazole, 250 mg of clarithromycin, and 400 mg of metronidazole twice a day for 7 days. At least 1 month after the completion of treatment, the mice were sacrificed and their stomachs were cultured for H. pylori . The susceptibilities of isolates to metronidazole were assessed by agar dilution determination of the MICs. Mixed populations of metronidazole-resistant and -sensitive strains were isolated from 70% of mice treated with 400 mg of metronidazole TID. The ratio of resistant to sensitive strains was 1:100, and the MICs for the resistant strains varied from 8 to 64 μg/ml. In the second experiment, H. pylori was eradicated from 70% of mice treated with eradication therapy alone, compared to 25% of mice pretreated with metronidazole ( P 0.01). Mice still infected after treatment with metronidazole and eradication therapy contained mixed populations of metronidazole-resistant and -sensitive isolates in a ratio of 1:25. These results demonstrate that H. pylori readily acquires resistance to metronidazole in vivo and that prior exposure of the organism to metronidazole is associated with failure of eradication therapy. H. pylori -infected mice provide a suitable model for the study of resistance mechanisms in H. pylori and will be useful in determining optimal regimens for the eradication of resistant strains.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.IMMUNI.2018.03.003
Abstract: Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.
Publisher: Wiley
Date: 07-1999
DOI: 10.1046/J.1365-2958.1999.01478.X
Abstract: Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/C000164C
Abstract: The formation of bismuth(III) complexes of carboxylates and benzoates derived from the 1 : 3 reaction of BiPh(3) with the common non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen, naproxen, ibuprofen, mefenamic acid, diflunisal, 5-chlorosalicylic acid, fenbufen, sulindac, tolfenamic acid and flufenamic acid, has been achieved using both solvent-free and solvent-mediated methods. The thermochemical profiles of the solvent-free reactions were studied using DSC-TGA. All reactions produced the tris-substituted complexes of general formula [BiL(3)](n), with the complexes derived from ketoprofen and sulindac having an additional single bismuth bound H(2)O molecule in the inner coordination sphere. The complexes are stable in air over a period of six months, do not undergo significant decomposition when suspended overnight in water, but decompose in 1 M HCl solution to release the free acid form of the NSAID. All ten complexes show excellent in vitro activity against Helicobacter pylori with MIC values of > or = 6.25 microg mL(-1).
Publisher: Future Medicine Ltd
Date: 08-2010
DOI: 10.2217/FMB.10.84
Abstract: Helicobacter pylori colonizes a large proportion of the world’s population, with infection invariably leading to chronic, lifelong gastritis. While the infection often persists undiagnosed and without causing severe pathology, there are a number of host, bacterial and environmental factors that can influence whether infection provokes a mild inflammatory response or results in significant morbidity. Intriguingly, the most virulent H. pylori strains appear to deliberately induce the epithelial signaling cascades responsible for activating the innate immune system. While the reason for this remains unclear, the resulting adaptive immune responses are largely ineffective in clearing the bacterium once infection has become established and, as a result, inflammation likely causes more damage to the host itself.
Publisher: Springer International Publishing
Date: 2019
DOI: 10.1007/978-3-030-15138-6_7
Abstract: The human pathogen Helicobacter pylori interacts intimately with gastric epithelial cells to induce inflammatory responses that are a hallmark of the infection. This inflammation is a critical precursor to the development of peptic ulcer disease and gastric cancer. A major driver of this inflammation is a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI), present in a subpopulation of more virulent H. pylori strains. The cagPAI T4SS specifically activates signalling pathways in gastric epithelial cells that converge on the transcription factor, nuclear factor-κB (NF-κB), which in turn upregulates key immune and inflammatory genes, resulting in various host responses. It is now clear that H. pylori possesses several mechanisms to activate NF-κB in gastric epithelial cells and, moreover, that multiple signalling pathways are involved in these responses. Two of the dominant signalling pathways implicated in NF-κB-dependent responses in epithelial cells are nucleotide-binding oligomerisation domain 1 (NOD1) and a newly described pathway involving alpha-kinase 1 (ALPK1) and tumour necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA). Although the relative roles of these two pathways in regulating NF-κB-dependent responses still need to be clearly defined, it is likely that they work cooperatively and non-redundantly. This chapter will give an overview of the various mechanisms and pathways involved in H. pylori induction of NF-κB-dependent responses in gastric epithelial cells, including a 'state-of-the-art' review on the respective roles of NOD1 and ALPK1/TIFA pathways in these responses.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5DT02259B
Abstract: Bismuth( iii ) hydroxamate complexes of varying composition all show powerful bactericidal activity toward Helicobacter pylori .
Publisher: Springer Japan
Date: 2016
Start Date: 2011
End Date: 05-2017
Amount: $480,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2021
End Date: 12-2023
Amount: $429,700.00
Funder: Australian Research Council
View Funded ActivityStart Date: 09-2012
End Date: 12-2015
Amount: $270,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 07-2022
End Date: 06-2025
Amount: $635,000.00
Funder: Australian Research Council
View Funded Activity