RESUMEN
Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or ß-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells.
RESUMEN
AIM: To identify glycosylation-related genes in the HT29 derivative cell line, HT29-MTX-E12, showing differential expression on infection with Helicobacter pylori (H. pylori). METHODS: Polarised HT29-MTX-E12 cells were infected for 24 h with H. pylori strain 26695. After infection RNA was isolated from both infected and non-infected host cells. Sufficient infections were carried out to provide triplicate samples for microarray analysis and for qRT-PCR analysis. RNA was isolated and hybridised to Affymetrix arrays. Analysis of microarray data identified genes significantly differentially expressed upon infection. Genes were grouped into gene ontology functional categories. Selected genes associated with host glycan structure (glycosyltransferases, hydrolases, lectins, mucins) were validated by real-time qRT-PCR analysis. RESULTS: Infection of host cells was confirmed by the isolation of live bacteria after 24 h incubation and by PCR amplification of bacteria-specific genes from the host cell RNA. H. pylori do not survive incubation under the adopted culture conditions unless they associate with the adherent mucus layer of the host cell. Microarray analysis identified a total of 276 genes that were significantly differentially expressed (P < 0.05) upon H. pylori infection and where the fold change in expression was greater than 2. Six of these genes are involved in glycosylation-related processes. Real-time qRT-PCR demonstrated significant downregulation (1.8-fold, P < 0.05) of the mucin MUC20. REG4 was heavily expressed and significantly downregulated (3.1-fold, P < 0.05) upon infection. Gene ontology analysis was consistent with previous studies on H. pylori infection. CONCLUSION: Gene expression data suggest that infection with H. pylori causes a decrease in glycan synthesis, resulting in shorter and simpler glycan structures.
Asunto(s)
Infecciones por Helicobacter/patología , Helicobacter pylori/fisiología , Interacciones Huésped-Patógeno , Mucinas/metabolismo , Proteínas Asociadas a Pancreatitis/metabolismo , Polisacáridos/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Glicosilación , Células HT29 , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Análisis por MicromatricesRESUMEN
Studies of the interaction of bacteria with mucus-secreting cells can be complemented at a more mechanistic level by exploring the interaction of bacteria with purified mucins. Here we describe a far Western blotting approach to show how C. jejuni proteins separated by SDS PAGE and transferred to a membrane or slot blotted directly onto a membrane can be probed using biotinylated mucin. In addition we describe the use of novel mucin microarrays to assess bacterial interactions with mucins in a high-throughput manner.
Asunto(s)
Proteínas Aviares/metabolismo , Proteínas Bacterianas/metabolismo , Far-Western Blotting/métodos , Campylobacter jejuni/metabolismo , Ensayos Analíticos de Alto Rendimiento , Mucinas/metabolismo , Animales , Proteínas Aviares/química , Proteínas Bacterianas/química , Biotina/química , Campylobacter jejuni/química , Pollos , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes/química , Humanos , Mucinas/química , Análisis por Matrices de Proteínas , Unión Proteica , Mapeo de Interacción de Proteínas , Coloración y Etiquetado/métodosRESUMEN
Due to the recent rapid expansion in our understanding of the composition of the gut microflora and the consequences of altering that composition the question of how bacteria colonise mucus layers and interact with components of mucus, such as mucin, is now receiving widespread attention. Using a combination of mucus secreting cells, and a novel mucin microarray platform containing purified native mucins from different sources we recently demonstrated that two gastrointestinal pathogens, Helicobacter pylori and Campylobacter jejuni, colonise mucus by different mechanisms. This result emphasizes the potential for even closely related bacteria to interact with mucus in divergent ways to establish successful infection. Expanding the use of the mucin arrays described in the study to other microorganisms, both pathogenic and commensal, should lead to the discovery of biologically important motifs in bacterial-host interactions and complement the use of novel in vitro cell models, such as mucus secreting cell lines.
Asunto(s)
Campylobacter jejuni/patogenicidad , Mucosa Gástrica/microbiología , Helicobacter pylori/patogenicidad , Mucosa Intestinal/microbiología , Mucinas/metabolismo , Moco/metabolismo , Animales , HumanosRESUMEN
Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.
Asunto(s)
Campylobacter jejuni/patogenicidad , Mucosa Gástrica/microbiología , Helicobacter pylori/patogenicidad , Mucosa Intestinal/microbiología , Mucinas/metabolismo , Moco/metabolismo , Animales , Infecciones por Campylobacter/metabolismo , Campylobacter jejuni/metabolismo , Técnica del Anticuerpo Fluorescente , Mucosa Gástrica/metabolismo , Células HT29 , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Análisis por MicromatricesRESUMEN
Helicobacter pylori colonises the gastric mucosa of humans. The majority of organisms live in mucus. These organisms are an important reservoir for infection of the underlying epithelium. Cell culture models for H. pylori infection do not normally possess a mucus layer. The interaction of H. pylori with TFF1, a member of the trefoil factor family found in gastric mucin, is mediated by lipopolysaccharide. To test the hypothesis that the interaction of H. pylori with TFF1 promotes mucus colonization we characterised the interaction of H. pylori with a mucus secreting cell line, HT29-MTX-E12. An isogenic mutant of H. pylori with truncated core oligosaccharides was produced and binding to TFF1 and ability to colonise HT29-MTX-E12 cells determined. The adherent mucus layer of HT29-MTX-E12 cells contained the gastric mucin MUC5AC and trefoil factors, TFF1 and TFF3. H. pylori was found within the mucus layer in discrete clusters and in close association with TFF1. It also interacted with the membrane bound mucin MUC1 and replicated when co-cultured with the cells. An isogenic mutant of H. pylori with a truncated LPS core did not interact with TFF1, and colonization of HT29-MTX-E12 cells was reduced compared to the wild-type strain (p<0.05). Preincubation of cells with wild type LPS but not with truncated LPS resulted in reduced colonization by H. pylori. These results demonstrate that the interaction of TFF1 with H. pylori is important for colonization of gastric mucus and the core oligosaccharide of H. pylori LPS is critical for this interaction to occur. HT29-MTX-E12 cells are a useful system with which to study the interaction of bacteria with mucosal surfaces and the effect of such interactions on mediating colonization.
Asunto(s)
Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Helicobacter pylori/patogenicidad , Línea Celular , Helicobacter pylori/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Mucina 5AC/metabolismo , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Factor Trefoil-1 , Factor Trefoil-3 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The Cu(I) catalysed cycloaddition reaction of azides and alkynes has been used to generate a series of divalent GlcNAc clusters with both α and ß configurations. These glycoclusters can be considered as potential mimetics of an anti Helicobacter pylori hexasaccharide as they present two GlcNAc residues grafted onto a core scaffold. Two bivalent compounds based on α-O-GlcNAc were identified that selectively reduced the viability of H. pylori. These compounds showed activity towards different strains of H. pylori (Pu4 vs P12). The activity of the oligosaccharide mimetics is speculated to be due to the GlcNAc residues being able to adopt spatial arrangements accessible to the anti H. pylori hexasaccharide which may be important for activity.
Asunto(s)
Acetilglucosamina/química , Antibacterianos/farmacología , Materiales Biomiméticos/farmacología , Glicoconjugados/farmacología , Helicobacter pylori/efectos de los fármacos , Oligosacáridos/química , Alquinos/química , Antibacterianos/síntesis química , Antibacterianos/química , Azidas/química , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/química , Conformación de Carbohidratos , Catálisis , Cobre/química , Glicoconjugados/síntesis química , Glicoconjugados/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Campylobacter jejuni is the leading cause of acute bacterial infectious diarrhea in humans. Unlike in humans, C. jejuni is a commensal within the avian host. Heavily colonized chickens often fail to display intestinal disease, and no cellular attachment or invasion has been demonstrated in-vivo. Recently, researchers have shown that the reason for the attenuation of C. jejuni virulence may be attributed to the presence of chicken intestinal mucus and more specifically chicken mucin. Since mucins are heavily glycosylated molecules this observation would suggest that glycan-based compounds may act as anti-infectives against C. jejuni. Considering this, we have investigated naturally sourced foods for potential anti-infective glycans. Bovine colostrum rich in neutral and acidic oligosaccharides has been identified as a potential source of anti-infective glycans. In this study, we tested oligosaccharides isolated and purified from the colostrum of Holstein Friesian cows for anti-infective activity against a highly invasive strain of C. jejuni. During our initial studies we structurally defined 37 bovine colostrum oligosaccharides (BCO) by HILIC-HPLC coupled with exoglycosidase digests and off-line mass spectroscopy, and demonstrated the ability of C. jejuni to bind to some of these structures, in-vitro. We also examined the effect of BCO on C. jejuni adhesion to, invasion of and translocation of HT-29 cells. BCO dramatically reduced the cellular invasion and translocation of C. jejuni, in a concentration dependent manner. Periodate treatment of the BCO prior to inhibition studies resulted in a loss of the anti-infective activity of the glycans suggesting a direct oligosaccharide-bacterial interaction. This was confirmed when the BCO completely prevented C. jejuni binding to chicken intestinal mucin, in-vitro. This study builds a strong case for the inclusion of oligosaccharides sourced from cow's milk in functional foods. However, it is only through further understanding the structure and function of milk oligosaccharides that such compounds can reach their potential as food ingredients.
Asunto(s)
Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/patogenicidad , Pollos/microbiología , Calostro/química , Oligosacáridos/farmacología , Animales , Antiinfecciosos/farmacología , Campylobacter jejuni/fisiología , Bovinos , Intestinos/microbiología , Mucinas/farmacología , Moco/fisiología , VirulenciaRESUMEN
Blood-stage malarial parasites (Plasmodium falciparum) digest large quantities of host haemoglobin during their asexual development in erythrocytes. The haemoglobin digestion pathway, involving a succession of cleavages by various peptidases, appears to be essential for parasite development and has received much attention as an antimalarial drug target. A variety of peptidase inhibitors that have potent antimalarial activity are believed to inhibit and/or kill parasites by blocking haemoglobin digestion. It has not however been established how such a blockage might lead to parasite death. The answer to this question should lie in identifying the affected physiological function, but the purpose of excess haemoglobin digestion by P. falciparum has for many years been the subject of debate. The process was traditionally believed to be nutritional until Lew et al. [Blood 2003;101:4189-94] suggested that it is linked to volume control of the infected erythrocyte and is necessary to prevent premature osmotic lysis of the host cell. Their model predicts that sufficient inhibition of haemoglobin degradation should result in premature haemolysis. In this study we examined the downstream effects of reduced haemoglobin digestion on osmoprotection and nutrition. We found that inhibitors of haemoglobinases (plasmepsins, falcipains and aminopeptidases) did not cause premature haemolysis. The inhibitors did however block parasite development and this effect corresponded to a strong inhibition of protein synthesis. The effect on protein synthesis (i) occurred at inhibitor concentrations and times of exposure that were relevant to parasite growth inhibition, (ii) was observed with different chemical classes of inhibitor, and (iii) was synergistic when a plasmepsin and a falcipain inhibitor were combined, reflecting the well-established antimalarial synergism of the combination. Taken together, the results suggest that the likely primary downstream effect of inhibition of haemoglobin degradation is amino acid depletion, leading to blockade of protein synthesis, and that the parasite probably degrades globin for nutritional purposes.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Cisteína Endopeptidasas/metabolismo , Eritrocitos/parasitología , Hemoglobinas/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Hemólisis , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Protozoarias/metabolismoRESUMEN
Malaria is a disease in desperate need of new chemotherapeutic approaches. Certain microtubule inhibitors, including vinblastine and taxol, have highly potent activity against malarial parasites and disrupt the normal microtubular structures of intra-erythrocytic parasites at relevant concentrations. While these inhibitors are useful tools, their potential as anti-malarial drugs is limited by their high toxicity to mammalian cells. In contrast, two classes of antimitotic herbicide, namely dinitroanilines (e.g. trifluralin and oryzalin) and phosphorothioamidates (e.g. amiprophosmethyl), exhibit moderate activity against the major human malarial parasite Plasmodium falciparum in culture but very low mammalian cytotoxicity. We examined the dynamics and kinetics of uptake and subcellular compartmentation of [14C]trifluralin in comparison with [3H]vinblastine. We wished to determine whether the relatively modest activity of trifluralin was the consequence of poor uptake into parasite cells. Trifluralin accumulated in parasite-infected erythrocytes to approximately 300 times the external concentration and vinblastine at up to approximately 110 times. Accumulation into uninfected erythrocytes was much lower. Uptake of trifluralin was rapid, non-saturable and readily reversed. It appears that the hydrophobic nature of trifluralin leads to accumulation largely in the membranes of the parasite, reducing the levels in the soluble fraction and limiting access to its microtubular target. By contrast, vinblastine accumulated predominantly in the soluble fraction and uptake was saturable and mostly irreversible, consistent with binding predominantly to tubulin. The results indicate that synthesis of more polar trifluralin derivatives may be a promising approach to designing microtubule inhibitors with more potent antimalarial activity.
Asunto(s)
Antimaláricos/metabolismo , Eritrocitos/metabolismo , Plasmodium falciparum/fisiología , Trifluralina/metabolismo , Vinblastina/metabolismo , Animales , Antimaláricos/farmacología , Células Cultivadas , Eritrocitos/parasitología , Humanos , Microtúbulos/metabolismo , Plasmodium falciparum/efectos de los fármacos , Trifluralina/farmacologíaRESUMEN
Microtubules play important roles in cell division, motility and structural integrity of malarial parasites. Some microtubule inhibitors disrupt parasite development at very low concentrations, but most of them also kill mammalian cells. However, the dinitroaniline family of herbicides, which bind specifically to plant tubulin, have inhibitory activity on plant cells but are relatively non-toxic to human cells. Certain dinitroanilines are also inhibitory to various protozoal parasites including Plasmodium. Here we demonstrate that the dinitroanilines trifluralin and oryzalin inhibited progression of erythrocytic Plasmodium falciparum through schizogony, blocked mitotic division, and caused accumulation of abnormal microtubular structures. Moreover, radiolabelled trifluralin interacted with purified, recombinant parasite tubulins but to a much lesser extent with bovine tubulins. The phosphorothioamidate herbicide amiprophos-methyl, which has the same herbicidal mechanism as dinitroanilines, also had antimalarial activity and a similar action on schizogony. These data suggest that P. falciparum tubulin contains a dinitroaniline/phosphorothioamidate-binding site that is not conserved in humans and might be a target for new antimalarial drugs.