Different stages of the infection cycle are enriched for Campylobacter strains with distinct phenotypes and levels of fluoroquinolone resistance.

Campylobacter species are the leading cause of bacterial diarrhoea worldwide and consumption of contaminated chicken meat is the most common route of infection. Chickens can be infected with multiple strains of Campylobacter and during the infection cycle this pathogen must survive a wide variety of environments. Numerous studies have reported a high degree of genetic variability in this pathogen that can use antigenic and phase variation to alter the expression of key phenotypes. In this study the phenotypic profile of isolates from freshly slaughtered chickens, chicken products in the supermarket and stool samples from infected patients were compared to identify phenotypic changes during the passage of the bacteria through the infection cycle. Isolates from different stages of the infection cycle had altered phenotypic profiles with isolates from human stool samples showing a lower ability to form a biofilm and an increased ability to associate with epithelial cells in vitro. Resistance to fluoroquinolones was found in all cohorts but at a much higher occurrence (94%) in isolates from supermarket chicken. Isolates displaying high levels of resistance to fluoroquinolones also were more likely to display a higher tolerance to growth in the presence of oxygen. In conclusion, isolates with specific phenotypes appear to be overly represented at different stages of the infection cycle suggesting that environmental stresses may be enriched for strains with these phenotypes.

Investigating the Interaction of Helicobacter pylori with the Gastric Mucosa.

Helicobacter pylori chronically infects the gastric mucosa of humans and diseases associated with infection include gastritis, peptic ulceration, and development of gastric cancer. The organism displays a distinct tropism for the gastric mucosa of humans and for the gastric mucin MUC5AC. While the majority of organisms are found in the mucus layer overlying the epithelial cells in the stomach, adherence of the organism to the gastric epithelium is necessary for the development of disease. The interaction of H. pylori with epithelial cells results in subversion of host cell signaling and induction of an inflammatory response. Factors that influence the outcome of infection include host genetics, environmental factors, and the phenotype of the infecting strain. In this chapter, we describe cell culture assays to assess the interaction of H. pylori with epithelial cells, immunofluorescent staining to detect H. pylori in infected human gastric biopsy specimens and the use of flow cytometry to detect mucin binding to H. pylori.

Exploiting the MUC5AC Antigen for Noninvasive Identification of Pancreatic Cancer.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with 89Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. 89Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer 89Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

The Interaction of Helicobacter pylori with TFF1 and Its Role in Mediating the Tropism of the Bacteria Within the Stomach.

Helicobacter pylori colonises the human stomach and has tropism for the gastric mucin, MUC5AC. The majority of organisms live in the adherent mucus layer within their preferred location, close to the epithelial surface where the pH is near neutral. Trefoil factor 1 (TFF1) is a small trefoil protein co-expressed with the gastric mucin MUC5AC in surface foveolar cells and co-secreted with MUC5AC into gastric mucus. Helicobacter pylori binds with greater avidity to TFF1 dimer, which is present in gastric mucus, than to TFF1 monomer. Binding of H. pylori to TFF1 is mediated by the core oligosaccharide subunit of H. pylori lipopolysaccharide at pH 5.0-6.0. Treatment of H. pylori lipopolysaccharide with mannosidase or glucosidase inhibits its interaction with TFF1. Both TFF1 and H. pylori have a propensity for binding to mucins with terminal non-reducing α- or ß-linked N-acetyl-d-glucosamine or α-(2,3) linked sialic acid or Gal-3-SO42-. These findings are strong evidence that TFF1 has carbohydrate-binding properties that may involve a conserved patch of aromatic hydrophobic residues on the surface of its trefoil domain. The pH-dependent lectin properties of TFF1 may serve to locate H. pylori deep in the gastric mucus layer close to the epithelium rather than at the epithelial surface. This restricted localisation could limit the interaction of H. pylori with epithelial cells and the subsequent host signalling events that promote inflammation.

The Role of Host Genetic Polymorphisms in Helicobacter pylori Mediated Disease Outcome.

The clinical outcome of infection with the chronic gastric pathogen Helicobacter pylori is not the same for all individuals and also differs in different ethnic groups. Infection occurs in early life (<3 years of age), and while all infected persons mount an immune response and develop gastritis, the majority of individuals are asymptomatic. However, up to 10-15% develop duodenal ulceration, up to 1% develop gastric cancer (GC) and up to 0.1% can develop gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The initial immune response fails to clear infection and H. pylori can persist for decades. H. pylori has been classified as a group one carcinogen by the WHO. Interestingly, development of duodenal ulceration protects against GC. Factors that determine the outcome of infection include the genotype of the infecting strains and the environment. Host genetic polymorphisms have also been identified as factors that play a role in mediating the clinical outcome of infection. Several studies present compelling evidence that polymorphisms in genes involved in the immune response such as pro and anti-inflammatory cytokines and pathogen recognition receptors (PRRs) play a role in modulating disease outcome. However, as the number of studies grows emerging confounding factors are small sample size and lack of appropriate controls, lack of consideration of environmental and bacterial factors and ethnicity of the population. This chapter is a review of current evidence that host genetic polymorphisms play a role in mediating persistent H. pylori infection and the consequences of the subsequent inflammatory response.

Resident bacteria in breast cancer tissue: pathogenic agents or harmless commensals?

Breast cancer is the second most common cancer in women. Recent evidence identifies a unique microbiome in breast tissue; a site previously thought to be sterile. The identification that this microbiome varies considerably from healthy subjects to cancer patients has prompted investigations into the role of specific bacterial species in oncogenesis. Indeed, certain bacteria have been shown to aid cancer development in vitro by promoting genomic instability, invasion, and chemotherapy resistance. However, the in vivo role of the breast microbiome in cancer appears to be more complex, involving numerous interactions between its constituent species and host cells. As such, reduced abundances of species which exert a protective effect against oncogenesis have come into focus and there is an emerging consensus that states of microbial dysbiosis, in which the normal balance of bacterial species is altered, can contribute to the development of cancer. This review summarizes the findings to date from the available literature pertaining to the microbiome in breast cancer and outlines areas worthy of further investigation.

Binding of Helicobacter pylori to Human Gastric Mucins Correlates with Binding of TFF1.

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.

The use of stool specimens reveals Helicobacter pylori strain diversity in a cohort of adolescents and their family members in a developed country.

Helicobacter pylori infection occurs within families but the transmission route is unknown. The use of stool specimens to genotype strains facilitates inclusion of complete families in transmission studies. Therefore, we aimed to use DNA from stools to analyze strain diversity in H. pylori infected families. We genotyped H. pylori strains using specific biprobe qPCR analysis of glmM, recA and hspA. Concentration of H. pylori organisms before DNA isolation enhanced subsequent DNA amplification. We isolated H. pylori DNA from 50 individuals in 13 families. Tm data for at least 2 of the 3 genes and sequencing of the glmM amplicon were analyzed. Similar strains were commonly found in both mothers and children and in siblings. However, 20/50 (40%) individuals had multiple strains and several individuals harbored strains not found in other family members, suggesting that even in developed countries sources of infection outside of the immediate family may exist. Whether infection occurs multiple times or one transmission event with several strains occurs is not known but future studies should aim to analyze strains from children much closer to infection onset. The presence of multiple stains in infected persons has implications for antibiotic sensitivity testing and treatment strategies.

Glycosylation-related gene expression in HT29-MTX-E12 cells upon infection by Helicobacter pylori.

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.

Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC.

There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions.

Methods to Assess the Direct Interaction of C. jejuni with Mucins.

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.

Assays to Study the Interaction of Campylobacter jejuni with the Mucosal Surface.

Mucosal colonization and overcoming the mucosal barrier are essential steps in the establishment of infection by Campylobacter jejuni. The interaction between C. jejuni and host cells, including binding and invasion, is thought to be the key virulence factor important for pathogenesis of C. jejuni infections in animals or humans. The intestinal mucosal barrier is composed of a polarized epithelium covered by a thick adherent mucus gel layer. There is a requirement for cell culture assays of infection to accurately represent the in vivo mucosal surface. In this chapter, we describe the use of a number of cell culture models and the use of polarized in vitro organ culture to examine the interaction of C. jejuni with mucosal surfaces.

A Preliminary Study Examining the Binding Capacity of Akkermansia muciniphila and Desulfovibrio spp., to Colonic Mucin in Health and Ulcerative Colitis.

BACKGROUND: Akkermansia muciniphila and Desulfovibrio spp. are commensal microbes colonising the mucus gel layer of the colon. Both species have the capacity to utilise colonic mucin as a substrate. A. muciniphila degrades colonic mucin, while Desulfovibrio spp. metabolise the sulfate moiety of sulfated mucins. Altered abundances of these microorganisms have been reported in ulcerative colitis (UC). However their capacity to bind to human colonic mucin, and whether this binding capacity is affected by changes in mucin associated with UC, remain to be defined. METHODS: Mucin was isolated from resected colon from control patients undergoing resection for colonic cancer (n = 7) and patients undergoing resection for UC (n = 5). Isolated mucin was purified and printed onto mucin microarrays. Binding of reference strains and three clinical isolates of A. muciniphila and Desulfovibrio spp. to purified mucin was investigated. RESULTS: Both A. muciniphila and Desulfovibro spp. bound to mucin. The reference strain and all clinical isolates of A. muciniphila showed increased binding capacity for UC mucin (p < .005). The Desulfovibrio reference strain showed increased affinity for UC mucin. The mucin binding profiles of clinical isolates of Desulfovibrio spp. were specific to each isolate. Two isolates showed no difference in binding. One UC isolate bound with increased affinity to UC mucin (p < .005). CONCLUSION: These preliminary data suggest that differences exist in the mucin binding capacity of isolates of A. muciniphila and Desulfovibrio spp. This study highlights the mucin microarray platform as a means of studying the ability of bacteria to interact with colonic mucin in health and disease.

Expression and characterization of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines.

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.

Factors that mediate colonization of the human stomach by Helicobacter pylori.

Helicobacter pylori (H. pylori) colonizes the stomach of humans and causes chronic infection. The majority of bacteria live in the mucus layer overlying the gastric epithelial cells and only a small proportion of bacteria are found interacting with the epithelial cells. The bacteria living in the gastric mucus may act as a reservoir of infection for the underlying cells which is essential for the development of disease. Colonization of gastric mucus is likely to be key to the establishment of chronic infection. How H. pylori manages to colonise and survive in the hostile environment of the human stomach and avoid removal by mucus flow and killing by gastric acid is the subject of this review. We also discuss how bacterial and host factors may together go some way to explaining the susceptibility to colonization and the outcome of infection in different individuals. H. pylori infection of the gastric mucosa has become a paradigm for chronic infection. Understanding of why H. pylori is such a successful pathogen may help us understand how other bacterial species colonise mucosal surfaces and cause disease.

Interaction of microbes with mucus and mucins: recent developments.

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.

Copper promotes TFF1-mediated Helicobacter pylori colonization.

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.

Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response.

In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3'sialyllactose and 6'sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3'sialyllactose did not enhance adhesion. However, treatment with 6'sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3'- and 6'-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3'- and 6'-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6'sialyllactose, and 3'sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.

Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins.

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.