Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Dunne C

PROVIDER: S-EPMC5590460 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Dunne Ciara C McDermot Anthony A Anjan Kumar K Ryan Aindrias A Reid Colm C Clyne Marguerite M

Bioengineering (Basel, Switzerland) 20170415 2

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 <i>Helicobacter pylori</i> 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 ...[more]

PMID: 28952513

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Project description:BackgroundHelicobacter pylori is a human stomach pathogen, naturally-competent for DNA uptake, and prone to homologous recombination. Extensive homoplasy (i.e., phylogenetically-unlinked identical variations) observed in H. pylori genes is considered a hallmark of such recombination. However, H. pylori also exhibits a high mutation rate. The relative adaptive role of homologous recombination and mutation in species diversity is a highly-debated issue in biology. Recombination results in homoplasy. While convergent mutation can also account for homoplasy, its contribution is thought to be minor. We demonstrate here that, contrary to dogma, convergent mutation is a key contributor to Helicobacter pylori homoplasy, potentially driven by adaptive evolution of proteins.ResultsOur present genome-wide analysis shows that homoplastic nonsynonymous (amino acid replacement) changes are not typically accompanied by homoplastic synonymous (silent) variations. Moreover, the majority of the codon positions with homoplastic nonsynonymous changes also contain different (i.e. non-homoplastic) nonsynonymous changes arising from mutation only. This indicates that, to a considerable extent, nonsynonymous homoplasy is due to convergent mutations. High mutation rate or limited availability of evolvable sites cannot explain this excessive convergence, as suggested by our simulation studies. Rather, the genes with convergent mutations are overrepresented in distinct functional categories, suggesting possible selective responses to conditions such as distinct micro-niches in single hosts, and to differences in host genotype, physiology, habitat and diet.ConclusionsWe propose that mutational convergence is a key player in H. pylori's adaptation and extraordinary persistence in human hosts. High frequency of mutational convergence could be due to saturation of evolvable sites capable of responding to selection pressures, while the number of mutable residues is far from saturation. We anticipate a similar scenario of mutational vs. recombinational genome dynamics or plasticity for other naturally competent microbes where strong positive selection could favor frequent convergent mutations in adaptive protein evolution.

Project description:Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.

Project description:Helicobacter pylori colonizes the stomach of half of the human population. Most H. pylori are located in the mucus layer, which is mainly comprised by glycosylated mucins. Using mass spectrometry, we identified 631 glycans (whereof 145 were fully characterized and the remainder assigned as compositions) on mucins isolated from 14 Helicobacter spp.-infected and 14 Helicobacter spp.-noninfected stomachs. Only six identified glycans were common to all individuals, from a total of 60 to 189 glycans in each individual. An increased number of unique glycan structures together with an increased intraindividual diversity and larger interindividual variation were identified among O-glycans from Helicobacter spp.-infected stomachs compared with noninfected stomachs. H. pylori strain J99, which carries the blood group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), and the LacdiNAc-binding adhesin, bound both to Lewis b (Leb)-positive and Leb-negative mucins. Among Leb-positive mucins, H. pylori J99 binding was higher to mucins from Helicobacter spp.-infected individuals than noninfected individuals. Statistical correlation analysis, binding experiments with J99 wt, and J99ΔbabAΔsabA and inhibition experiments using synthetic glycoconjugates demonstrated that the differences in H. pylori-binding ability among these four groups were governed by BabA-dependent binding to fucosylated structures. LacdiNAc levels were lower in mucins that bound to J99 lacking BabA and SabA than in mucins that did not, suggesting that LacdiNAc did not significantly contribute to the binding. We identified 24 O-glycans from Leb-negative mucins that correlated well with H. pylori binding whereof 23 contained α1,2-linked fucosylation. The large and diverse gastric glycan library identified, including structures that correlated with H. pylori binding, could be used to select glycodeterminants to experimentally investigate further for their importance in host-pathogen interactions and as candidates to develop glycan-based therapies.

Dataset Information

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

Publications

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

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