Dataset Information

Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.

ABSTRACT: Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential for chick colonization by C. jejuni . While it is now clear that PglH catalyzes multiple transfer reactions, the mechanism whereby the reactions cease after the addition of just three GalNAc residues has yet to be understood. To address this issue, a series of mechanistic biochemical studies was conducted with purified native PglH. This enzyme was found to follow a processive mechanism under initial rate conditions; however, product inhibition and product accumulation led to PglH release of intermediate products prior to complete conversion to the native ultimate product. Point mutations of an essential EX(7)E sequence motif were used to demonstrate that a single active site was responsible for all three transferase reactions, and a homology model with the mannosyltransferase PimA, from Mycobacteria smegmatis , establishes the requirement of the EX(7)E motif in catalysis. Finally, increased binding affinity with increasing glycan size is proposed to provide PglH with a counting mechanism that does not allow the transfer of more than three GalNAc residues. These results provide important mechanistic insights into the function of the glycosyl transfer polymerase that is related to the virulence of C. jejuni .

SUBMITTER: Troutman JM

PROVIDER: S-EPMC2736683 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.

Troutman Jerry M JM Imperiali Barbara B

Biochemistry 20090301 12

Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential f ...[more]

PMID: 19159314

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Project description:Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed DeltagalT and DeltacstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (DeltawaaF and DeltalgtF but not DeltagalT or DeltacstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (DeltacstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The DeltawaaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

Project description:AimThis study was conducted to test the ability of a carvacrol-based formulation (Phodé, France) to decrease the C. jejuni caecal load in inoculated broiler chickens and to study the impact of the C. jejuni inoculation alone or combined with the product, on the caecal microbiota.Methods and resultsOn day 1, chickens were either fed a control feed or the same diet supplemented with a carvacrol-based product. On day 21, the carvacrol-supplemented chickens and half of the non-supplemented chickens were inoculated with C. jejuni (108 CFU). Quantitative PCR was used to quantify C. jejuni in chicken caecal samples and 16S rRNA gene sequencing was carried out at 25, 31 and 35 days of age. A significant decrease of 1.4 log of the C. jejuni caecal load was observed in 35-day-old chickens supplemented with the product, compared to the inoculated and unsupplemented group (p < 0.05). The inoculation with C. jejuni significantly increased the population richness, Shannon and Simpson diversity and altered beta-diversity. Compared to the control group, the C. jejuni inoculation causes significant changes in the microbiota. The carvacrol-based product associated with C. jejuni inoculation increased the diversity and strongly modified the structure of the microbial community. Functional analysis by 16S rRNA gene-based predictions further revealed that the product up-regulated the pathways involved in the antimicrobial synthesis, which could explain its shaping effect on the caecal microbiota.ConclusionsOur study confirmed the impairment of the caecal bacterial community after inoculation and demonstrated the ability of the product to reduce the C. jejuni load in chickens. Further investigations are needed to better understand the mode of action of this product to promote the installation of a beneficial microbiota to its host.Significance and impact of the studyResults suggested that this product could be promising to control C. jejuni contamination of broilers.

Dataset Information

Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.

Publications

Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.

Similar Datasets

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