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Abstract
Campylobacter fetus is an emerging pathogen and causative agent in veterinary disease. N-linked protein glycosylation by the Pgl system in Campylobacter jejuni is important for host colonization and adherence to intestinal cells. Interestingly, the Pgl system in C. fetus produces two distinct N-glycans in a ratio of 4:1. Production of multiple N-glycans is consistent in all non-thermotolerant Campylobacters, but the mechanism facilitating this differentiation is unknown. Annotation of the C. fetus pgl locus indicated the presence of two unannotated pgl glycosyltransferases and a putative bacteriophage three-component glucosylation system. Here, we characterized these genes and their physiological functions.
Mutations in C. fetus pglJ and pglX glycosyltransferases provided insight into the biosynthesis of N-acetyl glucosamine (GlcNAc) termini, versus the N-acetyl galactosamine terminus of C. jejuni. Functional transfer of these genes into Escherichia coli produced only the minor N-glycan, indicating a need for further studies into this biochemical pathway. Proteomic analysis of the pgl mutants indicated effects consistent with C. jejuni, including decreased motility and increased antibiotic sensitivity. In addition, we discovered a novel connection between protein N-glycosylation, H2-uptake hydrogenase complex HynABC, and metal homeostasis.
The three-component glucosylation system showed similarity with the putative bacteriophage gtr glucosylation system, canonically involved in adding glucose to the O-antigen of host lipopolysaccharide (LPS). N-glycan analysis of a gtr-negative strain indicated that it plays no role in N-glycosylation. In vitro analysis of C. fetus GtrB indicated that it solely transfers glucose (Glc); however, mutagenesis of the gtr operon resulted in the loss of a -GlcNAc-3--GlcNAc- cap. It is currently unclear how Glc is modified into a GlcNAc residue. Moreover, the mutant had no detectable S-layer and decreased serum resistance. Binding between S-layer protein (sap) and LPS occurs via the N-terminus and is dependent on sap-type and LPS serotype. Strikingly, the gtr operon correlates exclusively with all sequenced type A sap-/sero-type strains. Further work will assess whether Sap-LPS binding occurs through the gtr-dependent α-GlcNAc-3-α-GlcNAc-LPS cap.
This body of work connects glycosylation to important mechanisms of pathogenesis and host survival in a non-model Campylobacter species. Additionally, it provides novel insights into a system for studying N-glycan diversity.
Mutations in C. fetus pglJ and pglX glycosyltransferases provided insight into the biosynthesis of N-acetyl glucosamine (GlcNAc) termini, versus the N-acetyl galactosamine terminus of C. jejuni. Functional transfer of these genes into Escherichia coli produced only the minor N-glycan, indicating a need for further studies into this biochemical pathway. Proteomic analysis of the pgl mutants indicated effects consistent with C. jejuni, including decreased motility and increased antibiotic sensitivity. In addition, we discovered a novel connection between protein N-glycosylation, H2-uptake hydrogenase complex HynABC, and metal homeostasis.
The three-component glucosylation system showed similarity with the putative bacteriophage gtr glucosylation system, canonically involved in adding glucose to the O-antigen of host lipopolysaccharide (LPS). N-glycan analysis of a gtr-negative strain indicated that it plays no role in N-glycosylation. In vitro analysis of C. fetus GtrB indicated that it solely transfers glucose (Glc); however, mutagenesis of the gtr operon resulted in the loss of a -GlcNAc-3--GlcNAc- cap. It is currently unclear how Glc is modified into a GlcNAc residue. Moreover, the mutant had no detectable S-layer and decreased serum resistance. Binding between S-layer protein (sap) and LPS occurs via the N-terminus and is dependent on sap-type and LPS serotype. Strikingly, the gtr operon correlates exclusively with all sequenced type A sap-/sero-type strains. Further work will assess whether Sap-LPS binding occurs through the gtr-dependent α-GlcNAc-3-α-GlcNAc-LPS cap.
This body of work connects glycosylation to important mechanisms of pathogenesis and host survival in a non-model Campylobacter species. Additionally, it provides novel insights into a system for studying N-glycan diversity.