Genetic Analysis of Lipooligosaccharide Core Biosynthesis in Campylobacter jejuni 81-176

Abstract

We report isolation and characterization of Campylobacter jejuni 81-176 lgtF and galT lipooligosaccharide (LOS) core mutants. It has been suggested that the lgtF gene of C. jejuni encodes a two-domain glucosyltransferase that is responsible for the transfer of a ␤-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a ␤-1,2-glucose residue on Hep II. A site-specific mutation in the lgtF gene of C. jejuni 81-176 resulted in expression of a truncated LOS, and complementation of the mutant in trans restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a ␤-1,4-glucose on Hep I and a ␤-1,2-glucose on Hep II. Mutation of another gene, galT, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in C. jejuni 81-176, resulted in loss of the ␤-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core waaC mutant. These studies have important implications for the role of LOS in the pathogenesis of Campylobacter-mediated infection.

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

“…The results of mutations to the C. jejuni 81-176 LOS length have varied; mutagenesis of cgtA lead to increased invasion of cell layers, whereas large truncation mutants had decreased invasion (50,51). In our study, adherence of the C. jejuni 11168 ΔcgtA/neuB was increased compared with wild type.…”

Effects of SequentialCampylobacter jejuni81-176 Lipooligosaccharide Core Truncations on Biofilm Formation, Stress Survival, and Pathogenesis

“…1A (Fig. 1A) (41,43,66). waaV is uncharacterized in C. jejuni but exhibits homology to various putative bacterial glycosyltransferases.…”

“…The second mutant harbored an insertion in lgtF, encoding a two-domain glucosyltransferase responsible for the addition of ␤-1,4 glucose to HepI and ␤-1,2 glucose to HepII (Fig. 1A and B) (43).…”

“…Various C. jejuni LOS mutants have also been found to exhibit enhanced sensitivities to certain antimicrobial substances (37,43,53,56), and two severely truncated mutants (a strain 81-176 ⌬waaC mutant and a strain 11168 mutant with a large LOS gene cluster deletion) are defective for host cell invasion (43,56). However, the latter two mutants also produce altered CPS and lipid A, respectively (42,56), and an invasion defect previously ascribed to a ⌬waaF mutant (42) was subsequently shown to be due to sensitivity of that mutant to the detergent used for host cell lysis (43). Thus, although previous work has shown that the C. jejuni LOS is important as a pathogenesis factor, the relative contributions of specific oligosaccharide core structures to particular pathogenesis-related properties have not been described, nor has a direct role for the LOS in host colonization been demonstrated.…”

The Polysaccharide Capsule of Campylobacter jejuni Modulates the Host Immune Response

“…Both primers introduced a BamHI site at the 5= ends, indicated by the underlining. The resulting plasmid was subjected to transposon mutagenesis using an in vitro Tn5-based transposition system (Epicentre, Madison, WI) with a chloramphenicol resistance (Cm r ; cat) cassette, and the insertion points were identified by sequencing individual insertions with primers within the cassette, as previously described (10)(11)(12). A clone with a nonpolar insertion into gene CJJ81176_1415 (mpnC in Fig.…”