Mutation ofwaaC, Encoding Heptosyltransferase I inCampylobacter jejuni81-176, Affects the Structure of both Lipooligosaccharide and Capsular Carbohydrate

Abstract

Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first L-glycero-D-manno-heptose residue to 3-deoxy-D-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

Creation of a Large Deletion Mutant ofCampylobacter jejuniReveals that the Lipooligosaccharide Gene Cluster Is Not Required for Viability

“…Absence of the LOS outer core is detrimental to C. jejuni growth and undermines outer membrane integrity. The inability of the NCTC11168⌬32-52 large deletion mutant to invade Caco-2 further supports a role for LOS in adherence and invasion (7,8,13).…”

“…No differences in growth rate have been seen for C. jejuni waaC, htrB, or waaF mutants (8,16) (Millar and Ketley, unpublished), yet recovery of the NCTC11168⌬32-52 mutant after electroporation was noted to take much longer than is normal. Figure 5 shows the growth of the NCTC11168⌬32-52 mutant compared to the parent strain over 48 h; the mutant initially has a reduced growth rate.…”

“…1), and modulation of LOS structure arises due to gene content diversity, allelic variation, and high-frequency sequence variation of homopolymeric tracts (10). The function of some genes in the cluster has been elucidated, and individual mutations in waaC and waaF have yielded deep rough mutants (7,8,16). Here, we describe the construction and phenotype of a mutant in which the core LOS biosynthesis gene cluster has been removed.…”

Phase-Variable Changes in the Position of O -Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance

“…Interestingly, CPS from the CJJ1420R CJJ1435 Ϫ mutant also contained a majority of 6d-altro-Hep in place of the typical 3-O-methyl-6d-altro-Hep normally found in 81-176. The reason for this change remains uncertain, but a similar shift in Hep composition in the 81-176 CPS was observed previously in a deep rough LOS mutant (31).…”

“…31 P-NMR analyses of the CPS used to generate the conjugate used to generate the hyperimmune serum used in this study indicated the presence of MeOPN-2-Gal only, and at the time of its synthesis, we were unaware of the additional modifications (data not shown). Moreover, subsequent examination of the seed strain used to produce the vaccine revealed that the levels of expression of CJJ1420 and CJJ1435 were similar to those reported here for the laboratory stock of 81-176 (data not shown).…”