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. 2000 May;68(5):2594-601.
doi: 10.1128/IAI.68.5.2594-2601.2000.

The galE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence

B N Fry  1 , S Feng, Y Y Chen, D G Newell, P J Coloe, V Korolik
Affiliations

The galE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence

B N Fry et al. Infect Immun. 2000 May.

Abstract

Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complemented galE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-type Salmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuni encoded this epimerase. We constructed a C. jejuni galE mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galE gene in the synthesis of C. jejuni LPS. The galE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

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Figures

FIG. 1
FIG. 1
Alignment of the deduced amino acid sequences for GalE from C. jejuni (Cj), Salmonella serovar Typhimurium (St), H. influenzae (Hi), E. coli (Ec), and N. meningitidis (Nm), performed with the program Macaw. Shaded letters indicate identical amino acids. The conserved NAD-binding domain (GxxGxxG [46]) and essential amino acids are boxed.
FIG. 2
FIG. 2
LPS analysis by tricine SDS-PAGE of LPS prepared from serovar Typhimurium strains. LPS was visualized by silver staining. Lanes: 1, SL761; 2, control SL761(pB1); 3, SL761(pBF84And47); 4, parent strain SL696.
FIG. 3
FIG. 3
Southern blot analysis of chromosomal DNA preparations from C. jejuni wild-type and galE mutant strains. Chromosomal DNAs were digested with HindIII, resolved by agarose gel electrophoresis, and probed with plasmid pBF84Bnd17 containing the C. jejuni galE gene. Lane 1, wild-type strain 81116; lane 2, strain 81116galEa; lane 3, strain 81116galEb.
FIG. 4
FIG. 4
LPS separation by Tricine SDS-PAGE. (A) Silver-stained LPS samples; (B) immunoblot with C. jejuni 81116 LPS antiserum. Lanes: 1, parent strain 81116; 2, strain 81116galEa; 3, strain 81116galEb.
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