Differential Distribution of the wlaN and cgtB Genes, Associated with Guillain-Barré Syndrome, in Campylobacter jejuni Isolates from Humans, Broiler Chickens, and Wild Birds

Abstract

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOS^SIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOS^SIAL and the ability to invade eukaryotic cells.

Keywords: Campylobacter; Guillain-Barré syndrome; cgtB; lipooligosaccharide; wlaN.

Figure 1

Prevalence of wlaN and cgtB genes in C. jejuni strains from different origins. H, human patients; B, broiler chickens; W, wild birds. Statistical analyses were performed using Pearson’s chi-squared test (R Studio software). p < 0.005 was considered statistically significant (indicated by an asterisk).

Figure 2

G-tract characterization within the wlaN and cgtB genes. (A) Homopolymeric G-tracts identified in wlaN and cgtB genes from human (H), broiler chicken (B), and wild bird strains (W). The different homopolymeric variants identified are indicated and the number of strains is shown between parentheses. (B) Sequence of the intragenic homopolymeric G-tracts (indicated by red lines) of the wlaN gene of the strains H11, H52, W20, and B50. The number of G residues within the G-tract are indicated between parentheses. (C) Sequence of the intragenic homopolymeric G-tracts (indicated by red lines) of the cgtB gene of the strains H58 and W27. The number of G residues within the G-tracts are indicated between parentheses. (D) Detection of LOS^SIAL in purified LOS samples from cultures of the indicated strains grown onto CBA plates for 48 h at 42 °C. LOS^SIAL was detected by Western blot using HRP-CT. Below, the presence for each strain of either the wlaN or cgtB gene is indicated and the G-tract variant detected. (E) Silver-stained 15% SDS-PAGE of the same samples of purified LOS as in D. (F) Coomassie-stained 15% SDS-PAGE of whole cell extracts from the cultures used to obtain the purified LOS samples analyzed in D and E. In E and F, the migration of the 10 and 15 kDa proteins from the molecular mass marker are indicated.

Figure 3

Analysis of LOS from C. jejuni strains grown at either 37 °C or 42 °C. (A). Detection of LOS^SIAL in purified LOS samples from cultures of the indicated strains grown onto CBA plates for 48 h at either 37 °C or 42 °C. LOS^SIAL was detected by Western blot using HRP-CT. (B). Silver-stained 15% SDS-PAGE of the same samples of purified LOS as in (A). (C). Coomassie-stained 15% SDS-PAGE of whole cell extracts from the cultures used to obtain the purified LOS samples analyzed in (A,B). In (B,C), the migration of the 10 and 15 kDa proteins from the molecular mass marker is indicated.

Figure 4

Relation between invasion index and LOS^SIAL production in C. jejuni strains. The invasion index was calculated as the percentage of intracellular cells from the total cell-associated bacteria after 3 h infection of Caco-2 cells with the indicated strains. The average and standard deviation of three independent experiments are shown. Grey and black bars indicate strains that express or do not express LOS^SIAL, respectively.