The gastrointestinal pathogen Campylobacter jejuni metabolizes sugars with potential help from commensal Bacteroides vulgatus

Abstract

Although the gastrointestinal pathogen Campylobacter jejuni was considered asaccharolytic, >50% of sequenced isolates possess an operon for L-fucose utilization. In C. jejuni NCTC11168, this pathway confers L-fucose chemotaxis and competitive colonization advantages in the piglet diarrhea model, but the catabolic steps remain unknown. Here we solved the putative dehydrogenase structure, resembling FabG of Burkholderia multivorans. The C. jejuni enzyme, FucX, reduces L-fucose and D-arabinose in vitro and both sugars are catabolized by fuc-operon encoded enzymes. This enzyme alone confers chemotaxis to both sugars in a non-carbohydrate-utilizing C. jejuni strain. Although C. jejuni lacks fucosidases, the organism exhibits enhanced growth in vitro when co-cultured with Bacteroides vulgatus, suggesting scavenging may occur. Yet, when excess amino acids are available, C. jejuni prefers them to carbohydrates, indicating a metabolic hierarchy exists. Overall this study increases understanding of nutrient metabolism by this pathogen, and identifies interactions with other gut microbes.

Fig. 1. C. jejuni and B. vulgatus use of l-fucose and mucin degradation.

a Fucosidase activity detected by cleavage of 4-nitrophenyl-α-l-fucopyranoside measured by absorbance at 405 nm after 18 h incubation with the substrate under appropriate bacterial growth conditions (gray bars are at t = 0 h and white bars are at t = 18 h). Values represent means of three technical replicates and error bars show one standard deviation. Values from each replicate are overlaid as dots. The asterisk indicates a significant increase in absorbance (p = 0.02) compared to t = 0. b Ratios of C. jejuni (Cj) wildtype and fucP mutant in co-culture with B. vulgatus (Bv) and mucin (Muc) in minimal essential medium α. Values of bars represent means from eight biological replicates and error bars show one standard of the mean. Values from each replicate are overlaid as dots. Asterisks denote significance with p-values denoted in parentheses as determined by two-way ANOVA with Tukey post-test. c GC-MS chromatogram and spectrum (inset) of l-fucose released from mucin treated with B. vulgatus outer membrane vesicles (OMVs). d GC-MS chromatogram and spectrum (inset) of the l-fucose standard used to confirm the identity of l-fucose in the OMV-treated mucin sample. Fragment ions (Fuc1 and 2) are highlighted in red and exist in both α- and β- configurations and correspond to expected fragmentation patterns of fucose illustrated in Supplementary Fig. 1b.

Fig. 2. FucX crystal structure.

a The tetrameric organization of FucX observed in complex with NADP⁺. Two monomers are shown in blue and yellow cartoon representation and the other two in grey with transparent solvent accessible surfaces. Bound NADP⁺ is shown as green sticks. b Cartoon representation of the FucX monomer with β-strands shown in yellow, α-helices shown in purple, and loops shown in gray. Bound NADP⁺ is shown as green sticks. c Structural alignment of C. jejuni FucX (in blue) in complex with NADP⁺ (in cyan) with the homolog from Burkholderia multivorans (4GVX, in yellow) in complex with NADP+ (in orange) and l-fucose (in gray). d Close-up of the active site alignment of FucX (in blue) in complex with NADP+ (in cyan) with the homolog from B. multivorans (4GVX, in yellow) in complex with NADP+ (in orange) and L-fucose (in gray). Dashed lines indicate hydrogen bonds. E) Chair conformations of α-l-fucose and β-d-arabinose.

Fig. 3. Phenotypic effects of d-arabinose.

a d-arabinose (25 mM) enhances wild-type C. jejuni 11168 growth (gray bars) relative to growth in unsupplemented minimal essential medium alpha (white bars) and this growth advantage is lost in the key metabolic mutants of the l-fucose pathway. Values of bars represent means (n = 4 and error bars show one standard deviation). Values of individual replicates are overlaid as dots. Asterisks indicate a significant increase in growth in the presence of d-arabinose and p-values from a two-tailed paired Student’s t-test are indicated in parentheses. b C. jejuni 11168 wild-type displays chemotaxis (indicated by pink color development and + sign) to d-arabinose and this is lost in a fucX mutant strain. Similarly, C. jejuni 81–176 wildtype does not show chemotaxis to l-fucose and complementation with fucX confers this ability. l-fucose serves as a FucX-dependent control, L-serine serves as an independent positive control, and PBS serves as a negative control. c d-arabinose reduces transport of radiolabelled l-fucose into fucose-utilizing cells (11168 wildtype and 81–176Ωfuc) more than other aldoses in both uninduced (white bars) and cells pre-grown with 20 mM l-fucose (gray bars). Values represent means of two biological and two technical replicates, standard deviations are indicated by error bars, and values from individual replicates are overlaid as dots.

Fig. 4. l-fucose uptake by C. jejuni 11168 grown in the presence of alternate carbon sources.

a ³H-l-fucose uptake rates in cells of the wildtype and b the fucR::cm mutant grown in medium supplemented with different concentrations (5 = 5:1 ratio supplement to fucose, 1 = 1:1 ratio supplement to fucose, 0.2 = 1:5 ratio supplement to fucose) of other carbon sources in the presence (gray) or absence (white) of l-fucose is shown. ser, l-serine; asp, aspartic acid; glu, glutamic acid. Each bar represents the mean value from two biological and two technical replicates, standard deviations are indicated by error bars.

Fig. 5. Predicted l-fucose/d-arabinose metabolic pathway.

Model was developed based on homology to other characterized enzymes. The activity of FucX has been experimentally verified as has the production of l-fuconic acid. Inner molecules trace the products formed by l-fucose catabolism and outer molecules are from d-arabinose catabolism. The letters underneath each enzyme name denote the mutant growth phenotype (G = mutant has a growth enhancement on l-fucose and d-arabinose, N = no growth enhancement as determined either in the present or previous study).