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.

   Subject terms: Pathogens, Glycobiology
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   Garber et al. solved the structure of the fucose dehydrogenase FucX of
   the gastrointestinal pathogen Campylobacter jejuni. Although C. jejuni
   lacks fucosidases and prefers amino acids to carbohydrates, Bacteroides
   vulgatus enhances the growth of C. jejuni in vitro in the presence of
   mucin, suggesting that this pathogen may obtain sugars from the
   commensal microbiota.

Introduction

   Campylobacter jejuni is a common commensal in chickens and often
   transmitted to humans through consumption of undercooked or
   contaminated food products where it causes gastrointestinal infections
   that are generally self-limiting or treatable with antibiotics.
   However, post-infectious complications including Guillain-Barré
   syndrome, irritable bowel syndrome, reactive arthritis^[54]1, and
   growth stunting^[55]1,[56]2 can occur. Additionally, increasing rates
   of C. jejuni antibiotic resistance, particularly to fluoroquinolones,
   is a growing concern^[57]3.

   C. jejuni was once considered asaccharolytic since it lacks key enzymes
   from the Entner-Doudoroff and pentose phosphate pathways for
   carbohydrate metabolism. Most of its nutrition is derived from the
   amino acids serine, aspartate, asparagine, glutamate, and proline,
   which are readily abundant in the chicken gastrointestinal tract^[58]4.
   Additionally, some, but not all strains can utilize
   glutamine^[59]4,[60]5. Tricarboxylic acid cycle intermediates and short
   chain fatty acids regularly found in the gut as by-products of
   microbial metabolism are also used by C. jejuni^[61]4,[62]5 and their
   spatial distribution may be important for niche establishment in both
   commensal and pathogenic systems^[63]6.

   Carbohydrate utilization pathways have recently been described in
   certain Campylobacter species. For example, few isolates of C. coli and
   C. jejuni subsp. doylei possess enzymes for the full Entner-Doudoroff
   pathway and thus metabolize glucose^[64]7,[65]8. Also, we identified a
   functional fuc locus for l-fucose utilization (cj0480c-cj0489 in C.
   jejuni NCTC11168) that exists in ~65% of sequenced
   strains^[66]9,[67]10. Many structures in the gastrointestinal tract
   contain l-fucose, including mucins^[68]11, blood group antigens^[69]12,
   the capsules, and glycoproteins of microbial species such as
   Bacteroides^[70]13, dietary plant polysaccharides^[71]14, and human
   milk oligosaccharides^[72]15 in breastfed infants. l-fucose plays an
   important role in the health of the host and the maintenance of the
   associated microbiota, including microbiota-mediated resistance to
   pathogens^[73]16, protection from Crohn’s disease^[74]17, and as a
   functional receptor for cholera toxin^[75]18. l-fucose is also an
   important nutrient for the microbiota, whether the bacteria can obtain
   it directly from the host^[76]17,[77]19,[78]20 or rely on scavenging
   from their neighbors^[79]21. Furthermore, fucosylated structures can be
   important adherence targets^[80]17, which has led to several studies
   reporting that human milk oligosaccharides may protect against
   infection by acting as binding decoys^[81]15. Bacteroides spp. are
   known to secrete fucosidases and can influence host fucosylation to
   maximize fucose utilization for nutrition^[82]19. However, C. jejuni
   does not encode any obvious fucosidase homologs suggesting that C.
   jejuni may forage on l-fucose released by other members of the
   microbiota^[83]22,[84]23 as has been observed for Salmonella enterica
   serovar Typhimurium, Clostridium difficile^[85]24, and
   enterohemorrhagic Escherichia coli^[86]21.

   l-fucose enhances C. jejuni growth in minimal media and provides the
   strain with a colonization advantage in a piglet model of diarrheal
   disease^[87]9. Furthermore, we showed that strains possessing the fuc
   locus (fuc+) strains chemotax towards fucose, and the putative
   dehydrogenase Cj0485 is necessary for this response^[88]25. The fucose
   permease encoded by cj0486 (fucP) complements an Escherichia coli fucP
   mutant^[89]9. However, l-fucose metabolism in C. jejuni does not rely
   upon ATP or GTP^[90]9. Thus, in C. jejuni, l-fucose must be catabolized
   by a pathway different from the common phosphorylation-dependent
   pathways found in E. coli, Bacteroides thetaiotaomicron^[91]19,
   Lactobacillus rhamnosus^[92]26, S. Typhimurium, and Klebsiella
   pneumoniae^[93]27 and it is unlikely that l-fucose is used in C. jejuni
   capsular polysaccharides via the GDP-activation-dependent mechanism
   described for Bacteroides^[94]28.

   This work examines the crosstalk that may occur between commensal
   microbes and C. jejuni to enable l-fucose scavenging and provides
   evidence that C. jejuni can metabolize l-fucose released by commensal
   Bacteroides vulgatus. We also characterize the putative dehydrogenase
   Cj0485 through crystallography, enzymology, and multiple biological
   assays to confirm its function as a fucose dehydrogenase, which we have
   named FucX. This enzyme is also capable of reducing d-arabinose and is
   the sole component encoded by the fuc operon necessary for chemotaxis
   to both sugars. Nuclear magnetic resonance (NMR) and mutagenesis
   studies provide further insight into the mechanism of l-fucose
   breakdown and allowed us to propose a pathway for l-fucose and
   d-arabinose catabolism. We also investigate the impact of l-fucose on
   carbon source preferences by C. jejuni. Overall this study provides