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Abstract
Campylobacter jejuni (Cj) is a gastrointestinal pathogen causing diarrhea in humans. Over 60% of sequenced Cj isolates possess fuc operon necessary for L-fucose metabolism and providing colonization advantage in piglet model of diarrheal disease. Human breastmilk contains high concentrations of human milk oligosaccharides (HMOs). HMOs are extensively fucosylated and L-fucose is readily released by gut commensals allowing L-fucose concentrations to reach up to 4-5mg/g feces. We previously reported that in fecal samples of infants from low-to-middle income countries, the proportion of Cj/Campylobacter coli in breastfed infants with diarrhea was significantly higher than non-breastfed infants. Furthermore, isolation of asaccharolytic Cj was significantly higher than saccharolytic Cj in breastfed infants. We hypothesized that L-fucose chemotaxis attracts saccharolytic Cj towards L-fucose released from HMOs and the bacteria are subsequently cleared from the gut. Our lab reported that FucX, L-fucose dehydrogenase, is necessary for L-fucose chemotaxis in Cj. My studies demonstrated that FucX alters the NADP+/NADPH ratio by converting the NADP+ cofactor to NADPH when oxidizing L-fucose to L-fuconolactone. Subsequently, NADPH reduces the FAD cofactor, associated with the CetABC energy taxis system, causing Cj to move toward the perceived energy source.
To understand what pathways are involved in Cj metabolism of human breastmilk, we incubated Cj11168 and Cj81-176 in 100% milk and analyzed their transcriptome. The results showed increased expression of pathways for the utilization of amino acids, iron, and ribosomal proteins, but Cj could not persist in milk. Thus, we evolved them with human breastmilk until the strains showed stable growth in milk. Genome sequencing of evolved strains showed mutations in the acyl carrier protein, AcpP and major outer membrane porin, PorA in both strains. Introduction of acpP and porA mutations into the wildtype strains indicated that acpP mutations play a more prominent role in Cj resistance to human milk. Furthermore, proteinaceous compounds in milk were found to inhibit Cj growth with fats possibly having a synergistic effect. Fractionation experiments are planned to identify the inhibiting compound(s) in human breastmilk, and oral gavaging studies in our diarrheal mouse model will aim to understand the impact of human breastmilk and L-fucose on Cj colonization.