Characterizing Human Milk Oligosaccharides Utilization by Infant Gut Bacteroides Species

Human milk oligosaccharides (HMOs) are a family of glycans found in breastmilk. Despite being t​​he third-largest solid component in breastmilk, HMOs are indigestible by infants, and they serve as food for the infant gut bacteria. Most research thus far has focused on Bifidobacterium species, yet there is increasing evidence that additional microbes in the infant gut, such as Bacteroides, are able to break-down HMOs as well. Here, we developed an optimized system for isolating Bacteroides strains from infant stool samples. We then examined the HMO utilization capacity of multiple Bacteroides isolates by performing growth curves on six common HMOs (2’-FL, DFL, 3’-SL, 6’-SL, LNT, LNnT), identifying variation across strains. Isolates displayed similar growth characteristics on similarly-structured HMOs, like sialylated or fucosylated sugars.

We chose to focus here on a Bacteroides dorei isolate that was able to utilize the test HMOs. Next, we performed RNA-sequencing on B. dorei cultures, comparing the transcriptional profile in minimal media supplemented with glucose or HMOs. B. dorei exhibits an extensive and generalized response to HMOs, up-regulating several shared glycoside hydrolase (GH) families across all conditions. This response pattern to HMOs has yet to be described in other commensals of the infant gut. Additionally, we found that polysaccharide utilization locus (PUL) 32 was the only PUL for which all genes were consistently expressed, especially for sialylated carbohydrates. To further prove the significance and function of PUL32, we established a CRISPR-based genetic manipulation system for Bacteroides in our lab and knocked-out PUL32 in B. dorei.