High-Throughput Single-Cell Sequencing of B. Fragilis Populations in the Mouse Gut

[1] Christine Stark, Freeman Lan [2], Ophelia Venturelli [2]
1. North Carolina State University, 2. University of Wisconsin - Madison

Background:
B. fragilis is a gram-negative gut commensal with eight capsular polysaccharides which could be
expressed depending upon promoter expression. Capsular polysaccharides are essential as they
contribute to the harmfulness of many gut pathogens as these capsular polysaccharides are able
to hide cell-surface components which could trigger an immune response from the host [1]. This
prompts the question regarding which capsular polysaccharides are selected for when the host
undergoes various stressors. Through the use of high-throughput single-cell sequencing,
promoter inversion and show was determined to be diverse in B. fragilis populations due to many
capsular polysaccharide promoter states [2].

Results:
On day 29 the mice were inoculated with communities, thus increasing competition and selection
within the mouse gut; consequently, variation decreased. By day 33 compositions with initially
high capsular polysaccharide A proportions begin to experience less selection. This indicates a
chance that capsular polysaccharide A does not play a significant role in the mouse gut.
Furthermore, by day 33 those with low capsular polysaccharide H proportions begin to
experience increased selection; thus, indicating the plausibility of capsular polysaccharide H
playing a prevalent role in the mouse gut. Furthermore, capsular polysaccharides B and E seem
to have a relationship as both proportions individually are low in the mouse gut as time
progresses; however, when capsular polysaccharides B and E promoters are turned on in the
same composition the proportion is exceptionally high. These results were confirmed when
sequencing of the biological replicate occurred. On day 43, ATC is introduced into the mice's
water supply. Furthermore, the introduction of ATC induced promoter scrambling resulting in
increased variation.

Conclusions:
When looking at the data as a whole there is a chance that skewing occurred in the analysis
process as those compositions containing ambiguities either in the form of “X” or “U” were
removed. This could greatly affect the proportions and could account for some of the
unexpected results. The next steps for this project include running the DoTA-seq workflow on
the other experimental groups. Once compositions are obtained for all the experimental groups
big picture conclusions can be made in regard to how the state/health of the mouse gut impacts
capsular polysaccharide composition proportions.