**4. CST Regulation of Gut Microbiota**

*4.1. Microbiomes in Colonic Mucosa versus Feces*

Recent studies have identified a larger role of gut microbiota in gut-immune homeostasis and in intestinal pathology. The human intestinal microbiota is dominated by five phyla: high-abundant (>80%) (1) Bacillota (aka Firmicutes) and (2) Bacteroidota; lessabundant (3) Actinomycetota (aka Actinobacteria), (4) Pseudomonadota (aka Proteobacteri), and (5) Verrucomicrobiota [76] as compared to four phyla in mice: high-abundant Bacteroidota, Bacillota, Deferribacterota, and Pseudomonadota [77]; and (4) low-abundant Actinomycetota and Verrucomicrobiota compared to humans. In mouse colonic mucosa samples, 19 phyla were identified [78] (Figure 6). Although CST failed to alter bacterial populations in the four high-abundant phyla, it altered colonic mucosa-associated bacterial community composition at lower taxonomic levels, including orders Bacteroidales, Clostridiales, and YS2, and Families Chitinophagaceae, Clostridiaceae, Coriobacteriaceae, Pseudomonadaceae, Rikenellaceae, and Ruminococcaceae [78]. While CST increased the relative abundance of Bacteroidota, it caused a marked decrease in the Bacillota population (Figure 6). *Bacteroides* and *Parabacteroides* species, representing ~25% of the colonic microbiota, transform simple and complex sugars into volatile short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate [79–81], which are absorbed in the colon as a nutrient [82,83]. In addition to colonic nutrients, SCFAs are well established for their roles in accelerating gut transit time via the release of serotonin [84,85]. SCFAs also release glucagon-like peptide 1 from the enteroendocrine L-cells [86–88] and improve insulin sensitivity [89–91]. *Bacteroides thetaiotaomicron* produces significant amounts of glycosylhydrolases, which prevent obesity [92]. Other *Bacteroides* species are also reported to prevent obesity and increase insulin sensitivity [93,94]. Furthermore, *Bacteroides fragilis* produces zwitterionic polysaccharide, which activates CD4+ T cells to produce interleukin 10 (IL-10). IL-10 plays crucial roles in preventing abscess formation and other unchecked inflammatory responses [95,96]. The functional correlation between different CST mutants across species and their respective microbiota has remained elusive.


**Figure 6.** Abundance of bacterial species in mucosal and fecal samples in WT and CST-KO mice in presence or absence of CST as well as after fecal microbial transplant. Green arrows indicate CST effects; red arrows indicate FMT effects.

#### *4.2. Microbiomes in CST Knockout (CST-KO) Mice and Inflammation*

CST knockout (CST-KO) mice were generated in 2018 and are: insulin-resistant on a normal chow diet [97], hyperadrenergic [98], hypertensive [98], and with a leaky gut [99]. The microbiome in CST-KO mice was found to be quite different in composition than its WT littermates [99]. Microbial richness revealed a significant decrease in CST-KO compared to WT mice [100]. (Figure 7). Surprisingly, Verrucomicrobiota population was very low in CST-KO mice, indicating low levels of *Akkermansia* species. Since *A. muciniphila* modulates obesity by regulating metabolism and energy homeostasis to improve insulin sensitivity and glucose homeostasis [101], low Verrucomicrobiota population possibly contributed to the insulin resistance reported for CST-KO mice [97].
