increased expression of PSA in vivo(Fig. 2C). While phase variation of capsular polysaccharides is known to influence general in vivo fitness of B. fragilis(29, 30), these udies identify a pathway for transcriptional regulation of specific polysaccharides in the context of mucosal colonization We modeled single-strain stability using a horizontal transmission assay, wherein co-housing animals respectively harboring isogenic strains of wild-type B. fragilis resulted in minimal strain transmission from one animal to another( Fig. 2D, S2A). This intra-species colonization resistance is provided through bacterial occupation of a species-specific nutrient or spatial niche(26). However, as previously reported (26), if mice are colonized initially with B. fragilis Accf animals were permissive to co-colonization by wild-type B fragilis after co-housing(Fig. 2E, S2B), indicating a CCF-dependent defect in niche saturation Mice harboring a mutant in the biosynthesis genes for PSC(APSC) showed highly variable co-colonization by wild-type bacteria( Fig. 2F, S2C). Interestingly, we observed an unexpected increase in expression of the PSB biosynthesis genes in this mutant (Fig. 21), which may compensate for the loss of Psc. We generated a strain defective in synthesizing both PSB and PSC (APSB/C), and mice mono-associated with the double mutant were consistently unable to maintain colonization resistance(Fig. 2G, S2D-F), though the strain retained ccfexpression(fig. S2G) Despite lack of competition in a mono- colonized setting and equal levels of colonization in the colon lumen(fig. S2H), the B fragilis Accfand APSB/C strains were defective in colonization of the ascending colon mucus(Fig. 21), reflecting impaired saturation of the mucosal niche. Accordingly, when we maged the PSB/C strain in vivo employing TEM, though the capsule was not as thin as in B. fragilis Accf(fig. S2I and ) the hallmark epithelial aggregation phenotype was abrogated compared to wild-type bacteria(fig. S2K and L). Therefore, we conclude that the CCF system regulates capsule expression to mediate B. fragilis mucosal colonization and single To investigate host responses contributing to mucos transcriptome of the ascending colon during colonization with wild-type B. fragilis or B Accf Remarkably, 7 of the 14 differentially expressed genes encode immunoglobulin responses in Accfcolonized mice(fig. S3A), indicating that changes in mucosal association regulation by ccfaffects IgA recognition of bacteria(31-33). In fecal samples from mono- diminished in Accfand APSB/C strains(Fig. 3B, 3C, and S4A). We observed no difference between these strains in the induction of total fecal IgA(Fig. 3D), reflecting equivalent imulation of nonspecific IgA production (10, 34, 35). To test bacteria-specific responses, IgA extracted from feces of mice mono-colonized with B. fragilis was evaluated for binding to bacteria recovered from mono-colonized Rag/* mice(in vivo-adapted, yet IgA-free bacteria). Western blots of bacterial lysates showed that strong IgA reactivity to capsular polysaccharides was abrogated in the Accf and APSB/C strains( Fig. 3E and F). Although IgA can be polyreactive(10, 34, 35), binding to lysates of Bacteroides was species-specific (fig. S4B)and required induction of IgA following bacterial colonization(fig. $4C and D) Accordingly, in a whole bacteria binding assay, IgA induced by wild-type bacteria cience. Author manuscript; available in PMC 2018 November 18increased expression of PSA in vivo (Fig. 2C). While phase variation of capsular polysaccharides is known to influence general in vivo fitness of B. fragilis (29, 30), these studies identify a pathway for transcriptional regulation of specific polysaccharides in the context of mucosal colonization. We modeled single-strain stability using a horizontal transmission assay, wherein co-housing animals respectively harboring isogenic strains of wild-type B. fragilis resulted in minimal strain transmission from one animal to another (Fig. 2D, S2A). This intra-species colonization resistance is provided through bacterial occupation of a species-specific nutrient or spatial niche (26). However, as previously reported (26), if mice are colonized initially with B. fragilis Δccf, animals were permissive to co-colonization by wild-type B. fragilis after co-housing (Fig. 2E, S2B), indicating a CCF-dependent defect in niche saturation. Mice harboring a mutant in the biosynthesis genes for PSC (ΔPSC) showed highly variable co-colonization by wild-type bacteria (Fig. 2F, S2C). Interestingly, we observed an unexpected increase in expression of the PSB biosynthesis genes in this mutant (Fig. 2H), which may compensate for the loss of PSC. We generated a strain defective in synthesizing both PSB and PSC (ΔPSB/C), and mice mono-associated with the double mutant were consistently unable to maintain colonization resistance (Fig. 2G, S2D–F), though the strain retained ccf expression (fig. S2G). Despite lack of competition in a mono￾colonized setting and equal levels of colonization in the colon lumen (fig. S2H), the B. fragilis Δccf and ΔPSB/C strains were defective in colonization of the ascending colon mucus (Fig. 2I), reflecting impaired saturation of the mucosal niche. Accordingly, when we imaged the ΔPSB/C strain in vivo employing TEM, though the capsule was not as thin as in B. fragilis Δccf (fig. S2I and J), the hallmark epithelial aggregation phenotype was abrogated compared to wild-type bacteria (fig. S2K and L). Therefore, we conclude that the CCF system regulates capsule expression to mediate B. fragilis mucosal colonization and single strain stability. To investigate host responses contributing to mucosal colonization, we defined the transcriptome of the ascending colon during colonization with wild-type B. fragilis or B. fragilis Δccf. Remarkably, 7 of the 14 differentially expressed genes encode immunoglobulin variable chains (Fig. 3A and table S2). We did not observe any elevation of immune responses in Δccf–colonized mice (fig. S3A), indicating that changes in mucosal association are not caused by inflammation. Accordingly, we tested whether capsular polysaccharide regulation by ccf affects IgA recognition of bacteria (31–33). In fecal samples from mono￾colonized animals, wild-type B. fragilis was highly coated with IgA, which was significantly diminished in Δccf and ΔPSB/C strains (Fig. 3B, 3C, and S4A). We observed no difference between these strains in the induction of total fecal IgA (Fig. 3D), reflecting equivalent stimulation of nonspecific IgA production (10, 34, 35). To test bacteria-specific responses, IgA extracted from feces of mice mono-colonized with B. fragilis was evaluated for binding to bacteria recovered from mono-colonized Rag1 −/− mice (in vivo-adapted, yet IgA-free bacteria). Western blots of bacterial lysates showed that strong IgA reactivity to capsular polysaccharides was abrogated in the Δccf and ΔPSB/C strains (Fig. 3E and F). Although IgA can be polyreactive (10, 34, 35), binding to lysates of Bacteroides was species-specific (fig. S4B) and required induction of IgA following bacterial colonization (fig. S4C and D). Accordingly, in a whole bacteria binding assay, IgA induced by wild-type bacteria Donaldson et al. Page 3 Science. Author manuscript; available in PMC 2018 November 18. Author Manuscript Author Manuscript Author Manuscript Author Manuscript