Impact of PepT1 deletion on microbiota composition and colitis requires multiple generations

Numerous studies of knockout mice find impacts on microbiota composition that influence host phenotype. However, such differences can vanish when KO mice are compared directly to WT littermates, suggesting these differences do not reflect the genetic deletion per se but microbiota composition drifting over generations. Hence, our hypothesis that absence of di/tri-peptide transporter PepT1 altered microbiota composition resulting in resistance to colitis compelled scrutiny. In this study, we used PepT1^-/- and WT founder mice bred separately for multiple generations. Such mice were then bred to each other to generate F1 PepT1^-/- and WT littermates, which were then bred within their genotype to generate F2, F3, and F4, offspring. Here we report that founder PepT1^-/- mice were, relative to their WT counterparts, resistant to DSS colitis. Such resistance was associated with alterations in gut microbiota, which, when transplanted to germfree mice, was sufficient to transfer resistance to colitis. Such differences were not observed when comparing F1 PepT1^-/- to F1 WT littermates but rather, returned gradually over subsequent generations such that, relative to their F4 WT controls, F4 PepT1^-/- displayed microbiota composition and colitis-resistant phenotype nearly identical to the founder PepT1^-/- mice. Our findings indicate a role for PepT1 in influencing microbiota composition and, consequently, proneness to colitis and cancer. Overall, our study indicates that littermate-controlled experiments can be insufficient for assessing microbiota-dependent phenotypes and prevent a full comprehension of genotype-driven phenomena. Rather, impact of a single genetic alteration on microbiota and host phenotype may take generations to manifest.