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.

对基因敲除小鼠的大量研究发现，微生物群组成会影响宿主表型。然而，当直接将 KO 小鼠与 WT 同窝小鼠进行比较时，这种差异可能会消失，这表明这些差异并不反映基因缺失本身，而是微生物群组成在几代人之间漂移。因此，我们的假设是缺乏二/三肽转运蛋白 PepT1 改变了微生物群组成，导致对结肠炎的抵抗力，这需要仔细审查。在这项研究中，我们使用了 PepT1 ^-/-和 WT 建立者小鼠，它们分别繁殖了多代。然后将此类小鼠彼此交配以产生 F1 PepT1 ^-/-和 WT 同窝仔，然后在其基因型内繁殖以产生 F2、F3 和 F4 后代。在这里我们报告创始人 PepT1 ^-/-相对于它们的 WT 对应物，小鼠对 DSS 结肠炎具有抗性。这种耐药性与肠道微生物群的改变有关，当移植到无菌小鼠时，肠道菌群的改变足以转移对结肠炎的耐药性。当比较 F1 PepT1 ^-/-与 F1 WT 同窝仔时，没有观察到这种差异，而是在随后的几代中逐渐恢复，因此，相对于他们的 F4 WT 对照，F4 PepT1 ^-/-显示的微生物群组成和结肠炎抗性表型几乎与创始人 PepT1 ^-/-老鼠。我们的研究结果表明 PepT1 在影响微生物群组成中的作用，从而影响结肠炎和癌症的易感性。总体而言，我们的研究表明，同窝小鼠控制的实验不足以评估微生物群依赖性表型并阻止对基因型驱动现象的全面理解。相反，单个基因改变对微生物群和宿主表型的影响可能需要几代人才能体现出来。