BACKGROUND & AIMS Colibactin-producing Escherichia coli (CoPEC) colonize the colon mucosa of a higher proportions of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice with and without IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by histology, immunoblot, and quantitative reverse transcription polymerase chain reaction analyses for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

中文翻译：

---

肠道上皮细胞的自噬抑制了在ApcMin / +小鼠中产生Colibactin的大肠杆菌诱导的大肠癌发生。

背景与目的产生colibactin的大肠杆菌（CoPEC）在患有结肠直肠癌（CRC）和未患有结肠直肠癌（CRC）的患者中，结肠结肠黏膜的比例更高，并在易感的CRC小鼠模型中促进结肠直肠癌的发生。自噬通过溶酶体降解细胞质含量，包括细胞内病原体，并调节肠内稳态。我们调查了抑制自噬是否会影响感染CoPEC的易感小鼠的结直肠癌发生。方法用从患者体内分离的CoPEC株（11G5株）或不产生大肠杆菌素的突变株（11G5ΔclbQ）感染人肠上皮细胞（IECs）（HCT-116）。使用小的干扰RNA在HCT-116细胞中降低了ATG5，ATG16L1和SQSTM1（也称为p62）的水平。将具有和没有IEC特异性破坏Atg16l1的ApcMin / +小鼠（ApcMin / + /Atg16l1ΔIEC）感染11G5或11G5ΔclbQ。从小鼠收集结肠组织，并分析肿瘤大小和数量，并通过组织学，免疫印迹和定量逆转录聚合酶链反应分析来分析自噬，DNA损伤，细胞增殖和炎症的标志物。我们通过定量逆转录聚合酶链反应分析了来自散发性CRC患者结肠癌粘膜组织中自噬中涉及的自噬蛋白的信使RNA（mRNA）的水平，其中散发性CRC患者被定植于或未定植于CoPEC。结果与没有这些细菌的结肠粘膜相比，具有CoPEC克隆的患者结肠粘膜具有更高的编码自噬蛋白的mRNA水平。用11G5感染培养的IEC会诱导自噬和DNA损伤修复，而使用11G5ΔclbQ则不会。击倒HCT-116细胞中的ATG5会增加细胞内11G5的数量，白介素（IL）6和IL8的分泌以及DNA双链断裂的标记，但DNA修复的标记减少，这表明自噬是细菌诱导的DNA损伤所必需的。修理。击倒HCT-116细胞中的ATG5可增加11G5诱导的衰老，促进未感染细胞的增殖。与ApcMin / +小鼠相比，ApcMin / + /Atg16l1ΔIEC小鼠发生的结肠肿瘤越来越少。但是，感染11G5后，与感染的ApcMin / +小鼠相比，ApcMin / + /Atg16l1ΔIEC小鼠出现了更多，更大的肿瘤，平均组织学评分显着提高。Il6，Tnf和Cxcl1 mRNA的水平升高，Il10 mRNA的水平降低，与感染11G5的ApcMin / +小鼠相比，在感染11G5的ApcMin / + /Atg1611ΔIEC小鼠的结肠粘膜中观察到了DNA标记和DNA双链断裂和增殖的增加标记。结论用CoPEC感染IEC和易感小鼠可促进自噬，这是预防结直肠肿瘤发生所必需的。IEC中ATG16L1的缺失会增加ApcMin / +小鼠中炎症，DNA损伤和细胞增殖的标志物，并增加结肠直肠肿瘤的发生。这些发现表明自噬对CoPEC感染的响应的重要性，并且可能为CRC和CoPEC定植的患者制定诱导自噬的策略。结论用CoPEC感染IEC和易感小鼠可促进自噬，这是预防结直肠肿瘤发生所必需的。IEC中ATG16L1的缺失会增加ApcMin / +小鼠中炎症，DNA损伤和细胞增殖的标志物，并增加结肠直肠肿瘤的发生。这些发现表明自噬对CoPEC感染的反应的重要性，并且可能为CRC和CoPEC定植的患者制定诱导自噬的策略。结论用CoPEC感染IEC和易感小鼠可促进自噬，这是预防结直肠肿瘤发生所必需的。IEC中ATG16L1的缺失会增加ApcMin / +小鼠中炎症，DNA损伤和细胞增殖的标志物，并增加结肠直肠肿瘤的发生。这些发现表明自噬对CoPEC感染的反应的重要性，并且可能为CRC和CoPEC定植的患者制定诱导自噬的策略。