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Abstract：
Attaching/effacing (A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagic Escherichia coli. The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1, followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of replication protein A 32 in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.IMPORTANCEOxidative DNA lesions play causative roles in colitis-associated colon cancer. Accumulating evidence shows strong links between attaching/effacing (A/E) pathogens and colorectal cancer (CRC). EspF is one of many effector proteins exclusive to A/E pathogens with defined roles in the induction of oxidative stress, double-strand breaks (DSBs), and repair dysregulation. Here, we found that EspF promotes reactive oxygen species generation and 8-oxoguanine (8-oxoG) lesions when the repair system is activated, contributing to sustained cell survival. However, infected cells exposed to EspF presented 8-oxoG, which results in DSBs and ssDNA accumulation when the cell cycle is arrested at the G2/M phase and the repair system is defective or saturated by DNA lesions. In addition, we found that EspF could intensify the accumulation of nuclear DNA lesions through oxidative and replication stress. Overall, our work highlights the involvement of EspF in DNA lesions and DNA damage response, providing a novel avenue by which A/E pathogens may contribute to CRC.
摘要：
附着/去除(A/E)病原体通过经由III型分泌系统(T3SS)将效应蛋白注射到宿主细胞中来诱导DNA损伤和结直肠癌。EspF是A/E病原体特有的T3SS依赖性效应蛋白之一，其中包括肠出血性大肠杆菌。EspF在诱导双链断裂（DSB）和修复蛋白SMC1磷酸化中的作用先前已得到证明。然而,损伤积累和DSB形成的过程仍然是神秘的，并且损坏响应还没有得到很好的理解。这里,我们首先显示了错配修复蛋白MutS同源物2（MSH2）和MSH6以及聚（ADP-核糖）聚合酶1的代偿性增加，在存在EspF的情况下威胁细胞存活。流式细胞术显示EspF将细胞周期阻滞在G2/M期以促进DNA修复。随后，8-氧鸟嘌呤（8-oxoG）病变，氧化损伤的标志，通过ELISA和免疫荧光检测，揭示了8-oxoG从细胞质到细胞核的积累。此外，确认了单链DNA（ssDNA）和DSB的状态。我们观察到EspF加速了DNA损伤的进程，包括8-oxoG和未修复的ssDNA,转化为DSB；这伴随着修复缺陷细胞中复制蛋白A32的磷酸化。总的来说，这些发现表明，EspF引发各种类型的DNA氧化损伤，损害DNA损伤反应，并可能导致基因组不稳定和细胞死亡，为EspF的致瘤潜力提供了新的见解。重要氧化性DNA损伤在结肠炎相关结肠癌中发挥致病作用.越来越多的证据表明，附着/去除（A/E）病原体与结直肠癌（CRC）之间存在紧密联系。EspF是A/E病原体独有的许多效应蛋白之一，在诱导氧化应激中具有明确的作用，双链断裂(DSB)，修复失调。这里,我们发现，当修复系统被激活时，EspF促进活性氧的产生和8-氧鸟嘌呤（8-oxoG）病变，有助于持续的细胞存活。然而,暴露于EspF的感染细胞呈现8-oxoG，当细胞周期停滞在G2/M期并且修复系统因DNA损伤而缺陷或饱和时，会导致DSB和ssDNA积累。此外,我们发现EspF可以通过氧化和复制应激增强核DNA损伤的积累。总的来说,我们的工作强调了EspF参与DNA损伤和DNA损伤反应，提供了一种新的途径，A/E病原体可能导致CRC。
