Yuting Fang, Muqing Fu, Xinyue Li, Bao Zhang, Chengsong Wan
{"title":"肠出血性大肠杆菌效应因子 EspF 触发肠上皮细胞 DNA 氧化损伤。","authors":"Yuting Fang, Muqing Fu, Xinyue Li, Bao Zhang, Chengsong Wan","doi":"10.1128/iai.00001-24","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>Escherichia coli</i>. 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.</p>","PeriodicalId":13541,"journal":{"name":"Infection and Immunity","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11003234/pdf/","citationCount":"0","resultStr":"{\"title\":\"Enterohemorrhagic <i>Escherichia coli</i> effector EspF triggers oxidative DNA lesions in intestinal epithelial cells.\",\"authors\":\"Yuting Fang, Muqing Fu, Xinyue Li, Bao Zhang, Chengsong Wan\",\"doi\":\"10.1128/iai.00001-24\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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 <i>Escherichia coli</i>. 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. 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引用次数: 0
摘要
附着/脱落(A/E)病原体通过 III 型分泌系统(T3SS)向宿主细胞注入效应蛋白,从而诱发 DNA 损伤和结直肠癌。EspF是A/E病原体(包括肠出血性大肠杆菌)独有的依赖T3SS的效应蛋白之一。之前已经证实了 EspF 在诱导双链断裂(DSB)和修复蛋白 SMC1 磷酸化中的作用。然而,损伤积累和 DSB 形成的过程仍是一个谜,损伤反应也不甚了解。在这里,我们首先发现了错配修复蛋白 MutS homolog 2(MSH2)和 MSH6 以及聚(ADP-核糖)聚合酶 1 的代偿性增加,随后急剧减少,这威胁到了存在 EspF 的细胞的存活。流式细胞仪显示,EspF 将细胞周期阻滞在 G2/M 阶段,以促进 DNA 修复。随后,用酶联免疫吸附和免疫荧光法测定了氧化损伤的标志物--8-氧鸟嘌呤(8-oxoG)病变,结果显示 8-oxoG 从细胞质积累到了细胞核。此外,单链 DNA(ssDNA)和 DSB 的状况也得到了证实。我们观察到,EspF 加快了 DNA 病变的进程,包括 8-oxoG 和未修复的 ssDNA,它们被转化为 DSB;在修复缺陷细胞中,这伴随着复制蛋白 A 32 的磷酸化。总之,这些研究结果表明,EspF 会引发各种类型的 DNA 氧化病变,损害 DNA 损伤反应,并可能导致基因组不稳定和细胞死亡,从而为了解 EspF 的致癌潜力提供了新的视角。越来越多的证据表明,附着/脱落(A/E)病原体与结直肠癌(CRC)之间存在密切联系。EspF是A/E病原体独有的多种效应蛋白之一,在诱导氧化应激、双链断裂(DSB)和修复失调方面具有明确的作用。在这里,我们发现当修复系统被激活时,EspF 会促进活性氧的生成和 8-氧鸟嘌呤(8-oxoG)病变,从而有助于细胞的持续存活。然而,当细胞周期停滞在G2/M期,修复系统出现缺陷或DNA病变饱和时,暴露于EspF的感染细胞会出现8-oxoG,导致DSB和ssDNA积累。此外,我们还发现 EspF 能通过氧化和复制应激加剧核 DNA 病变的积累。总之,我们的工作强调了EspF参与DNA病变和DNA损伤反应,为A/E病原体可能导致CRC提供了一条新途径。
Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells.
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.
期刊介绍:
Infection and Immunity (IAI) provides new insights into the interactions between bacterial, fungal and parasitic pathogens and their hosts. Specific areas of interest include mechanisms of molecular pathogenesis, virulence factors, cellular microbiology, experimental models of infection, host resistance or susceptibility, and the generation of innate and adaptive immune responses. IAI also welcomes studies of the microbiome relating to host-pathogen interactions.