scholarly journals Identification of a novel interaction between SMC1 DNA damage repair protein and Escherichia coli O157: H7 EspF using co-immunoprecipitation combined with mass spectrometry

2020 ◽  
Author(s):  
Muqing Fu ◽  
Ying Hua ◽  
Kaina Yan ◽  
Jia Li ◽  
Jiali Wu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Damage Repair ◽  
Colorectal Carcinogenesis ◽  
Host Cells ◽  
Phase Cell ◽  
Host Proteins ◽  
C Terminus ◽  
Damage Repair

Abstract Background: It is known that the enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interaction between EspF or its N/ C-terminus with host proteins are still unclear. Results: In this study, we used co-immunoprecipitation combined with mass spectrometry to screen EspF-interacting proteins. A total of 311 host proteins are detected. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus of EspF. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. We verify for the first time that SMC1 interacts with EspF and more likeliy by its C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1, known to reduce the S-phase cell cycle arrest and DNA damage repair. Surprisingly, we screen that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage through SMC1 phosphorylation.Conclusion: Taken together, this is the first study describing the interaction between EspF and SMC1. Our work lays a foundation for further research on directly EspF-mediated host cells’ DNA damage, apoptosis, and even colorectal carcinogenesis.

scholarly journals Identification of a novel interaction between SMC1 DNA damage repair protein and Escherichia coli O157: H7 EspF using co-immunoprecipitation combined with mass spectrometry

2020 ◽  
Author(s):  
Muqing Fu ◽  
Ying Hua ◽  
Kaina Yan ◽  
Jia Li ◽  
Jiali Wu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Dna Damage ◽  
Rna Splicing ◽  
Colorectal Carcinogenesis ◽  
Host Cells ◽  
Phase Cell ◽  
Host Proteins ◽  
C Terminus ◽  
Damage Repair

Abstract Background: It is known that the enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interaction between EspF, its N- or C-terminus, and host proteins, are still unclear. Results: In this study, we used co-immunoprecipitation combined with mass spectrometry to screen EspF-interacting proteins. A total of 311 host proteins are detected. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus of EspF. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. We verify here for the first time that SMC1 interacts with EspF and more strongly with its C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1, known to influence the S-phase cell cycle arrest and usually express during periods of DNA damage. Surprisingly, Mass spectrometry reveals that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage through SMC1 phosphorylation.Conclusion: Taken together, this is the first study describing the interaction between EspF and SMC1. Our work lays a foundation for further research on directly EspF-mediated host cells’ DNA damage, apoptosis, and even colorectal carcinogenesis.

scholarly journals Identification of a Novel Interaction Between SMC1 DNA Damage Repair Protein and Escherichia Coli O157: H7 EspF Using Co-Immunoprecipitation Combined With Mass Spectrometry

2020 ◽  
Author(s):  
Muqing Fu ◽  
Ying Hua ◽  
Jiali Wu ◽  
Zhikai Zhang ◽  
Jinyue Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Dna Damage ◽  
Enterohemorrhagic Escherichia Coli ◽  
Colorectal Carcinogenesis ◽  
Host Protein ◽  
Host Cells ◽  
Phase Cell ◽  
Host Proteins ◽  
C Terminus

The enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is known to be a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interactions between EspF, its N- or C-terminus, and host proteins are still unclear. In this work, we use co-immunoprecipitation combined with mass spectrometry (CoIP-MS) to screen the interactions between EspF/EspF-N/EspF-C terminus and host proteins. A total of 311 host proteins are analyzed. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. Surprisingly, MS results reveal that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage. Here, by western blot and immunofluorescence (IF), we verified that EspF can cause phosphorylation of H2AX and mediates cell multi-nuclearation and cell hypertrophy. Furthermore, we verify here for the first time that SMC1 interacts with EspF -C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1 is known to influence S-phase cell cycle arrest and usually increases during periods of DNA damage. Our work revealed a novel interaction between EspF and the host protein SMC1 and lays a foundation for further research on EspF-mediated host DNA damage, apoptosis, and even colorectal carcinogenesis.

scholarly journals EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells

2019 ◽  
Vol 116 (28) ◽  
pp. 14144-14153 ◽  
Author(s):  
Merrin Man Long Leong ◽  
Arthur Kwok Leung Cheung ◽  
Wei Dai ◽  
Sai Wah Tsao ◽  
Chi Man Tsang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Excision Repair ◽  
Dna Damage Repair ◽  
Promoter Hypermethylation ◽  
Host Cells ◽  
Independent Manner ◽  
Ebv Infection ◽  
Damage Repair

Epstein−Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members,MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression ofMLH1correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.

scholarly journals The Escherichia coli SOS Response: Much More than DNA Damage Repair

2021 ◽  
Author(s):  
Zdravko Podlesek ◽  
Darja Žgur Bertok
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Damage Repair ◽  
Sos Response ◽  
Genetic Adaptation ◽  
Repair Pathway ◽  
E Coli ◽  
Bacterial Pathogenicity ◽  
Damage Repair ◽  
Regulator Genes

The Escherichia coli SOS response is an inducible DNA damage repair pathway controlled by two key regulators, LexA, a repressor and RecA, an inducer. Upon DNA damage RecA is activated and stimulates self cleavage of LexA, leading to, in E. coli, derepresion of approximately 50 SOS genes. The response is triggered by exogenous and endogenous signals that bacteria encounter at a number of sites within the host. Nevertheless, besides regulating DNA damage repair the SOS response plays a much broader role. Thus, SOS error prone polymerases promote elevated mutation rates significant for genetic adaptation and diversity, including antibiotic resistance. Here we review the E. coli SOS response in relation to recalcitrance to antimicrobials, including persister and biofilm formation, horizontal gene tranfer, gene mobility, bacterial pathogenicity, as well SOS induced bacteriocins that drive diversification. Phenotypic heterogeneity in expression of the SOS regulator genes, recA and lexA as well as colicin activity genes is also discussed.

scholarly journals An Escherichia coli Effector Protein EspF May Induce Host DNA Damage via Interaction With SMC1

2021 ◽  
Vol 12 ◽  
Author(s):  
Muqing Fu ◽  
Song Liang ◽  
Jiali Wu ◽  
Ying Hua ◽  
Hanzong Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Host Cell ◽  
Foodborne Pathogen ◽  
Effector Proteins ◽  
Host Cells ◽  
Cell Counting ◽  
Damage Level ◽  
C Terminus

Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is an important foodborne pathogen that causes human diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. EspF is one of the most important effector proteins injected by the Type III Secretion System. It can target mitochondria and nucleoli, stimulate host cells to produce ROS, and promote host cell apoptosis. However, the mechanism of the host-pathogen interaction leading to host oxidative stress and cell cytotoxic effects such as DNA damage remains to be elucidated. Here, we used Cell Counting Kit-8 (CCK-8) assays and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) ELISA to study cell viability and DNA oxidative damage level after exposure to EspF. Western blot and immunofluorescence were also used to determine the level of the DNA damage target protein p-H2AX and cell morphology changes after EspF infection. Moreover, we verified the toxicity in intestinal epithelial cells mediated by EspF infection in vivo. In addition, we screened the host proteins that interact with EspF using CoIP-MS. We found that EspF may more depend on its C-terminus to interact with SMC1, and EspF could activate SMC1 phosphorylation and migrate it to the cytoplasm. In summary, this study revealed that EspF might mediate host cell DNA damage and found a new interaction between EspF and the DNA damage repair protein SMC1. Thus, EspF may mediate DNA damage by regulating the subcellular localization and phosphorylation of SMC1.

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