Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

Abstract

Aim:To screen host proteins that interact with enterohemorrhagic Escherichia coli O157:H7 EspF. Materials & methods: Flow cytometry and high-throughput sequencing were used to screen interacting proteins. Molecular function, biological processes and Kyoto Encyclopedia of Genes and Genomes pathways were studied using the DAVID online tool. Glutathione S-transferase pull down and dot blotting were used to verify the interactions. Results: 293 host proteins were identified to associate with EspF. They were mainly enriched in RNA splicing (p = 0.005), ribosome structure (p = 0.012), and involved in 109 types of signaling pathways. SNX9 and ANXA6 were confirmed to interact with EspF. Conclusion: EspF interacts with ANXA6; they may form a complex to manipulate the process of phagocytosis; EspF plays a highlighted pathogenic role in enterohemorrhagic E. coli infection process. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne causative agent in sporadic outbreaks of illness such as diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Severe symptoms may even lead to death [1][2][3][4]. Research into EHEC has mainly explored the development of attaching and effacing (A/E) intestinal lesions and the secretion of Shiga toxin in EHEC infection [5][6][7]. The protein EspF is one of the multifunctional effectors of A/E lesions induced by EHEC and enteropathogenic E. coli (EPEC). EspF participates in a number of damage processes in host cells, targeting mitochondria and nucleoli [6,8] and disrupting the tight junctions (TJs) of intestinal epithelial cells [9], leading to cytoskeletal rearrangements, actin polymerization and pedestal formation [10]. EspF thus emerges as the 'Swiss army knife' of a bacterial pathogen [11,12]. EHEC O157:H7 employs a type-III secretion system to export toxic factors and effector proteins to host cells after adhering to the brush border of epithelial cells [5,[13][14]. The production of virulence factors, subsequent invasion and diffusion, and the interaction of effector proteins with host proteins are key steps in EHEC O157:H7 pathogenesis. The mechanism by which EspF breaks through the intestinal epithelial barrier into host cells, the host proteins that EspF interacts with, and how cellular damage, and even apoptosis, result remain unclear. Studying the pathogen-host interaction process will increase the emerging knowledge about EHEC O157:H7 pathogenesis.The EspF protein has been shown to induce apoptosis after 'injection' into host cells [1]. The N-terminal (1-73 amino acids [aa]) of this approximately 27 kDa protein contains a secretory signal (1-20 aa), a mitochondrialtargeting signal (1-24 aa) and a nucleolar-targeting domain (21-74 aa); 73-248 aa consists of four proline-rich repeats (PRRs), each containing a eukaryotic cell sorting nexin 9 (SNX9) protein-binding site SH3 motif, an

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

“…Ying have found that EspF has the ability to interact with featured ribosomal proteins and to modulate the antiphagocytosis process by bonding with ANXA6 (Hua et al, 2018b). In our study, we found that SNX9, SNX18, 14-3-3 epsilon, ANXA6, ANXA2 and EHEC O157:H7 EspF proteins also interact.…”

Clever Cooperation: Interactions Between EspF and Host Proteins

“…EspF's role in causing diarrhea is the ability to disrupt TJs, but the precise mechanism of EspF's binding to host proteins to cause diarrhea has not been defined. In our previous research, we screened AQP7P2, a type of water channel protein that interacts with EspF (Hua et al, 2018 ). Their interaction may induce diarrhea by changing the activity of water molecule transport.…”

“…Recently, we screened and verified that Annexin A6 (Anxa6) interacts with EHEC O157:H7 EspF using Bimolecular Fluorescence Complementation (BiFC) technology for the first time (Hua et al, 2018 ). Anxa6 belongs to a highly conserved protein family characterized by calcium-dependent binding to phospholipids.…”

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“…As EspF is involved in TER disruption, we hypothesized that EspF would interact with a specific host protein to interfere with the pathogenic phenotype. In our previous study, we employed the bimolecular fluorescence complementation (BiFC) method to examine the interplay between EspF and host proteins, which provided specific candidates that interact with EspF (Hua et al, 2018b). Among these candidates, ANXA6 showed a strong positive reaction with EspF.…”

“…Despite the extensive evidence implicating EspF in the disruption of TJ barrier structure and function, a detailed molecular mechanism has not yet been elucidated. Our previous study demonstrated that host protein-Annexin A6 (ANXA6) co-precipitated with EspF (Hua et al, 2018b). ANXA6 is a scaffold protein that links the membrane microdomains with the cell cytoskeleton organization (Grewal et al, 2017).…”