Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2016
Publication Date: 2/22/2016
Citation: Sharma, V.K., Kudva, I.T., Bearson, B.L., Stasko, J.A. 2016. Contributions of EspA filaments and curli fimbriae in cellular adherence and biofilm formation of enterohemorrhagic Escherichia coli O157:H7. PLoS One. 11(2):e0149745. doi: 10.1371/journal.pone.0149745.
Interpretive Summary: Escherichia coli O157:H7 (O157) are Shiga toxin-producing bacteria that infect humans through the consumption of contaminated meats, dairy products, fresh produce and water. Human infections with O157 generally produce mild diarrhea but in children and elderly, the Shiga toxins produced by O157 could lead to bloody diarrhea, kidney malfunction, and even death. Cattle carry O157 in their intestines and, therefore, can shed these bacteria in their feces. Cattle feces are the major risk factors for the contamination of animal hides in feedlots, and subsequent carcass contamination at slaughter plants leading to the downstream contamination of meat products. In addition, manure represents a major risk factor for the contamination of water resources and environmental spread of O157 to food crops. According to CDC estimates, O157 causes over 73,000 human illnesses per year with over 2,000 cases of hospitalizations and 61 deaths. Combined economic losses due to human illnesses and food contamination are estimated at almost a billion dollars. Factors that promote colonization and persistence of O157 are highly diverse and are not completely understood. The aim of this study, therefore, was to use non-animal models, such as cultured epithelial cells and polystyrene surfaces, to determine the role of some of the structures produced by O157 bacteria for their attachment. By using these two adherence models, we showed that one of the tubular surface filaments is important for adherence of O157 bacteria to epithelial cells and the other promoted adherence to polystyrene plastic. These findings are important milestones in the understanding of the role of specific surface molecules in adherence of O157 to epithelial cells of animals and non-living surfaces outside of an animal. Adherence to epithelial cells is the first important step in colonization of cattle intestine, and similarly adherence to non-living surfaces could potential enhance survival of O157 bacteria in the environment and in animals through the formation of biofilms. Evaluation of these surface structures as constituents of vaccines are the potential applications in ongoing efforts for reducing colonization and persistence of O157 in cattle.
Technical Abstract: In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed increased adherence to HEp-2 cells and produced abundant biofilms. Transcriptional analysis revealed increased expression of espA as well as the csgA gene, which encodes curli fimbriae that are essential for biofilm formation. In the present study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to determine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and biofilm formation. In vitro adherence assays, conducted at 37 degree C in a tissue culture medium containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha espA and hha csgA espA mutants adhered to Hep-2 cells at higher levels only when complemented with an espA-expressing plasmid. Biofilm assays performed at 28 degree C in a medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA was not produced under these conditions. Despite production of detectable levels of EspA at 28 degree C or 37 degree C in media supplemented with 0.1% glucose, the biofilm formation occurred independent of EspA. These results indicate dependency of O157 adherence to epithelial cells on EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those found in the environment (low temperature with nutrient limitations) and in the digestive tract of an host animal (higher temperature and low levels of glucose).