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Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells

Abstract

Adhesion to epithelial cells1 and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC)2, a major cause of diarrhoea in travellers and children in developing countries3,4. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family5. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic E. coli6, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft7, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common pattern for bacterial adhesion, whereas participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.

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Figure 1: EtpA and flagella contribute to ETEC adhesion.
Figure 2: EtpA interacts with conserved regions of flagellins.
Figure 3: ETEC engage host cells through tips of flagella where EtpA interacts with highly conserved regions of flagellin.
Figure 4: ETEC adherence to epithelial cells in vitro and small intestinal colonization require the interaction of EtpA and flagellin.
Figure 5: Vaccination with flagellin inhibits ETEC infection in mice.

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Acknowledgements

We thank B. Westerlund-Wikström for supplying anti-flagellar antisera, K. Troughton of the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, for her assistance with transmission electron microscopy, and L. Boykins of the Integrated Microscopy Center at the University of Memphis for her assistance with scanning electron microscopy. We thank G. Byrne, H. Courtney, J. Dale, S. Dagogo-Jack and T. Strom for reading the manuscript. This research was supported by grants from the National Institutes of Health (National Center for Research Resources) RR16190-05, the Department of Veterans Affairs and funds from the University of Tennessee Microbial Pathogenesis Research Center.

Author Contributions K.R. performed adherence assays, transmission electron and confocal microscopy, mouse colonization studies, antibody purification and protein interaction studies. G.M.H. performed MALDI–TOF studies and D.J.H. assisted with mouse studies. J.L. performed cloning, sequencing and mutagenesis of the etpA locus. M.M.O. assisted with cloning, protein purification and protein interaction studies. J.M.F. assisted with study design, protein purification, linker mutagenesis, sample preparation, scanning electron microscopy, measurement of flagella and manuscript preparation.

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Correspondence to James M. Fleckenstein.

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University of Tennessee Research Foundation has applied for a patent related to studies included here.

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Roy, K., Hilliard, G., Hamilton, D. et al. Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature 457, 594–598 (2009). https://doi.org/10.1038/nature07568

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