Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin

Zachary T. Berndsen, Marjahan Akhtar, Mahima Thapa, Tim J. Vickers, Aaron Schmitz, Jonathan L. Torres, Sabyasachi Baboo, Pardeep Kumar, Nazia Khatoon, Alaullah Sheikh, Melissa Hamrick, Jolene K. Diedrich, Salvador Martinez-Bartolome, Patrick T. Garrett, John R. Yates, Jackson S. Turner, Renee M. Laird, Frédéric Poly, Chad K. Porter, Jeffrey CoppsAli H. Ellebedy, Andrew B. Ward*, James M. Fleckenstein*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryoEM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Original languageEnglish
Article numbere1012241
JournalPLoS Pathogens
Volume20
Issue number9 September
DOIs
StatePublished - Sep 2024
Externally publishedYes

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