Enterotoxigenic Escherichia coli–blood group A interactions intensify diarrheal severity
Pardeep Kumar, … , Mark Donowitz, James M. Fleckenstein
Pardeep Kumar, … , Mark Donowitz, James M. Fleckenstein
Published August 1, 2018; First published May 17, 2018
Citation Information: J Clin Invest. 2018;128(8):3298-3311. https://doi.org/10.1172/JCI97659.
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Categories: Research Article Infectious disease Vaccines

Enterotoxigenic Escherichia coli–blood group A interactions intensify diarrheal severity

Abstract

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A–specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Authors

Pardeep Kumar, F. Matthew Kuhlmann, Subhra Chakraborty, A. Louis Bourgeois, Jennifer Foulke-Abel, Brunda Tumala, Tim J. Vickers, David A. Sack, Barbara DeNearing, Clayton D. Harro, W. Shea Wright, Jeffrey C. Gildersleeve, Matthew A. Ciorba, Srikanth Santhanam, Chad K. Porter, Ramiro L. Gutierrez, Michael G. Prouty, Mark S. Riddle, Alexander Polino, Alaullah Sheikh, Mark Donowitz, James M. Fleckenstein

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Figure 6

EtpA and EtpA-expressing ETEC interact preferentially with the surface of blood group A enteroids.

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EtpA and EtpA-expressing ETEC interact preferentially with the surface o...
(A) Confocal imaging of EtpA binding to surface of polarized small intestinal enteroid monolayers from a blood group A subject. Differential interference contrast (DIC) image at bottom shows architecture of polarized enteroids on Transwell filter. Original magnification, ×63. (B) Kinetic ELISA quantification of biotinylated EtpA binding to surface of small intestinal enteroid cells from blood groups A, B, and O (n = 4 technical replicates representative of 3 separate experiments). *P < 0.05; **P < 0.01, ANOVA, Kruskal-Wallis. (C) Association of EtpA-expressing ETEC H10407 with A blood group on surface of small intestinal cells. Images represent volocity-processed confocal laser-scanning microscopy (CLSM) data of ETEC H10407 (red) to blood group A (green) and nuclei (blue). Lower panel shows polarized orientation of cells with bacteria adherent to the apical surfaces of enterocytes. Original magnification, ×63. (D) Top panel: CLSM of ETEC (green) and A blood group (red) on jejunal enteroids. Bottom panel: A blood group “footprints” (arrows) at sites of bacterial attachment. Data shown in C and D are representative of 3 experimental replicates. Original magnification, ×60. (E) Bacterial density on surface of small intestinal enteroids determined by CLSM quantification of H10407 (serotype O78). Quantitation based on imaging 10 fields per blood group, at ×20 magnification. *P = 0.039; #P < 0.0001, ANOVA, Kruskal-Wallis. (F) Adhesion of WT H10407 and etpA mutant bacteria to blood group A small intestinal enteroids (n = 5 technical replicates; representative of 3 biological replicates). Bars represent geometric mean values. (G) Production of cAMP by blood group A target enteroids following infection by WT (H10407) or the etpA mutant. Data represent total of 5 technical replicates from 2 separate experiments. (H) Production of cGMP by blood group A small intestinal enteroids following infection with WT (n = 6) or the etpA mutant (n = 4). Data are representative of 3 independent biological replicates. Inset graph indicates relative (mean ± SD, n = 6 technical replicates) production of heat stable toxin (ST) by the WT (blue bars) vs. the mutant (gray bars). **P < 0.01, Mann-Whitney U test, 2 tailed nonparametric testing.