Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Victor Yashunsky,Efrat Zlotkin-Rivkin,Leorah Kharilker,Eitan Erez Zahavi,Dan Davidov,Silvana Mercone,Michael Golosovsky,Eran Perlson,David Rund,Benjamin Aroeti,Naomi Melamed-Book

doi:10.1371/journal.pone.0078431

Abstract

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.

Highlights

Enteropathogenic Escherichia coli (EPEC) infection is a major cause of infant diarrhea in the developing world [1]
The surface plasmon approach is similar to the total internal reflection (TIRF) technique, since both are based on evanescent wave probing
A tight Madin Darby canine kidney (MDCK) cell monolayer cultured on a ZnS prism coated with a 20 nm-thick Au film was exposed to EPEC-wt infection in a flow chamber of the infrared surface plasmon resonance (IR-surface plasmon resonance (SPR)) set-up

Summary

Introduction

Enteropathogenic Escherichia coli (EPEC) infection is a major cause of infant diarrhea in the developing world [1]. The microbe colonizes the apical surface of the small intestine’s epithelial cells, where it forms characteristic attaching and effacing (A/E) lesions. EPEC utilizes a type-III secretion system (T3SS) to introduce bacterial effector proteins into its host epithelial cells. Several effectors have been implicated in brush border remodeling and the induction of the A/E effects, which significantly contribute to EPEC pathogenesis (recently reviewed in 2). These include effectors that promote local effacement of microvilli, intimate bacterial attachment to the host, and the induction of F-actin-rich protrusions beneath the adhering bacteria, often termed actin-rich pedestals [3].

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Results

Conclusion