Shiga toxin type-2 (Stx2) induces glutamate release via phosphoinositide 3-kinase (PI3K) pathway in murine neurons.

Fumiko Obata,Lauren M Hippler,Progyaparamita Saha,Dakshina M Jandhyala,Olga S Latinovic

doi:10.3389/fnmol.2015.00030

Abstract

Shiga toxin-producing Escherichia coli (STEC) can cause central nervous system (CNS) damage resulting in paralysis, seizures, and coma. The key STEC virulence factors associated with systemic illness resulting in CNS impairment are Shiga toxins (Stx). While neurons express the Stx receptor globotriaosylceramide (Gb3) in vivo, direct toxicity to neurons by Stx has not been studied. We used murine neonatal neuron cultures to study the interaction of Shiga toxin type 2 (Stx2) with cell surface expressed Gb3. Single molecule imaging three dimensional STochastic Optical Reconstruction Microscopy—Total Internal Reflection Fluorescence (3D STORM-TIRF) allowed visualization and quantification of Stx2-Gb3 interactions. Furthermore, we demonstrate that Stx2 increases neuronal cytosolic Ca2+, and NMDA-receptor inhibition blocks Stx2-induced Ca2+ influx, suggesting that Stx2-mediates glutamate release. Phosphoinositide 3-kinase (PI3K)-specific inhibition by Wortmannin reduces Stx2-induced intracellular Ca2+ indicating that the PI3K signaling pathway may be involved in Stx2-associated glutamate release, and that these pathways may contribute to CNS impairment associated with STEC infection.

Highlights

Shiga toxin-producing Escherichia coli (STEC) infection is an important cause of food poisoning, often associated with contaminated beef and leafy vegetables
In order to resolve Shiga toxin type 2 (Stx2)-Gb3 interactions, we applied 3D STORM-TIRF, which allows better resolution compared to standard confocal microscopy (i.e., 3D STORM TIRF resolves 20 nm along the x- and y-axes and 50 nm along the z-axis versus standard confocal microscopy which is limited to 200 nm resolution)
We demonstrate for the first time that cultured murine neurons express Gb3 and that Stx2 interacts with Gb3 displayed on the surface of the cell

Summary

Introduction

Shiga toxin-producing Escherichia coli (STEC) infection is an important cause of food poisoning, often associated with contaminated beef and leafy vegetables. In one cohort, 104 of 217 patients with complicated STEC infection developed neurological manifestations (Magnus et al, 2012). This outbreak was caused by a unique STEC strain with enteroaggregative properties but with the ability to express stx (Jandhyala et al, 2013). This outbreak alerts us both to the potential impact of new emerging infectious diseases, as well as to the importance of STEC-associated CNS disease. No specific therapy is available for prevention or remediation of STEC-associated CNS complications emphasizing the need for a better understanding of Stx2-associated CNS impairment

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Conclusion