Analysis of microdomain-association of Shiga toxin glycosphingolipid receptors in primary human endothelial cells

• Applicant: Professor Dr. Johannes Müthing
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2007 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 41775145

Infection with Shiga toxin (Stx) producing Escherichia coli (STEC) results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and the hemolytic-uremic syndrome (HUS). Stxs are believed to cause microvascular endothelial injury which is the primary histopathological event and pathophysiological process underlying hemorrhagic colitis and HUS. Stxs have been subdivided into two families, Stx1 and Stx2, and several variants (Stx1c, Stx1d, Stx2c, Stx2d, Stx2e, Stx2f). In most instances, there are as yet unknown receptor binding characteristics of these toxins. Although we have shown that the clinical outcome of an STEC infection depends, in large part, on the Stx type produced by the infecting strain, the reason for this differentiation is not well understood. To determine if these differences correlate with different receptorbinding capacities, we will purify the members of the Stx families and identify their receptors using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS), which we have recently applied to the definition of the detailed structural characterization of high- and low-affinity binding ligands of Stx1. Next, we will investigate if these receptors have different qualitative and quantitative distributions on endothelial cells. Because the fatty acid of globotriaosylceramide (Gb3Cer) can influence the endocytic pathway used, and because raft localization is required for efficient retrograde transport of Stx, we will characterize lipid rafts (isolated as detergent-resistant membranes, apical/basolateral membrane preparations, and caveolae) from human endothelial cells. We will specifically examine the composition of glycosphingolipids (GSLs) and GSL/cholesterol associated membrane proteins. This characterization will be performed in the context of Stx-cell binding interactions. By defining precise mechanisms by which different Stxs interact with their cellular targets, we hope to develop strategic preventive and therapeutic measures for STEC-mediated diseases.

• DFG Programme: Research Grants