Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains

Johanna Detzner,Helge Karch,Hans-Ulrich Humpf,Johannes Müthing,Elisabeth Krojnewski,Gottfried Pohlentz,Alexander Mellmann,Anna-Lena Klein

doi:10.3390/toxins13080529

Johanna Detzner, Helge Karch + Show 6 more

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https://doi.org/10.3390/toxins13080529

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Journal: Toxins	Publication Date: Jul 28, 2021
Citations: 6	License type: CC BY 4.0

Affiliation: University of Münster

Abstract

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic–uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic Escherichia coli (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD50) of 1.31 × 102 pg/mL and 1.66 × 103 pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.

Highlights

Shiga toxins (Stxs) are powerful bacteriogenic AB5 toxins and the primary virulence factors of human–pathogenic enterohemorrhagic Escherichia coli (EHEC), which represent a sublineage of Stx-producing Escherichia coli (STEC) [1] with emerging public health challenges [2]
Responsiveness toward Stx has been reported for primary human renal tubular epithelial cells [58,59,60,61,62]. These results suggest the involvement of kidney epithelial cells, beside the endothelial cells of the kidney microvasculature, in EHEC-hemolytic–uremic syndrome (HUS) supported by an appropriate mouse model, which has shown the direct contribution of tubular damage to Stx-mediated kidney failure [63,64]
Replicate 1 (R1) and replicate 2 (R2), were prepared from pHRPTEpiCs derived from early passages

Summary

Introduction

Shiga toxins (Stxs) are powerful bacteriogenic AB5 toxins and the primary virulence factors of human–pathogenic enterohemorrhagic Escherichia coli (EHEC), which represent a sublineage of Stx-producing Escherichia coli (STEC) [1] with emerging public health challenges [2]. Protection can be provided either by inhibiting the binding of Stx toward the cell surface using therapeutics based on chemical analogs of the Stx receptor [13,14,15], interfering of small-molecule inhibitors with any of the subsequent steps upon retrograde trafficking that act at the endosome/Golgi interface required for the toxin’s intracellular destructive effects [16,17,18], or blocking of transcriptional and translational inhibitors that may be of value in treating EHEC infections [19]

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