Abstract
Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.
Highlights
We report on the comprehensive structural analysis of the various lipoforms of Shiga toxin (Stx)-binding GSLs isolated from primary human renal cortical epithelial cells and their distribution to detergent-resistant membranes (DRMs) and non-DRMs used as counterparts for the liquid-ordered and the liquid-disordered membrane phase, respectively
The identified Stx receptor GSLs were further structurally characterized by thin-layer chromatography (TLC) overlay immunodetection using anti-Gb3Cer and antiGb4Cer antibodies combined with electrospray ionization mass spectrometry (ESI-MS)
The ambient phospholipids were analyzed in detail by MS1 and MS2 analysis, the latter performed by collision-induced dissociation (CID) experiments
Summary
Shiga toxins (Stxs) are potent bacterial exotoxins and the principal virulence factors of pathogenic enterohemorrhagic Escherichia coli (EHEC), a subset of Stx-producing E. coli (STEC) [1]. They are responsible for major clinical manifestations of STEC infections, including bloody diarrhea and life-threatening extraintestinal complications such as the hemolytic–uremic syndrome (HUS) and neurological disturbances as the most serious consequences of these infections [2,3,4,5]. An update on global outbreaks of STEC and its potential reservoirs has recently been published [6]. Subsequent endocytosis of the toxin–GSL complex and retrograde transport occurs by multifarious endocytic routes directing the toxin via early endosomes through the
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
