RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells

Abstract

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells' Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure. Gastrointestinal infections by enterohaemorrhagic Escherichia coli (EHEC), a highly pathogenic human subgroup of Shiga toxin (Stx)-producing Escherichia coli, cause haemorrhagic colitis and life-threatening extraintestinal complications such as haemolytic-uraemic syndrome (HUS) 1. HUS is characterised by the triad of thrombocytopenia, microangiopathic haemolytic anaemia, and acute renal failure and can also lead to neurological sequelae 2,3. Infections caused by EHEC are still a major global health threat, especially to children 4 , and antibiotic treatment is highly controversial 5,6. Thus, no conclusive treatment is available beyond supportive care. Mounting evidence has indicated that EHEC affects more cell types than originally thought. In the first step, EHEC attaches to the intestinal epithelium. There the cardinal virulence factor Stx-which occurs in two major types, Stx1 and Stx2, each of which have their own subtypes; subtype Stx2a is most often correlated with severe disease outcome 7-is translocated into the bloodstream by an incompletely understood mechanism 8. After dissemination through the bloodstream, Stx enters the kidney and primarily targets microvascular endothelial cells of the glomeruli 9,10 , but it may also target renal epithelial cells 11,12. The possible engagement of the renal epithelium in Stx intoxication was first indicated upon detecting the main Stx receptor, the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer; also known as CD77 [Galα1-4Galβ1-4Glcβ1-1Cer]), in the human kidney cortex and medulla 13. Later, more evidence was found, specifically renal tubular injuries by Stx 14 and Stx-induced apoptosis in kidney cortices of HUS patients and human renal tubular epithelial cells 15. Subsequently, various renal epithelial cell types were found to be Stx-sensitive 16-19. However, precisely how renal epithelial cells are affected by Stx is mostly unspecified. Stx belongs to the ...

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

“…Although a number of studies dealing with Stx-mediated injury of human renal epithelial cells have been published, the structural details of the Stx-binding GSLs of human kidney epithelial cells have been left open for a long time, and the receptors of Stx were only superficially known. The various lipoforms of the Stx-receptor GSLs Gb3Cer and Gb4Cer of the human kidney epithelial cell lines A498, ACHN, and Caki-2 have been recently characterized by us [ 65 , 114 ], while the Stx-binding GSLs of primary human renal epithelial cells are hitherto unknown. We now fill this lack of knowledge with a comprehensive structural characterization of the Stx receptors Gb3Cer and Gb4Cer of pHRCEpiCs.…”

“…A number of studies have shown that the renal cell lines HK-2 [ 54 , 55 , 56 , 57 , 58 , 59 ] and ACHN [ 60 , 61 , 62 , 63 , 64 ] derived from human tubule epithelium are sensitive to Stx, suggesting that injuries to this epithelium are involved in the development of acute renal failure in HUS. HK-2 and ACHN cells harbor globotriaosylceramide (Gb3Cer) [ 58 , 60 , 62 ], and the various lipoforms of Stx-binding Gb3Cer of the ACHN cell line have been recently identified [ 65 ]. Furthermore, Stx-mediated cytopathic action has also been shown for primary human renal glomerular and tubular epithelial cells [ 66 , 67 , 68 , 69 , 70 , 71 , 72 ], suggesting contribution of renal epithelial cells in Stx-mediated kidney failure as previously shown in a mouse model [ 73 , 74 ].…”

Valid Presumption of Shiga Toxin-Mediated Damage of Developing Erythrocytes in EHEC-Associated Hemolytic Uremic Syndrome

“…Last but not least, the emergence of new strains with rapidly aggressive virulence makes clinical and research initiatives in this field a high public health priority [ 278 , 279 , 399 ]. The stepping up of efforts directed toward the development of Stx therapeutics beyond neutralization is an additional challenge [ 500 ] with large global incidence for the years ahead to combat or, preferably, to prevent EHEC epidemic outbreaks [ 305 ].…”

Shiga Toxins: An Update on Host Factors and Biomedical Applications

“…The sensitivity of different cell lines to Stx depends not only on Gb3 expression levels but also on membrane microdomains, as well as other host factors involved in toxin trafficking [ 14 , 62 , 65 , 66 , 67 , 68 ]. For example, ACHN cells and Caki-2 cells, which are two human renal tubular adenocarcinoma cell lines, showed drastically different levels of sensitivity to Stx2: ACHN cells are highly sensitive while Caki-2 cells are not.…”

“…Further analysis shows that they have similar Gb3 levels. RNA sequencing analysis of the genes differentially expressed by ACHN and Caki-2 cells identified RAB5A, TRAPPC6B, and YKT6 as host cell factors required for the high sensitivity of ACHN to Stx2 [ 68 ].…”