scholarly journals Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx

2021 ◽  
Vol 22 (18) ◽  
pp. 10002
Author(s):  
Johanna Detzner ◽  
Charlotte Püttmann ◽  
Gottfried Pohlentz ◽  
Hans-Ulrich Humpf ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
Human Kidney ◽  
Human Colon ◽  
Enterohemorrhagic Escherichia Coli ◽  
Intestinal Lumen ◽  
Brain Endothelial Cells ◽  
Detergent Resistant Membranes ◽  
Globo Series ◽  
Major Target

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

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

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 139
Author(s):  
Johanna Detzner ◽  
Elisabeth Krojnewski ◽  
Gottfried Pohlentz ◽  
Daniel Steil ◽  
Hans-Ulrich Humpf ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Saturated Fatty Acids ◽  
Human Kidney ◽  
Enterohemorrhagic Escherichia Coli ◽  
Highly Sensitive ◽  
Liquid Ordered ◽  
Uremic Syndrome

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.

scholarly journals EnterohemorrhagicEscherichia coliinfection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells

2011 ◽  
Vol 301 (5) ◽  
pp. C1140-C1149 ◽  
Author(s):  
Valeriy Lukyanenko ◽  
Irina Malyukova ◽  
Ann Hubbard ◽  
Michael Delannoy ◽  
Edgar Boedeker ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
Molecular Mechanisms ◽  
Intestinal Epithelial Cells ◽  
Enterohemorrhagic Escherichia Coli ◽  
Intestinal Epithelial ◽  
Systemic Complications ◽  
E Coli ◽  
Systemic Spread ◽  
Uremic Syndrome

Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

scholarly journals Verotoxin 2 Enhances Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Intestinal Epithelial Cells and Expression of β1-Integrin by IPEC-J2 Cells

2010 ◽  
Vol 76 (13) ◽  
pp. 4461-4468 ◽  
Author(s):  
Bianfang Liu ◽  
Xianhua Yin ◽  
Yanni Feng ◽  
James R. Chambers ◽  
Aiguang Guo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Human Colon ◽  
Enterohemorrhagic Escherichia Coli ◽  
Β1 Integrin ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
O157 Strain ◽  
Human Laryngeal Carcinoma

ABSTRACT Verotoxin (VT) has been implicated in the promotion of adherence to and colonization of intestinal epithelial cells by enterohemorrhagic Escherichia coli (EHEC) O157:H7. The present study investigated the effect of VT2 on the adherence of EHEC O157:H7 strain 86-24 to porcine jejunal (IPEC-J2), human colon (CaCo-2), and human laryngeal carcinoma (HEp-2) cell lines and on the expression in IPEC-J2 cells of synthases for β1-integrin and nucleolin, both of which are implicated in bacterial adherence. The effect on expression of globotriaosylceramide (Gb3) synthase, the receptor for VT, was also examined. Data were obtained by adherence assays and quantitative reverse transcriptase PCR, using EHEC O157 strain 86-24, a vt2 deletion mutant, a vt2 phage-negative strain, and complemented mutants in which the vt2 gene was restored. Compared with the adherence of the parent and complemented mutant strains, the vt2-negative strains adhered significantly less to all three types of cells. Adherence of the wild-type EHEC strain to IPEC-J2 cells was accompanied by increased expression of β1-integrin, nucleolin, and Gb3 synthase. IPEC-J2 cells in association with wild-type EHEC O157:H7 or the complemented mutants expressed higher levels of β1-integrin than did cells in association with the vt2-negative strains or with no bacteria. Expression of nucleolin was decreased by association with the vt2-negative mutant, but complementation failed to restore wild-type expression. The data indicate that VT2 plays a role in the adherence of EHEC O157:H7 to intestinal epithelial cells, possibly by increasing the expression of the host receptor β1-integrin.

scholarly journals Shiga toxin remodels the intestinal epithelial transcriptional response to Enterohemorrhagic Escherichia coli

2020 ◽  
Author(s):  
Alyson R. Warr ◽  
Carole J. Kuehl ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Host Response ◽  
Diarrheal Disease ◽  
Rabbit Model ◽  
Innate Immune ◽  
Enterohemorrhagic Escherichia Coli ◽  
Transcriptional Responses ◽  
Immune Signaling

AbstractEnterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes diarrheal disease and the potentially lethal hemolytic uremic syndrome. We used an infant rabbit model of EHEC infection that recapitulates many aspects of human intestinal disease to comprehensively assess colonic transcriptional responses to this pathogen. Cellular compartment-specific RNA-sequencing of intestinal tissue from animals infected with EHEC strains containing or lacking Shiga toxins (Stx) revealed that EHEC infection elicits a robust response that is dramatically shaped by Stx, particularly in epithelial cells. Many of the differences in the transcriptional responses elicited by these strains were in genes involved in immune signaling pathways, such as IL23A, and coagulation, including F3, the gene encoding Tissue Factor. RNA FISH confirmed that these elevated transcripts were found almost exclusively in epithelial cells. Collectively, these findings suggest that within the intestine, Stx primarily targets epithelial cells, and that the potent Stx-mediated modulation of innate immune signaling skews the host response to EHEC towards type 3 immunity.Significance StatementEnterohemorrhagic Escherichia coli (EHEC) is a potentially lethal foodborne pathogen. During infection, EHEC releases a potent toxin, Shiga toxin (Stx), into the intestine, but there is limited knowledge of how this toxin shapes the host response to infection. We used an infant rabbit model of infection that closely mimics human disease to profile intestinal transcriptomic responses to EHEC infection. Comparisons of the transcriptional responses to infection by strains containing or lacking Stx revealed that this toxin markedly remodels how the epithelial cell compartment responds to infection. Our findings suggest that Stx biases the intestinal innate immune response to EHEC and provide insight into the complex host-pathogen dialogue that underlies disease.

scholarly journals Shiga Toxin Translocation across Intestinal Epithelial Cells Is Enhanced by Neutrophil Transmigration

2001 ◽  
Vol 69 (10) ◽  
pp. 6148-6155 ◽  
Author(s):  
Bryan P. Hurley ◽  
Cheleste M. Thorpe ◽  
David W. K. Acheson
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
Intestinal Epithelial Cells ◽  
Biologically Active ◽  
Intestinal Lumen ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Inflammatory Infiltration ◽  
E Coli ◽  
Intestinal Epithelia

ABSTRACT Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lackingeae and espB (eae- andespB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion thaneae- and espB-positive STEC or enteropathogenic E. coli. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.

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

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 529
Author(s):  
Johanna Detzner ◽  
Anna-Lena Klein ◽  
Gottfried Pohlentz ◽  
Elisabeth Krojnewski ◽  
Hans-Ulrich Humpf ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Kidney ◽  
Enterohemorrhagic Escherichia Coli ◽  
Cellular Damage ◽  
Membrane Microdomains ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular ◽  
The Impact

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.

scholarly journals Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

2009 ◽  
Vol 296 (1) ◽  
pp. G78-G92 ◽  
Author(s):  
Irina Malyukova ◽  
Karen F. Murray ◽  
Chengru Zhu ◽  
Edgar Boedeker ◽  
Anne Kane ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
Intestinal Epithelial Cells ◽  
Enterohemorrhagic Escherichia Coli ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Shiga Toxins ◽  
E Coli ◽  
Human Intestinal Epithelial Cells ◽  
Toxin Uptake

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

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