Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells

Abstract

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

Figure 1. Cells isolated from human pediatric kidneys cortex express characteristic endothelial markers.

HGEC seeded in gelatin coated glass coverslips and labeled with either a polyclonal rabbit anti-human VWF and then stained with a goat anti-rabbit FITC or with a FITC-monoclonal mouse anti-human PECAM-1. The expression of these proteins was analyzed by flow cytometry (A) and confocal microscopy (B), respectively.

Figure 2. Stx2 and SubAB modify HGEC morphology.

Cells were seeded in glass coverslips, treated or not with 10 ng/ml Stx2 or 3 µg/ml SubAB for 24 h and then stained with H and E. Morphology and number of HGEC was analyzed by light microscopy (×200 and ×400). The black arrows indicate the morphological changes as cell swelling (thick arrows) and cell elongation (thin arrows). HGEC areas were measured by using imageJ software. Results are expressed as means ± SEM of three experiments. One hundred percent represents values of cells control, Stx2 or SubAB vs Ctrl, *P <0.05.

Figure 3. Stx2 and SubAB decrease HGEC viability.

Cells placed in 96-well plates were exposed to 0.001 ng/ml to 100 ng/ml Stx2 (A) or 0.15 ng/ml to 1500 ng/ml SubAB (B) in growth-arrested conditions for 24, 48 and 72 h. Then, cells were incubated with neutral red for an additional 1 h at 37 °C in 5% CO₂. Absorbance of each well was read at 540 nm. One hundred percent represents cells incubated under identical conditions but without toxin treatment (Ctrl). Results are expressed as means ± SEM of five experiments, Stx2 or SubAB vs Ctrl, or 24 h vs 48 h vs 72 h, *P <0.05.

Figure 4. Gb3 is present on HGEC.

HGEC seeded in glass coverslips were labeled with an anti-human CD77 conjugated with FITC and visualized by confocal microscopy (×600) (A). Neutral glycolipids extracted from HGEC non-treated (Ctrl) or treated with C-9 (5 µM) for 48 h were subjected to TLC and visualized with orcinol (B). Gb3 was quantified after hydrolysis to galactose by an enzymatic fluorometric method and results are expressed as means ± SEM of three experiments, *P<0.05 (C).

Figure 5. Morphologic changes by Stx2 and SubAB relative to apoptosis and necrosis.

HGEC seeded in glass coverslips were exposed or not (Ctrl) to 10 ng/ml Stx2 or 3 µg/ml SubAB in growth-arrested conditions for 4, 6 and 24 h. After each period of time, % of necrotic and apoptotic cells was established morphologically by fluorescence microscopy after staining with acridine orange/ethidium (×200).

Figure 6. Stx2 and SubAB produce death cell through apoptotic mechanisms.

Annexin V-FITC/IP double staining assay was used to quantify necrosis and apoptosis by a flow cytometer. HGEC were treated with 10 ng/ml Stx2 (A) or 3 µg/ml SubAB (B) and labeled with annexin V-FITC/IP for 10 min. Results are expressed as means ± SEM of three experiments. *P<0.05 for necrosis vs apoptosis. A representative experiment is shown in panel C.