Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

Abstract

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

“…A small fraction of VT1 that rapidly reaches the ER within 1 h (Johannes et al, 1997;Sandvig et al, 1992) would explain the low translocation at this time. VT1 accesses the ER within 3-6 h (Khine et al, 2004), and [ 125 I]VTA 1 translocation reached a maximum at this time. Since the detected VT1 subunits remained associated throughout retrograde transport from the Golgi to the ER (Fig.…”

“…BFA fuses the transGolgi network (TGN) and endosomes (LippincottSchwartz et al, 1991), and collapses Golgi and ER (Lippincott-Schwartz et al, 1989). BFA protects VT1-treated cells from apoptosis (Fujii et al, 2003) and protein synthesis inhibition (Donta et al, 1995;Khine et al, 2004). BFA presumably protects cells through its collapse of the Golgi, thereby preventing further retrograde transport to the ER, from which translocation is presumed to occur.…”

“…BFA presumably protects cells through its collapse of the Golgi, thereby preventing further retrograde transport to the ER, from which translocation is presumed to occur. During BFA treatment, VT1 accumulates in the fused TGN/endosome (Khine et al, 2004), unable to proceed further. At low temperature (,20 u C), retrograde transport is also prevented and internalized cargo accumulates in endosomes, and transport to the lysosomes, TGN and Golgi is inhibited (Baravalle et al, 2005;Mallard et al, 1998;Punnonen et al, 1998).…”

Study on induction of apoptosis on HeLa and Vero cells by recombinant shiga toxin and its subunits

“…B subunit of Stx is supposed to be a convenient non-cytotoxic model ) of holotoxin internalization, and Stx A subunit lacks known trafficking motifs (Jackson et al 1999); the A subunit might also modify holotoxin transport via unknown motifs. Nevertheless, it has been known that holotoxin initial internalization will be A subunit-independent, and two pathways have been identified as applicable to holotoxin endocytosis (Khine et al 2004). Moreover, two distinct Gb3/ CD77 signaling pathways leading to apoptosis have been triggered by Anti-Gb3/CD77 mAb and Stx (Tétaud et al 2003).…”

“…Therefore, different intracellular routing may play a pivotal role in cell susceptibility and triggering a different pathway for apoptosis. Stxinduced apoptosis and cytotoxicity are distinct processes (Gordon et al 2000;Williams et al 1999), although the A subunit can be necessary for apoptosis (Khine et al 2004). In our study, the same result was also shown, as if A subunit can separately induce apoptosis morphologically in higher concentration by Hoechst staining and DNA laddering method.…”