Benzyl alcohol induces a reversible fragmentation of the Golgi apparatus and inhibits membrane trafficking between endosomes and the trans-Golgi network.

Benzyl alcohol (BnOH) is widely used as a component of foods, cosmetics, household products and medical products. It is generally considered to be safe for human use, however, it has been connected to a number of adverse effects, including hypersensitivity reactions and neonatal deaths. BnOH is a membrane fluidizing agent that can affect membrane protein activity and cellular processes such as ligand binding to cell surface receptors, endocytosis and degradation of lysosomal cargo. In this study, we examined the effects of BnOH on intracellular transport using Shiga toxin (Stx), diphtheria toxin (DT) and ricin. BnOH caused reduced toxicity of all three toxins at BnOH concentrations that cause membrane fluidization. The reduced toxicity of Stx and ricin was mainly due to inhibition of retrograde transport between endosomes and the trans-Golgi network as BnOH had small effects on cell association and endocytosis of ricin and Stx. Strikingly, BnOH also induced a reversible fragmentation of the Golgi apparatus.

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-ß-cyclodextrin (mßCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Vps11, a subunit of the tethering complexes HOPS and CORVET, is involved in regulation of glycolipid degradation and retrograde toxin transport.

We recently reported that ERM (ezrin, radixin, moesin) proteins are involved in intracellular sorting of Shiga toxin (Stx) and its receptor globotriaosylceramide (Gb3), and that depletion of ezrin and moesin reduced retrograde Golgi transport of Stx. In the same study, we found that knockdown of Vps11, a core subunit of both the homotypic fusion and protein sorting (HOPS) complex and the class C core vacuole/endosome tethering factor (CORVET), increased retrograde transport of Stx and could counteract the inhibiting effect of moesin and ezrin knockdown. In this study we demonstrate that Vps11 knockdown also leads to increased Stx toxicity as well as increased retrograde transport and toxicity of ricin. Additionally, we show that knockdown of Vps11 restores the reduced Gb3 level observed after moesin depletion.

The ERM proteins ezrin and moesin regulate retrograde Shiga toxin transport.

The ERM proteins (ezrin, radixin and moesin) are known for connecting the actin cytoskeleton to the plasma membrane. They have been found to associate with lipid rafts as well as to be important for endosomal sorting and receptor signaling. However, little is known about the role of ERM proteins in retrograde transport and lipid homeostasis. In this study, we show that ezrin and moesin are important for efficient cell surface association of Shiga toxin (Stx) as well as for its retrograde transport. Furthermore, we show that depletion of these proteins influences endosomal dynamics and seems to enhance Stx transport toward lysosomes. We also show that knockdown of Vps11, a subunit of the HOPS complex, leads to increased retrograde Stx transport and reverses the inhibiting effect of ezrin and moesin knockdown. Importantly, retrograde transport of the plant toxin ricin, which binds to both glycolipids and glycoproteins with a terminal galactose, seems to be unaffected by ezrin and moesin depletion.