Shiga Toxin Regulates Its Entry in a Syk-dependent Manner

Abstract

Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells. INTRODUCTIONThe bacterial toxin Shiga toxin (Stx) inhibits protein synthesis in cells after first binding to Gb3 at the cell surface. Then, the toxin is endocytosed, and thereafter it is transported retrogradely to the Golgi apparatus and the endoplasmic reticulum (ER) before being translocated to the cytosol where it inactivates the ribosomes enzymatically (for reviews, see . The toxin is composed of two moieties: the enzymatically active A-chain that is noncovalently attached to a stable pentamer of B-chains that binds to Gb3 and mediates the endocytic uptake of the toxin. In some cells, Stx is endocytosed mainly by clathrin-dependent endocytosis , although other mechanisms exist (Nichols et al., 2001;Lauvrak et al., 2004;Saint-Pol et al., 2004). Importantly, the toxin can induce its own transport to clathrin-coated pits (Sandvig et al., 1989). When Stx is added to HeLa cells at 0°C, the toxin becomes evenly distributed at the cell surface. However, after a short incubation at 37°C, the toxin moves to clathrin-coated pits and is internalized. In toxin-sensitive cells a larger fraction of the toxin seems to be internalized from clathrin-coated pits at the cell surface, and clathrin is required for endosome-to-Golgi transport of the toxin as well (Lauvrak et al., 2004;Saint-Pol et al., 2004). Because it has been shown by different groups that the fatty acid of Gb3 is important for efficient Golgi transport (Sandvig et al., 1994;Lingwood, 1999), the composition of Gb3 also may play a role for the endocytic pathway used. A raft localization of StxB was recently found to be required for efficient retrograde transport (Falguieres et al., 2001).In some hematopoetic cells, Stx has been shown to trigger a rapid signaling cascade that might lead to apoptosis (for review, see Cherla et al., 2003). In a number of other cell types induction of signaling leading to apoptosis seems to be mediated by the ribocytotoxic stress induced by the A-fr...

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

“…However, at 16 u C, protein synthesis inhibition remained steady at~50 % from 3 to 6 h of VT1 treatment and was not augmented by lactacystin proteosomal inhibition. This protein synthesis inhibition could result from signal transduction involving VTA or VT1 holotoxin, rather than from cytosolic translocation (Katagiri et al, 1999;Lauvrak et al, 2006;Mori et al, 2000), as VTB alone was incapable of reducing protein synthesis at 16 u C.…”

“…The VT1-Gb 3 complex is internalized by receptor-mediated endocytosis (Khine & Lingwood, 1994;Sandvig et al, 1989), both clathrin and caveolae dependent (Nichols et al, 2001). Following kinase-dependent endocytosis (Lauvrak et al, 2006), VT1 exploits a retrograde transport route to the Golgi and endoplasmic reticulum (ER) (Sandvig et al, 1992). This Golgi/ER retrograde transport pathway has been characterized using VTB (Johannes et al, 1997), and sorting along this pathway is also clathrin dependent (Lauvrak et al, 2004;Saint-Pol et al, 2004).…”

Lipid-Mediated Endocytosis

“…However, it is well documented that tyrosine-and ser/thr kinases are activated, and phosphatase inhibitors elevate vesicle formation (Richards et al 2002). In the case of STX and CTX, binding to the plasma membrane can activate Src-family kinases (SFKs) that copurify in detergent resistant membrane fractions (Katagiri et al 1999) and regulate endocytosis (Lauvrak et al 2006). …”

The Intracellular Journey of Shiga Toxins

“…Interestingly, it has been shown that Stx binding rapidly activates several kinases, such as the Src kinases Yes [28,29] and Lyn [30], the tyrosine kinase Syk [30,31], the serine/threonine kinase PKC [32], and the MAP kinase p38 [33]. The activity of some of these kinases is important for Stx entry and intracellular transport, suggesting that Stx is able to induce its own transport (see below).…”

“…clathrin and uptake by this pathway [31], however, the efficiency of clathrin-mediated toxin uptake is dependent on the concentration of cell surface-bound toxin molecules and the presence of the toxin A-chain [35]. The stimulated uptake seen at high concentrations of intact toxin, might be explained by increased aggregation of toxin-receptorcomplexes into large clusters that somehow enhance the recruitment to clathrin-coated pits.…”