Retrograde Shiga Toxin Trafficking Is Regulated by ARHGAP21 and Cdc42

Abstract

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus. INTRODUCTIONEnteritis caused by Shigella dysenteria and pathogenic strains of Escherichia coli is a global health threat. These bacteria secrete Shiga toxin that enters intestinal epithelial cells and kills them by blocking translation. In some cases, the toxin escapes the gut and targets the kidney and vascular endothelium resulting in hemolytic-uremic syndrome (Sandvig and van Deurs, 2000; O'Loughlin and RobinsBrowne, 2001;Proulx et al., 2001;Desch and Motto, 2007). Treatment options for Escherichia coli infection and hemolyticuremic syndrome are limited in part because of an incomplete understanding of the molecular mechanisms underlying Shiga toxin's trafficking within cells.Shiga toxin reaches the cytosol by using retrograde transport through the secretory pathway (Sandvig and van Deurs, 2002;Johannes and Popoff, 2008). Shiga toxin is a heteromultimeric protein containing one A subunit and five B subunits. The A subunit is an N-glycosidase that inhibits protein translation, whereas the Shiga toxin B subunits (STxBs) mediate intracellular targeting. STxB binds to the cell surface via a glycolipid receptor, globotriaosyl ceramide (Gb3). Entry is mediated by clathrin-dependent or -independent endocytosis (Lingwood, 1993;Sandvig and van Deurs, 2000). It is transported from early endosomes to the Golgi complex before undergoing COPI-independent retrograde transport to the endoplasmic reticulum (Mallard et al., 1998;Girod et al., 1999;Falguieres et al., 2001;Luna et al., 2002;Lauvrak et al., 2004;McKenzie et al., 2009). The A subunit exits the endoplasmic reticulum into the cytosol where it cleaves the rRNA (Obrig et al., 1985).Shiga toxin usurps several components of the constitutive trafficking machinery t...

Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function

“…(25) Characterized by malabsorption and secretory diarrhea, MVID is a known cause of IF. In addition to exocytosis, Rho GTPases can regulate endocytosis; this is exploited by toxins such as Shiga toxin, which employs macropinocytosis for enterocyte invasion, in addition to microtubule-assisted transport, another Cdc42-dependent cellular function (17). The fact that some cells in mTESI do coexpress lysozyme/Cdc42 and there are no differences in polarity that we have observed suggest that there is enough Cdc42 for normal intestinal epithelial cell development.…”

Transport of Influenza Virus Neuraminidase (NA) to Host Cell Surface Is Regulated by ARHGAP21 and Cdc42 Proteins

“…A previous study found that RhoA, Rac1, and Cdc42 each regulate distinct aspects of Shiga toxin trafficking from the cell surface to the Golgi apparatus (30). RhoA regulates the internalization of Shiga toxin, whereas Cdc42 regulates the retrograde motility of Shiga toxin to the juxtanuclear Golgi apparatus (30).…”

“…To start to elucidate the function of the ARHGAP21 in NA trafficking, HeLa cells were transiently co-transfected with NAcoding cDNA and a plasmid encoding a fragment of ARH-GAP21 containing the ARF binding and GAP domains as described previously (30). We observed that overexpression of ARHGAP21 caused pleiotropic defects in cell morphology but had no effect on cell viability (supplemental Fig.…”

“…Cdc42 activation assay was performed as described previously (30). Briefly, 293T cells transfected with plasmids expressing GFPCdc42 were infected with or without A/WSN/33 influenza virus (H1N1) for 10 h. The cells were harvested and solubilized with the lysis buffer.…”

“…ARHGAP21 Is Involved in Regulating Transport of NA to Cell Surface-Recent studies have shown that ARHGAP21 functions as a Cdc42-specific GAP and acts downstream of the small GTP binding ADP-ribosylation factor 1 (ARF1) to play an important role in regulating Golgi structure and function and in protein trafficking through the control of Cdc42 activity (30,31,(45)(46)(47). Because our experiments revealed that Cdc42(Q61L) promotes, but Cdc42(T17N) inhibits, NA trafficking to the plasma membranes, we sought to explore whether altering GTPase cycle had any effect on NA trafficking.…”