Cdc42 and the Actin-Related Protein/Neural Wiskott-Aldrich Syndrome Protein Network Mediate Cellular Invasion by Cryptosporidium parvum

Abstract

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.

Water flux in cell motility: Expanding the mechanisms of membrane protrusion

“…The formation of AQP9-induced filopodia was reduced by cotransfection with an inhibitory GTP-Cdc42-binding N-WASP CRIB synthetic analog [Loitto et al, 2007]. Further support for a Cdc42-AQP interaction comes from observations of a direct localization of Cdc42 to AQP1-enriched domains, shown in C parvum-induced membrane protrusions [Chen et al, 2004]. So, how is Cdc42 associated with the PKC activation/binding discussed above?…”

The Human Immunodeficiency Virus Type 1 Tat Protein EnhancesCryptosporidium parvum-Induced Apoptosis in Cholangiocytes via a Fas Ligand-Dependent Mechanism

“…In accordance with previous reports (14,37) The mechanisms by which FasL is translocated to the membrane and released from infected cells are unclear and currently under investigation. C. parvum activates kinase pathways in infected cells (10,11,16); we recently demonstrated that there is recruitment of transporters and channels to the site of infection (15), consistent with the active recruitment of vesicles to the plasma membrane. It therefore seems reasonable that C. parvum-induced initiation of vesicular trafficking and membrane insertion may culminate in the shedding of FasL-bearing membrane in directly infected cells.…”

Interaction of Cryptosporidium hominis and Cryptosporidium parvum with Primary Human and Bovine Intestinal Cells

“…Studies have shown that C. parvum induces host cell actin rearrangement upon invasion of different cell lines and that this process is necessary for invasion to occur (6,9,12,13).We have previously shown that this also occurs when C. hominis enters HCT-8 cells (17). Our demonstration of the colocaliza- A previous study has shown that treatment of primary bovine fallopian tube epithelial cells with genistein, staurosporine, suramin sodium salt, or wortmannin inhibited entry of C. parvum sporozoites (14).…”