Wsp1, a GBD/CRIB Domain-Containing WASP Homolog, Is Required for Growth, Morphogenesis, and Virulence of Cryptococcus neoformans

Abstract

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.

Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

“…We have demonstrated previously that the Cin1 and Wsp1 proteins regulate intracellular trafficking and that both proteins interact with Cdc42 through conserved domains (38,39). Here we present evidence for a Cin1-Cdc42-Wsp1 effector pathway.…”

“…Important oligonucleotide primers for PCR amplification and DNA plasmids are listed in Tables S2 and S3 in the supplemental material, respectively. All culture media and reagents were prepared as described previously (38,39).…”

“…Gene knockout alleles were generated by the split-marker method, and the resulting mutant strains were obtained as described previously (38,39). Auxotrophic and dominant selection marker genes, including URA5 (encoding orotidine monophosphate pyrophosphorylase), HPT (hygromycin phosphate transferase), NEO (neomycin phosphotransferase), and NAT (nourseothricin N-acetyltransferase) were all used in this study.…”

“…An interaction between Cin1 and Wsp1 was previously established using a yeast two-hybrid system (Invitrogen) for Cin1 and Wsp1 (38,39). RAC1 cDNA was digested with EcoRI and BglII from pGS1011 and was then inserted into pGADT7.…”

“…Cryptococcal cells were prepared and stained with rhodamine-conjugated phalloidin as described previously (38,39). For FM4-64 staining, cells grown in liquid YPD for 14 to 16 h (mid-to latelogarithmic phase) were harvested by centrifugation at 700 ϫ g for 1 min and were resuspended in ice-cold Hanks' buffered salt solution (HBSS; Invitrogen).…”