The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

Abstract

Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1⌬ mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1⌬ mutant occurs in the absence of major growth defects in rich medium and with classical virulenceassociated phenotypes.

Membrane Organization and Dynamics in Cell Polarity

“…In the fungus Cryptococcus neoformans, Cdc42 is mislocalized to the cytoplasm when the Rho GDI homolog is overexpressed (Price et al 2008). This is consistent with published data that recombinant Rdi1 or overexpressed Rdi1 in yeast can extract Rho proteins from the plasma membrane or vacuole membrane (Eitzen et al 2001;Tcheperegine et al 2005;Tiedje et al 2008).…”

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

“…Among the evolutionarily conserved Rasdependent genes, three genes, PXL1 (identification number 03907), RDI1 (03316), and BEM3 (04832), whose orthologs are known to be involved in the regulation of Rho-GTPase Cdc42 in S. cerevisiae, were notable since the Ras1-Cdc24 signaling pathway has been reported to be controlled by one of three Cdc42 homologs in C. neoformans (47). RDI1 and BEM3 encode a Rho-GDP dissociation inhibitor and a Rho-GTPaseactivating protein, respectively (52,80). Rdi1 plays a key role in controlling membrane localization and the normal function of Cdc42 and is required for full virulence of C. neoformans (52,80).…”

“…RDI1 and BEM3 encode a Rho-GDP dissociation inhibitor and a Rho-GTPaseactivating protein, respectively (52,80). Rdi1 plays a key role in controlling membrane localization and the normal function of Cdc42 and is required for full virulence of C. neoformans (52,80). Notably, in agreement with the role of Ras1 in the genotoxic stress response of C. neoformans (Fig.…”

Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis

“…Strains were incubated at 37°C in 5% CO 2 for 2 days in tissue culture wells with 2 ml of Dulbecco's modified Eagle's medium for induction of capsule as previously described (42), and images were visualized at ϫ100 magnification. Melanin production was verified following induction of growth on Niger seed agar (NSA) for 2 to 5 days at 30°C to visually confirm dark colonies.…”

“…We assessed ena1⌬, rub1⌬, and pik1⌬ mutant strains for survival of these yeasts within macrophages. The WT and the ena1⌬, rub1⌬, and pik1⌬ mutant strains were incubated overnight at 30°C at 150 rpm as inoculum for the macrophage survival assay, as previously described (11,42).…”