Small-molecule inhibitors of biofilm formation in laboratory and clinical isolates of Candida albicans.

Candida albicans cells have the ability to form biofilms on biotic and abiotic surfaces, such as indwelling medical devices. C. albicans cells can interconvert between budded and hyphal growth forms, herein termed the budded-to-hyphal transition (BHT), which is important for the formation of mature biofilms. Previous work identified 23 small organic molecules that could inhibit the BHT but did not affect C. albicans cell viability or budded cell growth. These BHT inhibitors were proposed to inhibit multiple signalling pathways regulating the BHT, many of which also regulate biofilm formation. However, only three of the BHT inhibitors, buhytrinA, ETYA and CGP-37157, were capable of inhibiting in vitro biofilm formation of wild-type laboratory C. albicans strains. When clinical C. albicans isolates were examined for their ability to form biofilms, only 11 of the 28 clinical isolates tested (39%) were capable of forming biofilms. Although buhytrinA, ETYA and CGP-37157 could inhibit the BHT of all 28 clinical isolates, they were only able to inhibit biofilm formation of a subset of these clinical isolates, with ETYA having 100% efficacy. These data indicate that the biofilm-forming capability of laboratory and clinical isolates of C. albicans, as well as the efficacy of BHT inhibitors against these different isolates, can differ dramatically. These differences between laboratory and clinical isolates should be an important aspect to consider when examining potentially new antifungal therapeutics.

Small molecule inhibitors of the Candida albicans budded-to-hyphal transition act through multiple signaling pathways.

The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.

Multiple proteins and phosphorylations regulate Saccharomyces cerevisiae Cdc24p localization.

Targeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP-tagged Cdc24 proteins or fragments was assayed in deletion mutants of Cdc24p-interacting proteins. The boi2Delta, ent2Delta, and hua1Delta mutants showed localization defects. The tos2Delta skg6Delta double mutant displayed aberrant pre-anaphase localization to the mother-bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.

Inhibitors of cellular signalling are cytotoxic or block the budded-to-hyphal transition in the pathogenic yeast Candida albicans.

The pathogenic yeast Candida albicans can grow in multiple morphological states including budded, pseudohyphal and true hyphal forms. The ability to interconvert between budded and hyphal forms, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans virulence, and is regulated by multiple environmental and cellular signals. To identify small-molecule inhibitors of known cellular processes that can also block the BHT, a microplate-based morphological assay was used to screen the BIOMOL-Institute of Chemistry and Cell Biology (ICCB) Known Bioactives collection from the ICCB-Longwood Screening Facility (Harvard Medical School, Boston, MA, USA). Of 480 molecules tested, 53 were cytotoxic to C. albicans and 16 were able to block the BHT without inhibiting budded growth. These 16 BHT inhibitors affected protein kinases, protein phosphatases, Ras signalling pathways, G protein-coupled receptors, calcium homeostasis, nitric oxide and guanylate cyclase signalling, and apoptosis in mammalian cells. Several of these molecules were also able to inhibit filamentous growth in other Candida species, as well as the pathogenic filamentous fungus Aspergillus fumigatus, suggesting a broad fungal host range for these inhibitory molecules. Results from secondary assays, including hyphal-specific transcription and septin localization analysis, were consistent with the inhibitors affecting known BHT signalling pathways in C. albicans. Therefore, these molecules will not only be invaluable in deciphering the signalling pathways regulating the BHT, but also may serve as starting points for potential new antifungal therapeutics.

Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae.

Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.

Small-molecule inhibitors of the budded-to-hyphal-form transition in the pathogenic yeast Candida albicans.

The pathogenic yeast Candida albicans can exist in multiple morphological states, including budded, pseudohyphal, and true hyphal forms. The ability to convert between the budded and hyphal forms, termed the budded-to-hyphal-form transition, is important for virulence and is regulated by multiple environmental and cellular signals. To identify inhibitors of this morphological transition, a microplate-based morphological assay was developed. With this assay, the known actin-inhibiting drugs latrunculin-A and jasplakinolide were shown to inhibit the transition in a dose-dependent and reversible manner. Five novel small molecules that reversibly inhibited the transition and hyphal elongation without affecting budded growth were identified. These molecules inhibited hyphal growth induced by Spider, Lee's, M199 pH 8, and 10% serum-containing media, with two molecules having a synergistic effect. The molecules also differentially affected the hyphal form-specific gene expression of HWP1 and endocytosis without disrupting the actin cytoskeleton or septin organization. Structural derivatives of one of the molecules were more effective inhibiters than the original molecule, while other derivatives had decreased efficacies. Several of the small molecules were able to reduce C. albicans-dependent damage to endothelial cells by inhibiting the budded-to-hyphal-form transition. These studies substantiated the effectiveness of the morphological assay and identified several novel molecules that, by virtue of their ability to inhibit the budded-to-hyphal-form transition, may be exploited as starting points for effective antifungal therapeutics in the future.

Cdc42p GTPase regulates the budded-to-hyphal-form transition and expression of hypha-specific transcripts in Candida albicans.

The yeast Candida albicans is a major opportunistic pathogen of immunocompromised individuals. It can grow in several distinct morphological states, including budded and hyphal forms, and the ability to make the dynamic transition between these forms is strongly correlated with virulence. Recent studies implicating the Cdc42p GTPase in hypha formation relied on cdc42 mutations that affected the mitotic functions of the protein, thereby precluding any substantive conclusions about the specific role of Cdc42p in the budded-to-hypha-form transition and virulence. Therefore, we took advantage of several Saccharomyces cerevisiae cdc42 mutants that separated Cdc42p's mitotic functions away from its role in filamentous growth. The homologous cdc42-S26I, cdc42-E100G, and cdc42-S158T mutations in C. albicans Cdc42p caused a dramatic defect in the budded-to-hypha-form transition in response to various hypha-inducing signals without affecting normal budded growth, strongly supporting the conclusion that Cdc42p has an integral function in orchestrating the morphological transition in C. albicans. In addition, the cdc42-S26I and cdc42-E100G mutants demonstrated a reduced ability to damage endothelial cells, a process that is strongly correlated to virulence. The three mutants also had reduced expression of several hypha-specific genes, including those under the regulation of the Efg1p transcription factor. These data indicate that Cdc42p-dependent signaling pathways regulate the budded-to-hypha-form transition and the expression of hypha-specific genes.

Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth.

The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.

Separate membrane targeting and anchoring domains function in the localization of the S. cerevisiae Cdc24p guanine nucleotide exchange factor.

The Saccharomyces cerevisiae Cdc24p guanine nucleotide exchange factor (GEF) activates the Cdc42p GTPase to a GTP-bound state. Cdc42p and Cdc24p co-localize at polarized growth sites during the cell cycle; and analysis of Cdc24p carboxyl-terminal truncation and site-specific mutations identified a 56-amino-acid domain as being necessary and sufficient for localization to these sites. This domain, however, was unable to anchor Cdc24p at these sites. Anchoring was restored by fusing the targeting domain to either the Cdc24p carboxyl-terminal PC domain that interacts with the Bem1p scaffold protein or the Cdc42p KKSKKCTIL membrane-anchoring domain. Mutant analysis and protein solubilization data indicated that anchoring required Bem1p, the Rsr1p/Bud1p GTPase, and the potential transmembrane protein YGR221Cp/Tos2p. These data are consistent with Cdc24p localization being a function of both membrane-specific targeting and subsequent anchoring within a multi-protein complex. Given the highly conserved roles of GEFs in Cdc42p signaling pathways, it is likely that similar targeting and anchoring mechanisms exist for Rho GEFs in other eukaryotes.

Saccharomyces cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of polarized growth.

The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.