Characterization of a novel separase-interacting protein and candidate new securin, Eip1p, in the fungal pathogen Candida albicans.

Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans, an important pathogen of humans, is essential for chromosome segregation. However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Esp1p-Interacting Protein1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APCCdc20-dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.

Depletion of the mitotic kinase Cdc5p in Candida albicans results in the formation of elongated buds that switch to the hyphal fate over time in a Ume6p and Hgc1p-dependent manner.

The fungal pathogen Candida albicans differentiates between yeast, hyphae and pseudohyphae in order to enhance survival in the human host. Environmental cues induce hyphal development and expression of hyphal-specific genes. Filaments also result from yeast cell cycle arrest, but the nature of these cells and their mechanisms of formation are less clear. We previously demonstrated that depletion of the mitotic polo-like kinase Cdc5p resulted in the production of filaments under yeast growth conditions that were distinct from hyphae with respect to several criteria, yet expressed hyphal-specific genes at later stages of development. In order to clarify the identity of these growth forms and their relationship to true hyphae, we conducted time course-based investigations of aspects of the polar growth machinery, which can distinguish cell types. During later stages of Cdc5p depletion, the myosin light chain Mlc1p demonstrated a Spitzenkörper-like localization in the tips of some filaments, and the Cdc42p GAP Rga2p became hyper-phosphorylated, as in true hyphae. Hyphal-specific genes HWP1, UME6 and HGC1 were strongly expressed at approximately the same time. HWP1 expression was dependent on Ume6p, and absence of Ume6p or Hgc1p influenced late-stage filament morphology and integrity. Finally, polarized growth and UME6 expression in Cdc5p-depleted cells were independent of the transcription factor Hms1p. Thus, depleting Cdc5p generates elongated buds that switch to a hyphal fate over time through a mechanism that involves UME6 and HGC1 induction, possibly in response to maintenance of polarized growth. The results expand on the multiple strategies with which C. albicans can modulate growth mode and expression of virulence determinants.

The Polo-like kinase PLKA in Aspergillus nidulans is not essential but plays important roles during vegetative growth and development.

The Polo-like kinases (Plks) are conserved, multifunctional cell cycle regulators that are induced in many forms of cancer and play additional roles in metazoan development. We previously identified plkA in Aspergillus nidulans, the only Plk investigated in filamentous fungi to date, and partially characterized its function through overexpression. Here, we report the plkA null phenotype. Surprisingly, plkA was not essential, unlike Plks in other organisms that contain a single homologue. A subset of cells lacking PLKA contained defects in spindle formation and chromosome organization, supporting some conservation in cell cycle function. However, septa were present, suggesting that PLKA, unlike other Plks, is not a central regulator of septation. Colonies lacking PLKA were compact with multibranched hyphae, implying a role for this factor in aspects of hyphal morphogenesis. These defects were suppressed by high temperature or low concentrations of benomyl, suggesting that PLKA may function during vegetative growth by influencing microtubule dynamics. However, the colonies also showed reduced conidiation and precocious formation of sexual Hülle cells in a benomyl- and temperature-insensitive manner. This result suggests that PLKA may influence reproduction through distinct mechanisms and represents the first example of a link between Plk function and development in fungi. Finally, filamentous fungal Plks have distinct features, and phylogenetic analyses reveal that they may group more closely with metazoan PLK4. In contrast, yeast Plks are more similar to metazoan proteins PLK1 to PLK3. Thus, A. nidulans PLKA shows some conservation in cell cycle function but may also play novel roles during hyphal morphogenesis and development.

Orthologues of the anaphase-promoting complex/cyclosome coactivators Cdc20p and Cdh1p are important for mitotic progression and morphogenesis in Candida albicans.

The conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G(1) phase. Candida albicans is an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterized C. albicans orthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets of S. cerevisiae cdc20 mutants, implying a distinct role in morphogenesis. In comparison, cdh1Δ/cdh1Δ cells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. The cdh1Δ/cdh1Δ phenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereas S. cerevisiae cdh1Δ yeast cells were reduced in size. Thus, C. albicans Cdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis in C. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.

G1/S transcription factor orthologues Swi4p and Swi6p are important but not essential for cell proliferation and influence hyphal development in the fungal pathogen Candida albicans.

The G(1)/S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe. In the fungal pathogen Candida albicans, G(1)/S regulation is not clear. To gain more insight into the G(1)/S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae. The mbp1Δ/Δ cells showed minor growth defects, whereas swi4Δ/Δ and swi6Δ/Δ yeast cells dramatically increased in size, suggesting a G(1) phase delay. Gene set enrichment analysis (GSEA) of transcription profiles revealed that genes associated with G(1)/S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4Δ/Δ swi6Δ/Δ and swi4Δ/Δ mbp1Δ/Δ strains were viable, in contrast to the situation in S. cerevisiae, and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans-host cell interactions. Since Swi4p and Swi6p influence G(1) phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G(1)/S regulatory machinery of C. albicans contains novel features and underscore the existence of a relationship between G(1) phase and morphogenetic switching, including hyphal development, in the pathogen.

Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans.

Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery.

Morphogenesis in Candida albicans.

Candida albicans is termed a dimorphic fungus because it proliferates in either a yeast form or a hyphal form. The switch between these forms is the result of a complex interplay of external and internal factors and is coordinated in part by polarity-regulating proteins that are conserved among eukaryotic cells. However, yeast and hyphal cells are not the only morphological states of C. albicans. The opaque form required for mating, the pseudohyphal cell, and the chlamydospore represent distinct cell types that form in response to specific genetic or environmental conditions. In addition, hyperextended buds can form as a result of various cell cycle-related stresses. Recent studies are beginning to shed light on some of the molecular controls regulating the various morphogenetic forms of this fascinating human pathogen.

Cell cycle arrest during S or M phase generates polarized growth via distinct signals in Candida albicans.

Treatments that perturb DNA synthesis or mitosis will activate checkpoints that prevent cell cycle progression and cell proliferation. In yeast-form cells of the fungal pathogen Candida albicans, exposure to hydroxyurea (HU) or shutting off expression of the polo-like kinase CaCDC5 blocked nuclear division and spindle elongation, but activated a highly polarized growth mode. We have used transcription profiling both to characterize the initiation and progression of this polar growth pattern and to determine how cell elongation may be linked to the cell cycle in C. albicans. Different gene expression patterns during early stages of cell elongation support the concept that CaCdc5p-depleted and HU-exposed cells were blocked at different stages of the cell cycle, and suggest that different signals may generate the common polarized growth phenotype. Consistent with this, BUB2 expression was modulated in CaCdc5p-depleted cells, and absence of BUB2 prevented the maintenance of cell polarization, resulting in multibudded, pseudohyphal cells with constrictions. In contrast, HU-induced filaments did not modulate or require BUB2, but were dependent on the GTPase Ras1p. However, at later stages of cell elongation, transcription profiles were more similar, and comparisons with serum-induced hyphae revealed that the cell cycle-arrested filaments expressed several targets of the hyphal signalling pathways. Thus, arresting the yeast cell cycle in S or M phase generates a polarized growth pattern through different mechanisms in C. albicans, and maintenance of the polar growth mode can ultimately lead to the expression of hyphal-associated cell wall and virulence-related factors, in the absence of any external stimuli.

A human-curated annotation of the Candida albicans genome.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Cyclin Cln3p links G1 progression to hyphal and pseudohyphal development in Candida albicans.

G1 cyclins coordinate environmental conditions with growth and differentiation in many organisms. In the pathogen Candida albicans, differentiation of hyphae is induced by environmental cues but in a cell cycle-independent manner. Intriguingly, repressing the G1 cyclin Cln3p under yeast growth conditions caused yeast cells to arrest in G1, increase in size, and then develop into hyphae and pseudohyphae, which subsequently resumed the cell cycle. Differentiation was dependent on Efg1p, Cph1p, and Ras1p, but absence of Ras1p was also synthetically lethal with repression of CLN3. In contrast, repressing CLN3 in environment-induced hyphae did not inhibit growth or the cell cycle, suggesting that yeast and hyphal cell cycles may be regulated differently. Therefore, absence of a G1 cyclin can activate developmental pathways in C. albicans and uncouple differentiation from the normal environmental controls. The data suggest that the G1 phase of the cell cycle may therefore play a critical role in regulating hyphal and pseudohyphal development in C. albicans.

The polo-like kinase PLKA is required for initiation and progression through mitosis in the filamentous fungus Aspergillus nidulans.

Polo-like kinases (PLK) function during multiple stages of mitotic progression and in cytokinesis. We identified and cloned a PLK homologue in Aspergillus nidulans, plkA, which is the first PLK reported in a filamentous fungus and the largest member of the PLK family to date. As plkA was essential, the effects of overexpression and localization of protein in living cells were explored to determine PLKA function. Overexpression of PLKA permitted hyphal formation, but blocked nuclear division in interphase. In NIMA or NIMT temperature-sensitive backgrounds, overexpression of PLKA impaired normal entry into mitosis upon release from restrictive temperature, supporting a role for PLKA during G2/M. In the few mitotic cells present, spindles were monopolar or disorganized, and chromatin condensation and segregation were impaired, suggesting additional roles for PLKA in spindle formation and in chromosome dynamics. Consistent with this, green fluorescent protein (GFP)-tagged PLKA could localize to the spb during interphase, and to the spb and nucleus throughout mitosis. Intriguingly, PLKA remained on the spb during telophase and into G1, in contrast to other PLK. In addition, spb localization was independent of NIMA function, unlike that demonstrated in Schizosaccharomyces pombe where PLK localization to the spb required the NIMA homologue Fin1. PLKA was not detected at cortical, septation-associated sites, and overexpression did not drive septum formation, also in contrast to that observed with other PLK. Therefore, PLKA is important for multiple events during mitosis, similar to PLK in higher organisms, but exhibits differences in size, localization and influence on septation/cytokinesis, suggesting additional novel regulatory features.

Potential link between the NIMA mitotic kinase and nuclear membrane fission during mitotic exit in Aspergillus nidulans.

We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans. The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated. TINC is a cytoplasmic protein. TINC homologues and a TINC-like protein (A. nidulans HETC) are conserved in other filamentous fungi. Neither deletion of tinC nor deletion of both tinC and A. nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (DeltaN-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell. Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses. Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA. Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective. The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A. nidulans. Additional experiments demonstrate that expression of DeltaN-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by DeltaN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth.

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.

Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition.

The ability of the pathogenic fungus Candida albicans to switch from a yeast to a hyphal morphology in response to external signals is implicated in its pathogenicity. We used glass DNA microarrays to investigate the transcription profiles of 6333 predicted ORFs in cells undergoing this transition and their responses to changes in temperature and culture medium. We have identified several genes whose transcriptional profiles are similar to those of known virulence factors that are modulated by the switch to hyphal growth caused by addition of serum and a 37 degrees C growth temperature. Time course analysis of this transition identified transcripts that are induced before germ tube initiation and shut off later in the developmental process. A strain deleted for the Efg1p and Cph1p transcription factors is defective in hyphae formation, and its response to serum and increased temperature is almost identical to the response of a wild-type strain grown at 37 degrees C in the absence of serum. Thus Efg1p and Cph1p are needed for the activation of the transcriptional program that is induced by the presence of serum.