The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that the process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with Cdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically to the affinity matrix, we used a substrate trapping mutant combined with mass spectrometry analysis using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% have not previously been identified as Cdc14 interactors in C. albicans or S. cerevisiae. In this set, 55 proteins are known from previous research in S. cerevisiae and S. pombe to play roles in the cell cycle, regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by re-activating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localised to the Spindle Pole Bodies (SPBs). Thus, we have greatly increased the number of proteins that physically interact with Cdc14 in C. albicans.

The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with Ca Cdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically bound to the affinity matrix we used an orthogonal approach of a substrate trapping mutant combined with mass spectrometry analysis using stable isotope labelling in cell culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% are novel. In this set, 53 proteins play known roles in the cell regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by re-activating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localized to the Spindle Pole Bodies (SPBs). Intriguingly, 83 proteins that only interacted with Cdc14 in yeast were significantly enriched in components of the ergosterol biosynthesis pathway targeted by azole anti-fungal drugs. Thus we have greatly expanded the set of known substrates of this key cell cycle regulator in C. albicans . Author summary Candida albicans is an important fungal pathogen causing life-threatening bloodstream infections in humans, as well as debilitating mucosal infections. Here we used Mass Spectroscopy to identify proteins that physically interacted with an enzyme called Cdc14. By removing phosphate groups from proteins, and thus regulating their function, this enzyme orchestrates the intricate molecular mechanisms of nuclear division to ensure that each daughter cell receives a full complement of chromosomes. C. albicans is unusual in the way that changes in chromosome number and composition are much more common than in other organisms. This suggests that the process of nuclear division may be more error prone in C. albicans . Our work identified 126 proteins that physically associate with Cdc14 and are thus potential substrates, including 53 proteins that we know are involved in many cell cycle processes that are necessary for nuclear and cell division. Thus, we have laid the ground work to study how changes in chromosomal composition may arise due to errors in nuclear division in this important pathogen. Unexpectedly, we found that Cdc14 may also act on proteins involved in the synthesis of ergosterol, a key lipid in the cell membrane. Azoles, a major class of antifungal drugs, inhibit the synthesis of ergosterol, so Cdc14 may also be involved in the action of azoles and thus one possible way in which drug resistance arises.

Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with MS analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and posttranslational modifications between cell populations. We develop and utilise SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that are auxotrophic for lysine are successfully labelled with both lysine and arginine stable isotopes. We find that prototrophic C. albicans preferentially uses exogenous arginine and down-regulates internal production, which allow it to achieve high incorporation rates. However, similar to other yeast, C. albicans is able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method is developed to correct for the incorporation of heavy proline. In addition, we utilise the developed SILAC labelling in C. albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14(PD).

The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

The exocyst, a conserved multiprotein complex, tethers secretory vesicles before fusion with the plasma membrane; thus it is essential for cell surface expansion. In both Saccharomyces cerevisiae and mammalian cells, cell surface expansion is halted during mitosis. In S. cerevisiae, phosphorylation of the exocyst component Exo84 by Cdk1-Clb2 during mitosis causes the exocyst to disassemble. Here we show that the hyphae of the human fungal pathogen Candida albicans continue to extend throughout the whole of mitosis. We show that CaExo84 is phosphorylated by Cdk1, which is necessary for efficient hyphal extension. This action of Cdk1 depends on the hyphal-specific cyclin Hgc1, the homologue of G1 cyclins in budding yeast. Phosphorylation of CaExo84 does not alter its localization but does alter its affinity for phosphatidylserine, allowing it to recycle at the plasma membrane. The different action of Cdk1 on CaExo84 and ScExo84 is consistent with the different locations of the Cdk1 target sites in the two proteins. Thus this conserved component of polarized growth has evolved so that its phosphoregulation mediates the dramatically different patterns of growth shown by these two organisms.

Trabajo presentado al Workshops Current Trends in Biomedicine: Comparative and Functional Genomics on Fungal Pathogens celebrado en la Universidad Internacional de Andalucia (Baeza, Espana) del 17 al 19 de Noviembbre de 2014.

SummaryCandida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans‐Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.

ABSTRACT In the hyphal tip of Candida albicans we have made detailed quantitative measurements of (i) exocyst components, (ii) Rho1, the regulatory subunit of (1,3)-β-glucan synthase, (iii) Rom2, the specialized guanine-nucleotide exchange factor (GEF) of Rho1, and (iv) actin cortical patches, the sites of endocytosis. We use the resulting data to construct and test a quantitative 3-dimensional model of fungal hyphal growth based on the proposition that vesicles fuse with the hyphal tip at a rate determined by the local density of exocyst components. Enzymes such as (1,3)-β-glucan synthase thus embedded in the plasma membrane continue to synthesize the cell wall until they are removed by endocytosis. The model successfully predicts the shape and dimensions of the hyphae, provided that endocytosis acts to remove cell wall-synthesizing enzymes at the subapical bands of actin patches. Moreover, a key prediction of the model is that the distribution of the synthase is substantially broader than the area occupied by the exocyst. This prediction is borne out by our quantitative measurements. Thus, although the model highlights detailed issues that require further investigation, in general terms the pattern of tip growth of fungal hyphae can be satisfactorily explained by a simple but quantitative model rooted within the known molecular processes of polarized growth. Moreover, the methodology can be readily adapted to model other forms of polarized growth, such as that which occurs in plant pollen tubes.

Fungi can grow in a variety of growth forms: yeast, pseudohyphae and hyphae. The human fungal pathogen Candida albicans can grow in all three of these forms. In this fungus, hyphal growth is distinguished by the presence of a Spitzenkörper-like structure at the hyphal tip and a band of septin bars around the base of newly evaginated germ tubes. The budding yeast Saccharomyces cerevisiae grows as yeast and pseudohyphae, but is not normally considered to show hyphal growth. We show here that in mating projections of both C. albicans and S. cerevisiae a Spitzenkörper-like structure is present at the growing tip and a band of septin bars is present at the base. Furthermore, in S. cerevisiae mating projections, Spa2 and Bni1 form a cap to the 3-dimensional ball of FM4-64 staining, exactly as previously observed in C. albicans hyphae, suggesting that the putative Spitzenkörper may be a distinct structure from the polarisome. Taken together this work shows that mating projections of both S. cerevisiae and C. albicans show the key characteristics of hyphal growth.

The fungus Candida albicans is often a benign member of the mucosal flora; however, it commonly causes mucosal disease with substantial morbidity and in vulnerable patients it causes life-threatening bloodstream infections. A striking feature of its biology is its ability to grow in yeast, pseudohyphal and hyphal forms. The hyphal form has an important role in causing disease by invading epithelial cells and causing tissue damage. This Review describes our current understanding of the network of signal transduction pathways that monitors environmental cues to activate a programme of hypha-specific gene transcription, and the molecular processes that drive the highly polarized growth of hyphae.

Candida albicans is the most common human fungal pathogen (Beck-Sague and Jarvis, 1993). It is normally a harmless commensal organism. However, it is a opportunistic pathogen for some immunologically weak and immunocompromised people. It is responsible for painful mucosal infections such as the vaginitis in women and oral-pharangeal thrush in AIDS patients. In certain groups of vulnerable patients it causes severe, life-threatening bloodstream infections and it causes severe, life-threatening bloodstream infections and subsequent infections in the internal organs. There are various fascinating features of the C. albicans life cycle and biology that have made the pathogen the subject of extensive research, including its ability to grow in unicellular yeast, psudohyphal, and hyphal forms (Fig. 1A); its ability to switch between different but stable phenotypic states, and the way that it retains the ability to mate but apparently loses the ability to go through meiosis to complete the sexual cycle. This research has been greatly facilitated by the derivation of the complete C. albicans genome sequence (Braun et al., 2005), the development of a variety of molecular tools for gene manipulation, and a store of underpinning knowledge of cell biology borrowed from the distantly related model yeast Saccharomyces cerevisiae (Berman and Sudbery, 2002; Noble and Johnson, 2007). This review will provide a brief overview of the importance of C. albicans as a public health issue, the experimental tools developed to study its fascinating biology, and some examples of how these have been applied.

Fungal hyphae show extreme polarized growth at the tip. Electron microscope studies have revealed a apical body called the Spitzenkörper that is thought to drive polarized growth. Studies of polarized growth in S. cerevisiae have identified the protein components of the polarized growth machinery, that are conserved in other fungi. Fusion of these proteins to GFP and its variants has for the first time allowed the localization of these proteins in real time to the hyphal tip without the need for drastic fixation procedures. Such studies showed that vesicle-associated proteins localize to the Spitzenkörper and identified a second compartment located at the tip surface composed of exocyst and other proteins that mediate the fusion of secretory vesicles with the plasma membrane.

Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkörper, which acts to focus delivery of the vesicles to the tip. Post-Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue-sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkörper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin-dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion.

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

ABSTRACT Candida albicans is a commensal organism that under certain circumstances can become pathogenic. During systemic infection C. albicans is disseminated via the circulation to distant organs, where it causes multiple organ failure. Despite the severity of systemic C. albicans infection, little is known about the mechanisms involved in the adhesion of this organism to the endothelium lining blood vessels. Previous studies have used static assays to examine adhesion. However, these do not realistically model blood vessels, where circulating C. albicans cells must adhere to the endothelium under conditions of flow and shear stress. Furthermore, there is conflicting evidence concerning the role played by yeast, pseudohyphal, and hyphal forms of C. albicans in adhesion to endothelium. To test the hypothesis that there may be differences in the abilities of these three morphogenic forms of C. albicans to adhere to endothelium under conditions of flow, we developed an in vitro flow adhesion assay. We found that all three forms of C. albicans rapidly bound to confluent endothelial cells under conditions of flow. Maximum adhesion was found at low shear stress levels similar to that found in postcapillary venules. Moreover, yeast forms bound in significantly greater numbers than did pseudohyphal and hyphal forms, respectively, contrasting with previous findings from static assays. These findings are consistent with recent in vivo data suggesting that yeast forms may be capable of adhering to the endothelium and migrating into the tissues before undergoing morphogenic change to cause tissue damage.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes. The genome sequences of six Candida species have been determined, and compared with those of Candida albicans, a marine yeast and baker's yeast. Candida species are the most common cause of opportunistic fungal infection in humans. The genomic comparisons reveal striking gene family expansions associated with pathogenic species. Other aspects of Candida biology, including evolution of the genetic code, and the architecture of mating and meiotic processes, can also be addressed in the interspecies comparisons. Candida species are the most common cause of opportunistic fungal infection worldwide. Here, the genomes of six Candida species are sequenced and compared with each other and with related pathogens and non-pathogens; providing insight into the genetic features that underlie the diversity of Candida biology, including pathogenesis and the architecture of mating and meiotic processes.