A functional link between hyphal maintenance and quorum sensing in Candida albicans

Morphogenesis in Candida albicans requires hyphal initiation and maintenance, and both processes are regulated by the fungal quorum sensing molecule (QSM) farnesol. We show that deletion of C. albicans EED1, which is crucial for hyphal extension and maintenance, led to a dramatically increased sensitivity to farnesol, and thus identified the first mutant hypersensitive to farnesol. Furthermore, farnesol decreased the transient filamentation of an eed1Δ strain without inducing cell death, indicating that two separate mechanisms mediate quorum sensing and cell lysis by farnesol. To analyze the cause of farnesol hypersensitivity we constructed either hyperactive or deletion mutants of factors involved in farnesol signaling, by introducing the hyperactive RAS1^G13V or pADH1‐CYR1^CAT allele, or deleting CZF1 or NRG1 respectively. Neither of the constructs nor the exogenous addition of dB‐cAMP was able to rescue the farnesol hypersensitivity, highlighting that farnesol mediates its effects not only via the cAMP pathway. Interestingly, the eed1Δ strain also displayed increased farnesol production. When eed1Δ was grown under continuous medium flow conditions, to remove accumulating QSMs from the supernatant, maintenance of eed1Δ filamentation, although not restored, was significantly prolonged, indicating a link between farnesol sensitivity, production, and the hyphal maintenance‐defect in the eed1Δ mutant strain.     

A G1 Cyclin Is Necessary for Maintenance of Filamentous Growth in Candida albicans

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.     

Deciphering fungal dimorphism: Farnesol's unanswered questions

Candida albicans excretes E,E‐farnesol as a virulence factor and quorum sensing molecule that prevents the yeast to hyphal conversion. Polke et al. (2016) identified eed1Δ/Δ as the first farnesol hypersensitive mutant of C. albicans. eed1Δ/Δ also excretes 10X more farnesol and while able to form hyphae, it cannot maintain hyphae. This mutant enables new research into unanswered questions, including the existence of potential farnesol receptors and transporters, regulation of farnesol synthesis, and relationships among farnesol, germ tube formation and hyphal maintenance. The eed1 farnesol hypersensitivity can be explained by higher internal concentrations of farnesol or lower thresholds for response. One possibility invokes misexpression of a transporter. Saccharomyces cerevisiae and C. albicans have transporters for farnesylated peptides, like the a‐factor pheromone, which could potentially also transport farnesol for virulence and quorum sensing. Significantly, these transporters are repressed in MTLa/MTLα C. albicans. An evolutionary pressure for C. albicans to become diploid could derive from its use of farnesol. Alternatively, maintenance of hyphal growth may increase the farnesol response threshold. Finally, Dpp1p, Dpp2p and Dpp3p are non‐specific pyrophosphatases responsible for farnesol synthesis. Changes in expression of these enzymes do not explain differences in farnesol levels implicating involvement of additional factors like a scaffolding molecule.     

Cytological interrelationships between the cell cycle and duplication cycle of Candida albicans

The cytology of nuclear division and septation in the yeast and hyphal phases of Candida albicans growing at 37 degrees C has been studied by fluorescence microscopy after staining of specimens with 4'6-diaminido-2-phenylindole (DAPI) and Calcofluor. Yeast and hyphal cells replicated their nuclei at about 18 min after the emergence of a bud or germ-tube. The site of nuclear division coincided with the future location of the septum in both forms. This occurred at the junction of the bud and parent yeast cell or 6.0 micron from the parent yeast in germ tubes which were formed in medium containing serum. The filamentous forms of a range of clinical and laboratory strains grown in a variety of germ tube-inducing media were all extensively vacuolated. Germ tube extension in all of these media was linear. It is suggested that there is little biosynthesis of cytoplasm during the initial stages of germ tube growth in this organism and that this accounts for the development of the large vacuoles and the linear growth kinetics.     

The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.     

Ras Signaling Is Required for Serum-Induced Hyphal Differentiation in Candida albicans

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the RAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 of Saccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the only RAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1 (ras1-2/ras1-3) are viable. The Candida ras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active RAS1(V13) allele manifest enhanced hyphal growth, whereas those expressing a dominant negative RAS1(A16) allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.     

Ras1-induced hyphal development in Candida albicans requires the formin Bni1

Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenk?rper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.     

Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.