Pph3 dephosphorylation of Rad53 is required for cell recovery from MMS-induced DNA damage in Candida albicans

The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.     

Effect of nonylphenol on growth of Neurospora crassa and Candida albicans.

The effects of the nonionic surfactant nonylphenol on the growth and morphologies of the filamentous fungus Neurospora crassa and the diploid yeast Candida albicans have been examined. Nonylphenol inhibited respiration and growth of N. crassa, effecting a 10-fold decrease in organism yield at 25 microM. Severe morphological defects were also induced: cell shape was abnormal and apical dominance was lost. Nonylphenol monoethoxylate (the parent compound of nonylphenol) was a less potent growth inhibitor and morphogen. The growth of the yeast form of C. albicans was sensitive to nonylphenol (inducing an order of magnitude decrease in specific growth rate with a 10-fold increase in dose concentration) but not nonylphenol monoethoxylate. Similarly, C. albicans ATP content was reduced and glucose-induced extracellular acidification was inhibited only by nonylphenol. Although estrogens may induce the dimorphic transition of C. albicans, nonylphenol (as an environmental estrogen mimic) failed to trigger germ tube formation under nonpermissive conditions and inhibited it under permissive conditions. The effects of nonylphenol are most readily explained as the result of uncoupling of respiration, which produces multiple physiological effects.     

Candida albicans, a distinctive fungal model for cellular aging study

The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.     

Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans

The pathogenic fungus Candida albicans is capable of responding to a wide variety of environmental cues with a morphological transition from a budding yeast to a polarized filamentous form. We demonstrate that the Ras homologue of C. albicans, CaRas1p, is required for this morphological transition and thereby contributes to the development of pathogenicity. However, CaRas1p is not required for cellular viability. Deletion of both alleles of the CaRAS1 gene caused in vitro defects in morphological transition that were reversed by either supplementing the growth media with cAMP or overexpressing components of the filament-inducing mitogen-activated protein (MAP) kinase cascade. The induction of filament-specific secreted aspartyl proteinases encoded by the SAP4-6 genes was blocked in the mutant cells. The defects in filament formation were also observed in situ after phagocytosis of C. albicans cells in a macrophage cell culture assay and, in vivo, after infection of kidneys in a mouse model for systemic candidiasis. In the macrophage assay, the mutant cells were less resistant to phagocytosis. Moreover, the defects in filament formation were associated with reduced virulence in the mouse model. These results indicate that, in response to environmental cues, CaRas1p is required for the regulation of both a MAP kinase signalling pathway and a cAMP signalling pathway. CaRas1p-dependent activation of these pathways contributes to the pathogenicity of C. albicans cells through the induction of polarized morphogenesis. These findings elucidate a new medically relevant role for Ras in cellular morphogenesis and virulence in an important human infectious disease.     

Candida albicans produces a cystatin-type cysteine proteinase inhibitor.

A cysteine proteinase inhibitor was found in culture media of Candida albicans. Purification to homogeneity of the inhibitor was performed by carboxymethyl-papain-Sepharose affinity, DE-52 ion-exchange, and reverse-phase high performance liquid chromatographies. The purified inhibitor had an M(r) of 15 kDa and a pI of 4.9. It was more stable to heat and pH than most proteins. The N-terminal sequence of the first 30 residues demonstrated high similarity with that of human cystatin A. Thus, C. albicans cysteine proteinase inhibitor seems to belong to the cystatin superfamily. The inhibitor activity of the yeast cellular form was 4.0 times higher than that of the hyphal cellular form in 7-day culture media. It is suggested that the inhibitor has regulatory functions similar to those of its counterpart proteinases in the invasion of host cells.     

A model for the germ tube formation and mycelial growth form of Candida albicans

A model based on morphological and ultrastructural evidence is presented which illustrates a novel and hitherto undescribed pattern of germ tube formation and hyphal growth in early and mature colonies of Candida albicans. Accordingly, most of the cytoplasm within the parent yeast cell migrates into and forward with the extending germ tubes and leaves behind an extensively vacuolated yeast cell. Growing hyphae similarly are subtended by migrating "slugs' of protoplasm and leave behind vacuolated intercalary compartments. The vacuolated cell compartments apparently must first regenerate their protoplasmic contents before producing branches or secondary germ tubes. This model is used to explain certain unusual features of the growth kinetics of the filamentous form of this organism.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans

Nitrogen starvation is one of the signals that induce Candida albicans, the major fungal pathogen of humans, to switch from yeast to filamentous growth. In response to nitrogen starvation, C. albicans expresses the MEP1 and MEP2 genes, which encode two ammonium permeases that enable growth when limiting concentrations of ammonium are the only available nitrogen source. In addition to its role as an ammonium transporter, Mep2p, but not Mep1p, also has a central function in the induction of filamentous growth on a solid surface under limiting nitrogen conditions. When ammonium is absent or present at low concentrations, Mep2p activates both the Cph1p-dependent mitogen-activated protein (MAP) kinase pathway and the cAMP-dependent signalling pathway in a Ras1p-dependent fashion via its C-terminal cytoplasmic tail, which is essential for signalling but dispensable for ammonium transport. In contrast, under ammonium-replete conditions that require transporter-mediated uptake Mep2p is engaged in ammonium transport and signalling is blocked such that C. albicans continues to grow in the budding yeast form. Mep2p is a less efficient ammonium transporter than Mep1p and is expressed at much higher levels, a distinguishing feature that is important for its signalling function. At sufficiently high concentrations, ammonium represses filamentous growth even when the signalling pathways are artificially activated. Therefore, C. albicans has established a regulatory circuit in which a preferred nitrogen source, ammonium, also serves as an inhibitor of morphogenesis that is taken up into the cell by the same transporter that mediates the induction of filamentous growth in response to nitrogen starvation.     

Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.     

The metabolic basis of Candida albicans morphogenesis and quorum sensing

Candida albicans is a polymorphic fungus that has the ability to rapidly switch between yeast and filamentous forms. The morphological transition appears to be a critical virulence factor of this fungus. Recent studies have elucidated the signal transduction pathways and quorum sensing molecules that affect the morphological transition of C. albicans. The metabolic mechanisms that recognize, and respond to, such signaling molecules and promote the morphological changes at a system level, however, remain unknown. Here we review the metabolic basis of C. albicans morphogenesis and we discuss the role of primary metabolic pathways and quorum sensing molecules in the morphogenetic process. We have reconstructed, in silico, the central carbon metabolism and sterol biosynthesis of C. albicans based on its genome sequence, highlighting the metabolic pathways associated with the dimorphic transition and virulence as well as pathways involved in the biosynthesis of important quorum sensing molecules.     

Characterization of binding of human fibrinogen to the surface of germ-tubes and mycelium of candida albicans

The binding of human fibrinogen to germ-tubes and mycelium of Candida albicans, forms usually found in infected tissues, was studied in vitro by an immunofluorescence assay. Binding was quantified by using 125I-labelled fibrinogen. The degree of binding differed according to the morphological form of the fungus. Binding relative to that of the yeast form was greater for mycelium (12-fold) than for germ-tube (7.7-fold). Pretreatment of yeasts with fragments D and E (terminal degradation products of fibrinogen) before fibrinogen binding showed that fragment D possessed a higher affinity for C. albicans than fragment E. Binding of fibrinogen was diminished when C. albicans was pretreated with 2-mercaptoethanol alone or in combination with pronase, or pretreated with alpha-mannosidase or trypsin. Binding was not decreased by pretreatment with pronase alone or chitinase. Inhibition experiments using C. albicans dialysed culture filtrate, C. albicans mannan, chitin, sugars or amino sugars were done by preabsorbing the fibrinogen with each of the above mentioned compounds. C. albicans dialysed culture filtrate inhibited the binding more strongly than C. albicans mannan. However, fibrinogen binding to C. albicans was not significantly reduced by mannose, several other sugars or chitin. These studies demonstrate the existence of a fibrinogen-binding factor (FBF) strongly associated with the surface of germ-tube and filamentous forms of C. albicans, and indicate a possible role for FBF in the pathogenicity of C. albicans.     

Demonstration of a septal pore in budding Candida albicans yeast cells

Electron microscopy of Candida albicans yeast cells grown in a peptone glucose broth at 37 degrees C revealed pores in the septum identical in appearance to those already described in the hyphal form of the fungus. The presence of septal pores in yeast cells may explain apparently synchronous post-septation events in parent and daughter cells, and emphasizes the close structural similarities between different morphological forms of C. albicans.     

Ashbya gossypii: a model for fungal developmental biology

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.     

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.     

Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi

The dimorphic fungus Candida albicans secretes farnesol, which acts as a quorum-sensing molecule and prevents the yeast to mycelium conversion. In this study we examined the effect of farnesol in the filamentous fungus Aspergillus nidulans. We show that externally added farnesol has no effect on hyphal morphogenesis; instead, it triggers morphological features characteristic of apoptosis. Additional experiments suggest that mitochondria and reactive oxygen species (ROS) participate in farnesol-induced apoptosis. Moreover, the effects of farnesol appear to be mediated by the FadA heterotrimeric G protein complex. Because A. nidulans does not secrete detectable amounts of farnesol, we propose that it responds to farnesol produced by other fungi. In agreement with this notion, growth and development were impaired in a farnesol-dependent manner when A. nidulans was co-cultivated with C. albicans. Taken together, our data suggest that farnesol, in addition to its quorum-sensing function that regulates morphogenesis, is also employed by C. albicans to reduce competition from other microbes.     

Hyphal formation of Candida albicans is controlled by electron transfer system

Most Candida albicans cells cultured in RPMI1640 medium at 37 degrees C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growth of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.