Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation

In Candida albicans, a fungal pathogen, the small G‐protein Ras1 regulates many important behaviors including white‐opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post‐translational lipid modifications. Here, we report that the levels of membrane‐associated, full‐length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha‐to‐yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane‐targeting domain. Ras1 cleavage was repressed by cAMP‐signalling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways.     

Cellophane surface-induced gene,VdCSIN1, regulates hyphopodium formation and pathogenesis via cAMP-mediated signalling in Verticillium dahliae

The soil-borne vascular pathogen Verticillium dahliae infects many dicotyledonous plants to cause devastating wilt diseases. During colonization, V. dahliae spores develop hyphae surrounding the roots. Only a few hyphae that adhere tightly to the root surface form hyphopodia at the infection site, which further differentiate into penetration pegs to facilitate infection. The molecular mechanisms controlling hyphopodium formation in V. dahliae remain unclear. Here, we uncovered a cellophane surface-induced gene (VdCSIN1) as a regulator of V. dahliae hyphopodium formation and pathogenesis. Deletion of VdCSIN1 compromises hyphopodium formation, hyphal development and pathogenesis. Exogenous application of cyclic adenosine monophosphate (cAMP) degradation inhibitor or disruption of the cAMP phosphodiesterase gene (VdPDEH) partially restores hyphopodium formation in the VdΔcsin1 mutant. Moreover, deletion of VdPDEH partially restores the pathogenesis of the VdΔcsin1 mutant. These findings indicate that VdCSIN1 regulates hyphopodium formation via cAMP-mediated signalling to promote host colonization by V. dahliae.     

Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe.

The adenylyl cyclase gene, cyr1, of Schizosaccharomyces pombe has been cloned. We have begun an analysis of the function and regulation of adenylyl cyclase by disrupting this gene and by over-expressing all or parts of this gene in various strains. cyr1- strains are viable and contain no measurable cyclic AMP. They conjugate and sporulate under conditions that normally inhibit wild-type strains. Strains containing the cyr1 coding sequences transcribed from the strong adh1 promoter contain greatly elevated adenylyl cyclase activity, as measured in vitro, but only modestly elevated cAMP levels. Such strains conjugate and sporulate less frequently than wild-type cells upon nutrient limitation. Strains which carry the wild-type cyr1 gene but that also express high levels of the amino terminal domain of adenylyl cyclase behave much like cyr1-strains, suggesting that the amino terminal domain can bind a positive regulator. A protein that copurifies with the adenylyl cyclase of S. pombe cross-reacts to antiserum raised against the S. cerevisiae adenylyl cyclase-associated regulatory protein, CAP.     

Candida albicans Cyr1, Cap1 and G‐actin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growth

A key virulence trait of Candida albicans is its ability to undergo the yeast‐to‐hyphal growth transition in response to environmental signals. This transition critically requires a rapid activation of the adenylyl cyclase Cyr1 to generate a cAMP spike. However, the identity of the signal sensors and mechanisms of signal processing and integration remain largely unclear. Recent evidence suggests that some sensors are embedded in Cyr1 itself. To test this hypothesis, we asked whether purified Cyr1 can respond to hyphal induction. Here, we report that Cyr1 co‐purifies with Cap1 and G‐actin as a tripartite complex which can increase cAMP synthesis in response to hyphal inducing signals in an actin‐dependent manner. Cap1 binds Cyr1 and G‐actin through its N‐ and C‐terminus respectively. Deleting the G‐actin binding sites or treating the complex with the actin toxin latrunculin A or cytochalasin A inhibits the activation of cAMP synthesis. Strains expressing Cap1 mutants lacking the G‐actin binding site are impaired in both cAMP synthesis and hyphal morphogenesis. Thus, our findings reveal an essentially intact sensor/effector apparatus composed of Cyr1, Cap1 and G‐actin. Furthermore, G‐actin's regulatory role in this apparatus may prove to be the missing link whereby cellular actin status knowingly influences cAMP‐mediated cellular processes.     

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.     

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (= Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.     

Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions

Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O₂. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.     

New findings on phosphodiesterases,MoPdeH and MoPdeL,in Magnaporthe oryzae revealed by structural analysis

The cyclic adenosine monophosphate (cAMP) signalling pathway mediates signal communication and sensing during infection‐related morphogenesis in eukaryotes. Many studies have implicated cAMP as a critical mediator of appressorium development in the rice blast fungus, Magnaporthe oryzae. The cAMP phosphodiesterases, MoPdeH and MoPdeL, as key regulators of intracellular cAMP levels, play pleiotropic roles in cell wall integrity, cellular morphology, appressorium formation and infectious growth in M. oryzae. Here, we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras. The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function. Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL, either singly or together, resulted in decreased cAMP hydrolysis activity of MoPdeH. All of the transformants exhibited phenotypes similar to that of the ΔMopdeH mutant, but also revealed that EAL and L1 play additional roles in conidiation, and that L1 is involved in infectious growth. We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis. The intracellular cAMP level negatively regulates Mps1‐MAPK and positively regulates Pmk1‐MAPK in the rice blast fungus. Our results provide new information to better understand the cAMP signalling pathway in the development, differentiation and plant infection of the fungus.     

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.     

A multi‐protein complex controls cAMP signalling and filamentation in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen of humans. The ability of the fungus to grow as both yeast and filamentous forms is essential for its pathogenicity. Morphogenesis of C. albicans is largely regulated through the secondary messenger cAMP, produced by the soluble adenylyl cyclase, Cyr1p. Recent evidence suggests that Cyr1p can be directly stimulated by environmental cues to increase cytoplasmic cAMP levels and thus promote hyphal development. In this issue of Molecular Microbiology, Zou et al. demonstrate that, in response to some environmental cues, Cyr1p functions as part of a tripartite complex additionally involving Cap1p and G‐actin. All three proteins in the complex are required to raise cytosolic cAMP levels after stimulation with serum and bacterial peptidoglycan. The formation of such a complex highlights the importance of precise regulation of Cyr1p activity in response to host environmental cues.     

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.     

Control of growth and differentiation by cyclic AMP in fungi

InSaccharomyces cerivisiae intracellular cAMP mediates environmental signals that regulate cellular metabolism and growth. The studies on the cAMP-requiring mutants and their suppressors in the yeast revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, stimulation of the phosphoinositide pathway and the post-meiotic stage of spourlation, and that inhibition of cAMP-dependent protein phosphorylation is required to go into the GO stage of and to induce meiotic division. Growth of some filamentous fungi was observed with significantly reduced levels of cAMP, suggesting that cAMP may not be essential for growth in some species of fungi. Germination of fungal spores, yeast-mycelium dimorphism and hyphal morphogenesis of several species of fungi were affected by cAMP. cAMP was involved in extension of hyphae, formation of hyphal aggregates and fruit body formation. Phosphorylation of cellular proteins is required in these processes, and the nature of these proteins phosphorylated by cAMP-dependent protein kinase is important to the understanding of the role of cAMP for growth and differentistion in fungal cells.     

Increased allocation to external hyphae of arbuscular mycorrhizal fungi under CO2 enrichment

Prunella vulgaris was inoculated with different arbuscular mycorrhizal fungi (AMF) and grown at two concentrations of CO₂ (ambient, 350 μl l⁻¹, and elevated, 600 μl l⁻¹) to test whether a plants response to elevated CO₂ is dependent on the species of AMF colonizing the roots. Using compartments accessible only to AMF hyphae but not to roots, we also tested whether elevated CO₂ affects the growth of external AMF hyphae. Plant biomass was significantly greater at elevated than at ambient CO₂; the biomass of the root system, for example, increased by a factor of 2. The colonization of AMF inside the root remained constant, indicating that the total AMF inside the root system also increased by a factor of 2. The length of external AMF hyphae at elevated CO₂ was up to 5 times that at ambient CO₂, indicating that elevated CO₂ promoted allocation of AMF biomass to the external hyphae. The concentration and content of phosphorus in the stolons differed significantly between ambient and elevated CO₂ but this resulted in either an increase or a decrease, according to which AMF isolate occupied the roots. We hypothesized that an increase in external hyphal growth at elevated CO₂ would result in increased P acquistion by the plant. To test this we supplied phosphorus, in a compartment only accessible to AMF hyphae. Plants did not acquire more phosphorus at elevated CO₂ when phosphorus was added to this compartment. Large increases in AMF hyphal growth could, however, play a significant role in the movement of fixed carbon to the soil and increase soil aggregation. Received: 28 March 1998 / Accepted: 27 August 1998     

Effect of endothelin-1 on lipolysis in rat adipocytes

Objective : To explore the role of endothelin‐1 (ET‐1) on lipid metabolism, we examined the effect of ET‐1 on lipolysis in rat adipocytes. Research Methods and Procedure : Adipocytes isolated from male Sprague‐Dawley rats, weighing 400 to 450 grams, were incubated in Krebs‐Ringer buffer with or without 10^?7 M ET‐1 for various times or with various concentrations of ET‐1 for 4 hours; then glycerol release into the incubation medium was measured. In addition, selective ET_AR and ET_BR blockers were used to identify the ET receptor subtype involved. We also explored the involvement of cyclic adenosine monophosphate (cAMP) in ET‐1‐stimulated lipolysis using an adenylyl cyclase inhibitor and by measuring changes in intracellular cAMP levels in response to ET‐1 treatment. To further explore the underlying mechanism of ET‐1 action, we examined the involvement of the extracellular signal‐regulated kinase (ERK)‐mediated pathways. Results : Our results showed that ET‐1 caused lipolysis in rat adipocytes in a time‐ and dose‐dependent manner. BQ610, a selective ET_AR blocker, blocked this effect. The adenylyl cyclase inhibitor, 2′, 5′‐dideoxyadenosine, had no effect on ET‐1‐stimulated lipolysis. ET‐1 did not induce an increase in intracellular cAMP levels. In addition, ET‐1‐induced lipolysis was blocked by inhibition of ERK activation using PD98059. Coincubation of cells with ET‐1 and insulin suppressed ET‐1‐stimulated lipolysis. Discussion : These findings show that ET‐1 stimulates lipolysis in rat adipocytes through the ET_AR and activation of the ERK pathway. The underlying mechanism is cAMP‐independent. However, this non‐conventional lipolytic effect of ET‐1 is inhibited by the anti‐lipolytic effect of insulin.     

The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development,lipid metabolism and virulence in Fusarium asiaticum

Septins are a highly conserved family of GTP‐binding proteins that contribute to many cellular and metabolic functions, including cell polarity, cytokinesis, cell morphogenesis and pathogenesis. In this study, we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus Fusarium asiaticum. The functions of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12, designated ΔFaCdc3‐5 and ΔFaCdc12‐71, respectively. The deletion mutants exhibited a reduced rate of mycelial growth, increased aerial hyphae formation, irregularly shaped hyphae, reduced conidiation and a lack of sexual reproduction in wheat kernels. Histochemical analysis revealed that the conidia and hyphae of ΔFaCdc3‐5 and ΔFaCdc12‐71 formed large lipid droplets (LDs). ΔFaCdc3‐5 and ΔFaCdc12‐71 also exhibited increased resistance to agents that induce osmotic stress and damage the cell membrane and cell wall. In addition, the hyphae and conidia of the two mutants formed fewer septa than those of the wild‐type and exhibited aberrant nuclear distribution. Pathogenicity assays showed that ΔFaCdc3‐5 and ΔFaCdc12‐71 exhibited reduced virulence on wheat spikelets, which was indirectly correlated with a reduced level of deoxynivalenol accumulation. All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12. These results indicate that FaCdc3 and FaCdc12 play a critical role in various cellular processes in F. asiaticum.