Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.     

Regulation of hyphal morphogenesis by Ras and Rho small GTPases

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa, develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans. The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.     

The mating projections of Saccharomyces cerevisiae and Candida albicans show key characteristics of hyphal growth

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 Spitzenk?rper-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 Spitzenk?rper-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 Spitzenk?rper 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.     

A Population-Based Morphologically Structured Model for Hyphal Growth and Product Formation in Streptomycin Fermentation

Summary A population model discriminating the hyphae according to the hyphal length and a morphologically structured model considering the specific function of different morphological forms of a hypha are combined together to describe mycelial growth, substrate consumption and secondary metabolite formation in streptomycin fermentation. In the population model, the growth modes of hyphae with different age or length are considered, while in the morphologically structured model, the morphological forms of hyphae and their functions in growth and metabolism are described. The population model and the morphologically structured model are interrelated by a branching function and a differentiation function. In the model, the growth rate of immature apical compartment is distinguished from those of matured ones, branching is proposed to occur only in the subapical region, and the hyphal compartment is assumed to synthesize secondary metabolites. The model is successfully applied to simulate the batch fermentation process of streptomycin production. The growth characteristics of filamentous microorganisms are also discussed using the model predictions.     

The DNA Damage Checkpoint Regulates a Transition between Yeast and Hyphal Growth in Schizosaccharomyces japonicus

Dimorphic yeasts change between unicellular growth and filamentous growth. Many dimorphic yeasts species are pathogenic for humans and plants, being infectious as invasive hypha. We have studied the determinants of the dimorphic switch in the nonpathogenic fission yeast Schizosaccharomyces japonicus, which is evolutionarily close to the well-characterized fission yeast S. pombe. We report that camptothecin, an inhibitor of topoisomerase I, reversibly induced the unicellular to hyphal transition in S. japonicus at low concentrations of camptothecin that did not induce checkpoint arrest and the transition required the DNA checkpoint kinase Chk1. Furthermore, a mutation of chk1 induced hyphal transition without camptothecin. Thus, we identify a second function for Chk1 distinct from its role in checkpoint arrest. Activation of the switch from single cell bipolar growth to monopolar filamentous growth may assist cells to evade the source of DNA damage.Yeasts and molds are major members of the kingdom Fungi. Molds grow as multicellular filamentous hyphae. On the other hand, yeasts propagate in a unicellular fashion by budding or by binary fission. However, many types of yeast can switch their growth modes, changing from unicellular growth to filamentous branching multicellular hyphae. This hyphal transition can be induced by a wide variety of environmental changes ranging from pH to the nature of the carbon source, and many species of dimorphic yeasts that are pathogenic for humans and plants are infectious in the hyphal form (15, 20).Hyphal transition is a simple mode of cellular differentiation program that is turned on upon environmental changes. The fungi may differentiate to adapt to the environmental challenges. Especially in the case of Candida albicans strains that infect humans, the hyphal transition may function as an action to resist against attack from macrophages or neutrophils. Hyphae are more difficult to phagocytose (16). It can also eventually kill macrophages if hyphal transition is triggered after ingestion by macrophage (14). Indeed, C. albicans cells that cannot form hyphae are avirulent. However, inducing hyphal growth in pathogenic yeasts is not always readily achievable in the laboratory, and genetic analysis of the hyphal growth phase and transition to this phase is often limited by the lack of appropriate tools. Thus, genetically tractable nonpathogenic dimorphic yeasts are attractive models for investigating invasive hypha.The nonpathogenic fission yeast Schizosaccharomyces japonicus is evolutionarily close to the well-characterized fission yeast Schizosaccharomyces pombe (5, 24). S. japonicus is dimorphic, transiting between unicellular and hyphal growth, and thus offers itself as an appropriate model to study this differentiation mechanism and the requirements of hyphal growth (25). In S. japonicus, hyphal growth occurs naturally on most solid medium and can occur over a range of nutrient conditions (26). It has been proposed that a gradient of nitrogen in the substrate is necessary to both initiate and direct hyphal growth in S. japonicus (26). In this report we establish conditions to induce hyphal growth in a microchamber in liquid media. In addition, we show that a low dose of the topoisomerase inhibitor camptothecin (CPT) induces hyphal differentiation under rich nutrient conditions and identify a role for the DNA damage checkpoint response in promoting the CPT-dependent transition from unicellular to hyphal growth. Genetic analysis demonstrates that this role of the checkpoint is distinct from checkpoint arrest, and we suggest it may provide an opportunity for S. japonicus to grow away from sources of genotoxic stress.     

Control of Dimorphism in Mucor by Hexoses: Inhibition of Hyphal Morphogenesis

In anaerobic cultures of Mucor rouxii, morphogenesis was strongly dependent on hexose concentration as well as pCO(2). At low levels of hexose or CO(2), or both, hyphal development occurred; at high levels, the fungus developed as yeast cells. Other dimorphic strains of Mucor responded similarly to hexose and CO(2) but differred in their relative sensitivity to these agents. Glucose was the most effective hexose in eliciting yeast development of M. rouxii; fructose and mannose were next; and galactose was last. The fungus may be grown into shapes covering its entire dimorphic spectrum simply by manipulating the hexose concentration of the medium. Thus, at 0.01% glucose, hyphae were exceedingly long and narrow; at higher sugar concentrations, the hyphae became progressively shorter and wider; finally, at about 8% glucose, almost all cells and their progeny were isodiametric (spherical budding cells). Such yeast development occurred without a manifested requirement for exogenous CO(2). The stimulation of yeast development by hexose is not an artifact due to increased production of metabolic CO(2) (hyphae or yeast cells released metabolic CO(2) at similar rates). Presumably, the effect was caused by some other hexose catabolite which interfered with hyphal morphogenesis (apical growth); deprived of its polarity, the fungus grew into spherical yeastlike shapes. Although 10% glucose inhibited the development of hyphae from germinating spores, it did not prevent the elongation of preformed hyphae. This suggests that hexose inhibits hyphal morphogenesis not by blocking the operation of the enzyme complex responsible for apical growth but by preventing its initiation; such inhibition may be regarded as a repression of hyphal morphogenesis.     

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.     

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.     

An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.     

白念珠菌的二型性转换：从共生菌到致病菌

白念珠菌Candida albicans是人体内的良性共生真菌,存在于宿主的口腔、表皮、胃肠道及阴道等处,在免疫能力低下的人群中可能引起严重的疾病。一般以二倍体的形式存在,且能在酵母、假菌丝和菌丝的状态之间转换。菌丝状态促进了白念珠菌的侵染能力,同时也可以使白念珠菌逃逸宿主的免疫攻击,在其对宿主的感染途径中起到了重要的作用。本综述将阐述白念珠菌菌丝形成的调控机制、菌丝的发育模式以及菌丝形态对宿主免疫系统的影响,并且简要介绍念珠菌属中热带念珠菌和耳念珠菌菌丝发育方面的相关研究。     

Hyphal Growth in the Fission Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe grows in a single-celled form or can mate and undergo meiosis and sporulation. Here we show that wild-type S. pombe can also differentiate to form elaborately branched hyphae which invade deep into solid medium. Branches appear in the hyphae adjacent to unseparated septa. Electron microscopy reveals unusual multivesicular structures within the hyphae. Nitrogen deprivation appears to be the main stimulus for hyphal growth. No mitogen-activated protein kinase is necessary for the response. Inhibition of cyclic AMP (cAMP) production or signaling prevents the response, and exogenous cAMP promotes it, suggesting that detection of a good carbon source is required for hyphal growth but not for mating.     

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenk?rper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.     

The SH3/PH domain protein AgBoi1/2 collaborates with the Rho-type GTPase AgRho3 to prevent nonpolar growth at hyphal tips of Ashbya gossypii

Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.     

Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Fungal growth during rice tapé fermentation

Fungal growth was quantified during Indonesian rice tapé fermentation using an agar-film technique following sample homogenization for 1 min at 25000 rev/min. After 72 h fermentation, mould hyphal length was 0·68 km/g, yeast hyphal length 2·1 km/g and the numbers of mould chlamydospores and single yeast cells were 14 times 10⁵/g and 5·1 times 10⁷/g respectively. The estimated fungal biomass in rice tapé after 72 h was 25 mg/g dry weight with 62% of this being mould hyphae, 24% mould chlamydospores, 13% yeast hyphae and 1% yeast cells.     

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.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

Effect of water potential on the development of an haploid strain of Sporisorium reilianum f.sp. zeae

Sporisorium reilianum f.sp. zeae, the causal agent of head smut, infects the roots of the maize plantlets. Little information is available concerning the development of the fungus in soil, although this saprophytic phase is an important part of the life cycle. This paper reports that water potential also affects hyphal induction, and this effect on the fungus may influence disease transmission. In response to a decrease in water potential from 0 to –1.52 MPa in presence of variable molecular weight polyethylene glycols, haploid hyphae develop from the haploid yeast. Hyphal extension is fastest at low water potentials (–1.2 MPa) controlled with high molecular weight polyethylene glycols, PEG-3350 and PEG-8000. Formation of parasitic dikaryotic hyphae following fusion between haploid hyphae was possible at low water potential (–1.2 MPa) and was not inhibited by water stress. These results are consistent with the hypothesis that the effects of low soil water potential on yeast–hyphal transition and hyphal growth facilitate the convergence of compatible haploid strains, and that this may increase disease severity.