Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth

Rho G‐proteins are critical for polarized growth, yet little is known about the dynamics of their activation during fungal filamentous growth. We first investigated the roles of Rho1 and Rho2 during Candida albicans filamentous growth. Our results show that Rho1 is required for invasive filamentous growth and that Rho2 is not functionally redundant with Rho1. Using fluorescent reporters, we examined the dynamics of the active form of Rho1 and Cdc42 during initiation and maintenance of hyphal growth. Quantitative analyses indicated that the distribution, but not the level, of these active G‐proteins is altered during initial polarization upon germ tube emergence. A comparison of the dynamics of these active G‐proteins during budding and hyphal growth indicates that a higher concentration of active Cdc42 was recruited to the germ tube tip than to the bud tip. During hyphal elongation, active Cdc42 remained tightly restricted to the hyphal tip, whereas active Rho1 was broadly associated with the apex and subsequently recruited to the cell division site. Furthermore, our data suggest that phosphoinositide‐bis‐phosphates are critical to stabilize active Rho1 at the growth site. Together, our results point towards different regulation of Cdc42 and Rho1 activity during initiation and maintenance of filamentous growth.     

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.     

Increased filamentous growth of Candida albicans in simulated microgravity

Knowledge of simulated microgravity （SMG）-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeasthyphal transition. The morphological response may have significant implications for astronauts＇ safety, as the fungal pathogen may become more virulent during spaceflight.     

Antagonistic interplay of Swi1 and Tup1 on filamentous growth of Candida albicans

Candida albicans is a polymorphic human opportunistic pathogen in which the Swi-Snf complex functions as an activator whereas Tup1 acts as a general repressor during the yeast-hyphae transition. In Saccharomyces cerevisiae, the interplay between the Swi-Snf complex and the Tup1-Ssn6 repressive complex regulates the balance between active and repressed chromatin structures of a number of genes. To study the interplay between Candida albicans Swi1 and Tup1 and their effects on morphogenesis, we analyzed phenotypes of swi1/swi1, tup1/tup1 and swi1/swi1 tup1/tup1 mutants under various growth conditions. The swi1/swi1 mutant failed to form true hyphae, whereas the tup1/tup1 mutant exhibited constitutive filamentous growth. Deletion of SWI1 in the tup1/tup1 mutant completely blocked hyphal growth under all the conditions examined. Under aerobic conditions, the swi1/swi1 tup1/tup1 mutant most resembled the swi1/swi1 mutant in phenotype, actin polarization and gene expression pattern. In invaded agar, the double mutant showed similar phenotypes as the swi1/swi1 mutant, while under embedded conditions, it grew as a pseudohypha-like form different from that of the wild-type strain, swi1/swi1 or tup1/tup1 mutants. These results suggest that Swi1 may play a dominant role by antagonizing the repressive effect of the Tup1 on hyphal development in C. albicans.     

The regulation of filamentous growth in yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.     

Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of Efg1p-mediated repression

Filamentation of Candida albicans occurs in response to many environmental cues. During growth within matrix, Efg1p represses filamentation and Czf1p relieves this repression. We propose that Czf1p interacts with Efg1p, altering its function. The complex regulation of filamentation may reflect the versatility of C. albicans as a pathogen.     

Negative regulation of the actin-regulating kinase Prk1p by patch localization-induced autophosphorylation

The Prk1 family of protein kinases are important regulators of endocytosis and actin cytoskeleton in some eukaryotic cells. In budding yeast, Prk1p phosphorylates numerous endocytic proteins including Pan1p and Sla1p. Prk1p has been observed to undergo autophosphorylation in vivo . In this study, we determined the sites and underlying role of the autophosphorylation. Two sites located in the noncatalytic region were identified to be the autophosphorylation sites. When the sites were mutated, the non-autophosphorylatable Prk1p phosphorylated Pan1p and Sla1p more efficiently than the wild-type kinase, suggesting a negative effect of the autophosphorylation. In addition, the dynamic properties of actin and the coat complex were also altered in the autophosphorylation mutant cells. Interestingly, the autophosphorylation of Prk1p was dependent on cortical localization of the kinase and could be induced by phosphorylated Sla1p. These results suggest that the autophosphorylation of Prk1p may represent a feedback mechanism possibly involved in fine-tuning the pace of progression during actin-coupled endocytosis.     

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip‐growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long‐range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three‐dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub‐apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short‐range “on‐site” secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.     

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.     

Accumulation of P-bodies in Candida albicans under different stress and filamentous growth conditions

Candida albicans is an opportunistic fungal pathogen that grows as budding yeast, pseudohyphal, and hyphal forms. In response to external signals, C. albicans switches rapidly among these forms. mRNA-containing cytoplasmic granules, termed processing bodies (P-bodies), have been reported to accumulate under various environmental stress conditions in diverse species from yeast to mammals. Here, we provide the first microscopic and genetic characterization of P-bodies in C. albicans. The core components of P-bodies, including the decapping machinery (Dcp2 and Dhh1), 5′–3′ exoribonuclease (Kem1/Xrn1), and the P-body scaffolding protein (Edc3), were identified and their localizations with respect to P-bodies were demonstrated. Various growth conditions, including glucose deprivation, hyperosmotic stress, and heat stress, stimulated the accumulation of P-bodies. In addition, we observed P-body aggregation during hyphal development. The deletion mutant strain edc3/edc3 had a defect in filamentation and exhibited a dramatic reduction in the number of P-bodies. These results suggest that Edc3 plays an essential role in the assembly and maintenance of P-bodies in C. albicans, and that the switch to filamentous growth appears to accompany P-body accumulation.