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.     

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.     

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.     

Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans

The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenk?rper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42(G12V), which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.     

In Candida albicans, the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization

In the development of hyphal germ tubes of Candida albicans, a band of septin forms at the base of the germ tube (basal septin band). Later, a septin ring forms, which organizes the first septum within the germ tube (septin ring). We have investigated the role of the Nim1 kinases, Gin4 and Hsl1, in the formation of these septin structures. We show that during germ tube formation, Gin4 is required for the organization of the septin ring but not the basal septin band. Hsl1 is not required for the formation of either septin rings or basal bands. Unexpectedly, we found that both gin4Delta and hsl1Delta mutants form pseudohyphae constitutively, in a fashion that in the case of gin4Delta, is partly independent of Swe1. Gin4-depleted pseudohyphae are unable to form hyphae when challenged with serum, but this can be overcome by ectopic expression of Gin4 from the MET3 promoter. Thus, Gin4 may regulate the developmental switch from pseudohyphae to hyphae.     

Fluorescent proteins illuminate the structure and function of the hyphal tip apparatus

Fungal hyphae show extreme polarized growth at the tip. Electron microscope studies have revealed a apical body called the Spitzenk?rper that is thought to drive polarized growth. Studies of polarized growth in S. cerevisiae have identified the protein components of the polarized growth machinery, that are conserved in other fungi. Fusion of these proteins to GFP and its variants has for the first time allowed the localization of these proteins in real time to the hyphal tip without the need for drastic fixation procedures. Such studies showed that vesicle-associated proteins localize to the Spitzenk?rper and identified a second compartment located at the tip surface composed of exocyst and other proteins that mediate the fusion of secretory vesicles with the plasma membrane.     

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.     

Lipid raft polarization contributes to hyphal growth in Candida albicans

The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans, these results suggest that membrane polarization may contribute to the pathogenesis of this organism.     

Functional characterization of Aspergillus nidulans homologues of Saccharomyces cerevisiae Spa2 and Bud6

The importance of polarized growth for fungi has elicited significant effort directed at better understanding underlying mechanisms of polarization, with a focus on yeast systems. At sites of tip growth, multiple protein complexes assemble and coordinate to ensure that incoming building material reaches the appropriate destination sites, and polarized growth is maintained. One of these complexes is the polarisome that consists of Spa2, Bud6, Pea2, and Bni1 in Saccharomyces cerevisiae. Filamentous hyphae differ in their development and life style from yeasts and likely regulate polarized growth in a different way. This is expected to reflect on the composition and presence of protein complexes that assemble at the hyphal tip. In this study we searched for polarisome homologues in the model filamentous fungus Aspergillus nidulans and characterized the S. cerevisiae Spa2 and Bud6 homologues, SpaA and BudA. Compared to the S. cerevisiae Spa2, SpaA lacks domain II but has three additional domains that are conserved within filamentous fungi. Gene replacement strains and localization studies show that SpaA functions exclusively at the hyphal tip, while BudA functions at sites of septum formation and possibly at hyphal tips. We show that SpaA is not required for the assembly or maintenance of the Spitzenk?rper. We propose that the core function of the polarisome in polarized growth is maintained but with different contributions of polarisome components to the process.     

The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization

The location of the septin ring in the germ tubes of Candida albicans hyphae and pseudohyphae was studied using an antibody to Saccharomyces cerevisiae Cdc11p. In pseudohyphae induced by growth at 35 degrees C in YEPD or Lee's medium, a septin ring formed at or near (mean 1.8 microm) the neck between the mother cell and the germ tube. This became double later in the cycle, and the first mitosis took place across the plane of this double ring. A septin ring also formed at the germ tube neck of developing hyphae induced by serum or growth on Lee's medium at 37 degrees C. However, at later times, this ring became disorganized and disappeared. A second double ring then appeared 10-15 microm (mean 12.5 microm) along the length of the germ tube. The nucleus subsequently migrated out of the mother cell into the germ tube, and the first mitosis took place across the plane of this second septin ring. The relocation of the septin ring in developing hyphae provides a clear-cut molecular distinction between hyphae and pseudohyphae. Commitment to one type of septin localization and mitosis was shown to occur early in the first mitotic cycle, well before evagination. Germ tubes of hyphae and pseudohyphae also have different widths. A point of commitment to germ tube width was also demonstrated, but occurred later in the cycle, approximately coincident with the time of evagination.     

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Delta mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.     

Candida albicans protein kinase CaHsl1p regulates cell elongation and virulence

Saccharomyces cerevisiae Hsl1p is a Ser/Thr protein kinase that regulates cell morphology. We identified Candida albicans CaHSL1 and analysed its function in C. albicans. Cells lacking CaHsl1p exhibited filamentous growth under yeast growth conditions with the filaments elongating more quickly than did those of the wild type under hyphal growth conditions, suggesting that it plays a role in the suppression of cell elongation. Green fluorescent protein-tagged CaHsl1p colocalized with a septin complex to the bud neck during yeast growth or to a potent septation site during hyphal growth, as expected from the localization in S. cerevisiae. However, the localization of the septin complex did not change in DeltaCahsl1, suggesting that CaHsl1p does not participate in septin organization. CaHsl1p was expressed in a cell cycle-dependent manner and, except for the G1 phase, phosphorylated throughout the cell cycle. In DeltaCahsl1 cells, the phosphorylation of a possible CaHsl1p target CaSwe1p decreased, while that of CaCdc28p at tyrosine18 increased. Either an extra copy of the tyrosine18-mutated CaCdc28p or deletion of CaSWE1 suppressed the cell elongation phenotype caused by CaHSL1 deletion. Furthermore, DeltaCahsl1 exhibited reduced virulence in the mouse systemic candidiasis model. Thus, the CaHsl1p-CaSwe1p-CaCdc28p pathway appears important in the cell elongation of both the yeast and hyphal forms and to the virulence of C. albicans.     

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.     

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.     

Polarized growth in fungi: symmetry breaking and hyphal formation

Cell shape is a critical determinant for function. The baker's yeast Saccharomyces cerevisiae changes shape in response to its environment, growing by budding in rich nutrients, forming invasive pseudohyphal filaments in nutrient poor conditions and pear shaped shmoos for growth towards a partner during mating. The human opportunistic pathogen Candida albicans can switch from budding to hyphal growth, in response to numerous environmental stimuli to colonize and invade its host. Hyphal growth, typical of filamentous fungi, is not observed in S. cerevisiae. A number of internal cues regulate when and where yeast cells break symmetry leading to polarized growth and ultimately distinct cell shapes. This review discusses how cells break symmetry using the yeast S. cerevisiae paradigm and how polarized growth is initiated and maintained to result in dramatic morphological changes during C. albicans hyphal growth.     

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.     

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.