Yan-Ming Wang

Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis

The human fungal pathogen Candida albicans switches from yeast to hyphal growth when exposed to serum or phagocytosed. However, the importance of this morphological switch for virulence remains highly controversial due to the lack of a mutant that affects hyphal morphogenesis only. Although many genes specifically expressed in hyphal cells have been identified and shown to encode virulence factors, none is required for hyphal morphogenesis. Here we report the first hypha‐specific gene identified, HGC1, which is essential for hyphal morphogenesis. Deletion of HGC1 abolished hyphal growth in all laboratory conditions tested and in the kidneys of systemically infected mice with markedly reduced virulence. HGC1 expression is co‐regulated with other virulence genes such as HWP1 by the cAMP/protein kinase A signaling pathway and transcriptional repressor Tup1/Nrg1. Hgc1 is a G1 cyclin‐related protein and co‐precipitated with the cyclin‐dependent kinase (Cdk) CaCdc28. It has recently emerged that cyclin/Cdk complexes promote other forms of polarized cell growth such as tumor cell migration and neurite outgrowth. C. albicans seems to have adapted a conserved strategy to control specifically hyphal morphogenesis.

Bacterial Peptidoglycan Triggers Candida albicans Hyphal Growth by Directly Activating the Adenylyl Cyclase Cyr1p

Human serum potently induces hyphal development of the polymorphic fungal pathogen Candida albicans, a phenotype that contributes critically to infections. The fungal adenylyl cyclase Cyr1p is a key component of the cAMP/PKA-signaling pathway that controls diverse infection-related traits, including hyphal morphogenesis. However, identity of the serum hyphal inducer(s) and its fungal sensor remain unknown. Our initial analyses of active serum fractions revealed signs of bacterial peptidoglycan (PGN)-like molecules. Here, we show that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can strongly promote C. albicans hyphal growth. Analogous to PGN recognition by the mammalian sensors Nod1 and Nod2 through their leucine-rich-repeat (LRR) domain, we show that MDPs activate Cyr1p by directly binding to its LRR domain. Given the abundance of PGN in the intestine, a natural habitat and invasion site for C. albicans, our findings have important implications for the mechanisms of infection by this pathogen.

The /`obligate diploid/' Candida albicans forms mating-competent haploids

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast–hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin‐dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase‐activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1‐dependent hyperphosphorylation. Using the analog‐sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Δ mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28‐dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.

CaSPA2 is important for polarity establishment and maintenance in Candida albicans

Saccharomyces cerevisiae Spa2p is a component of polarisome that controls cell polarity. Here, we have characterized the role of its homologue, CaSpa2p, in the polarized growth in Candida albicans. During yeast growth, GFP‐tagged CaSpa2p localized to distinct growth sites in a cell cycle‐dependent manner, while during hyphal growth it persistently localized to hyphal tips throughout the cell cycle. Persistent tip localization of the protein was also observed in Catup1Δ and Canrg1Δ, mutants constitutive for filamentous growth. Caspa2Δ exhibited defects in polarity establishment and maintenance, such as random budding and failure to confine growth to a small surface area leading to round cells with wide, elongated bud necks and markedly thicker hyphae. It was also defective in nuclear positioning, presumably a result of defective interactions between cytoplasmic microtubules with certain polarity determinants. The highly conserved SHD‐I and SHD‐V domains were found to be important and responsible for different aspects of CaSpa2p function. Caspa2Δ exhibited no virulence in the mouse systemic candidiasis model. Because of the existence of distinct growth forms and the easy control of the switch between them in vitro, C. albicans may serve as a useful model in cell polarity research.

Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice

Here, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SAC‐defective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild‐type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti‐C. albicans therapies.

Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation

The growing tips of Candida albicans hyphae are sites of polarized exocytosis. Mammalian septins have been implicated in regulating exocytosis and C. albicans septins are known to localize at hyphal tips, although their function here is unknown. Here, we report that C. albicans cells deleted of the exocyst subunit gene SEC3 can grow normal germ tubes, but are unable to maintain tip growth after assembly of the first septin ring, resulting in isotropic expansion of the tip. Deleting either of the septin genes CDC10 or CDC11 caused Sec3p mislocalization and surprisingly, also restored hyphal development in the sec3Δ mutant without rescuing the temperature sensitivity. Co-immunoprecipitation experiments detected association of the septin Cdc3p with the exocyst subunits Sec3p and Sec5p. Our results reveal that C. albicans hyphal development occurs through Sec3p-independent and dependent phases, and provide strong genetic and biochemical evidence for a role of septins in polarized exocytosis.

The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans

Formins are conserved eukaryotic proteins playing key roles in regulating cell polarity. We have characterized the roles of a formin CaBni1p in the polymorphic fungus Candida albicans. CaBni1p localized persistently to hyphal tips during hyphal growth but to distinct growth sites at different cell cycle stages during yeast growth. Cabni1Δ yeast cells exhibited several morphological defects, such as round and enlarged cells, widened bud necks and a random budding pattern. Although Cabni1Δ cells could still undergo yeast-hypha growth switch, the hyphae were markedly swollen. Cabni1Δ also showed defects in spindle and cytoplasmic microtubule orientation and positioning. Coincidentally, the spindle orientation protein CaKar9p in Cabni1Δ yeast cells appeared as multiple random cortical spots, in contrast to the single spot at the bud tip of many wild-type cells. Interestingly, several defects manifested in Cabni1Δ yeast cells were partially corrected during hyphal growth. We found that the second formin CaBnr1p was recruited to hyphal tips, while it localized only to the bud neck during yeast growth. This behavior of CaBnr1p may play a key role in correcting Cabni1Δ defects during hyphal growth. Cabni1Δ exhibited reduced virulence in mice. These results indicate that the formins play an important role in Candida albicans polarized growth and CaBni1ps function is required for virulence.

Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationary-phase entry and stress response in Candida albicans

The GTPase Ras1 activates the yeast‐to‐hypha transition in Candida albicans by activating cAMP synthesis. Here, we have characterized Ras2. Ras2 belongs to a group of atypical Ras proteins in some fungal species that share poor identity with other Ras GTPases with many variations in conserved motifs thought to be crucial for Ras‐associated activities. We find that recombinant Ras2 is enzymatically as active as Ras1. However, only RAS1 can rescue the lethality of the Saccharomyces cerevisiae ras1 ras2 mutant, suggesting functional divergence of the two genes. ras2Δ is normal in hyphal growth, but deleting RAS2 in the ras1Δ background greatly aggravates the hyphal defect, indicating that Ras2 also has a role in hyphal development. Strikingly, while RAS1 deletion causes a ∼20‐fold decrease in cellular cAMP, further deletion of RAS2 restores it to ∼30% of the wild‐type level. Consistently, while the ras1Δ mutant enters the stationary phase prematurely, the double mutant does so normally. Moreover, ras1Δ cells exhibit increased resistance to H2O2 and higher sensitivity to the heavy metal Co2+, whereas ras2Δ cells show the opposite phenotypes. Together, our data reveal a novel regulatory mechanism by which two antagonizing Ras GTPases balance each other in regulating multiple cellular processes in C. albicans.

Actinopolymorpha singaporensis gen. nov., sp. nov., a novel actinomycete from the tropical rainforest of Singapore.

A novel actinomycete was isolated from soil in the tropical rainforest of Singapore. The cells of this actinomycete are highly pleomorphic. In the early stages of growth, most cells are of irregular squarish shape and varied sizes. Cells remain attached after cell division, often forming chains or aggregates of a few cells. Cells at the end of a chain tend to elongate. With prolonged cultivation, cells show different degrees of elongation and enlargement, producing branched hyphae of uneven thickness. At the periphery of the colony, long hyphae form, which are divided into alternating segments of elongated cells and chains of squarish cells. This actinomycete is considerably salt-tolerant, able to grow in the presence of 15% NaCl. Chemotaxonomically, it contains LL-diaminopimelic acid (DAP) in the cell wall, type PI phospholipids and MK-9(H6) as the predominant menaquinone. 16S rDNA sequence analysis assigned this actinomycete to the family Nocardioidaceae, but its 16S rDNA shared no more than 91.2% sequence similarity with other members of the family. Based on phenotypic, chemotaxonomic and phylogenetic evidence, it is proposed that this actinomycete be classified as a new species in a new genus, Actinopolymorpha singaporensis gen. nov., sp. nov.