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.     

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.     

Function of Candida albicans adhesin Hwp1 in biofilm formation

Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.     

Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.     

白念珠菌生物被膜形成的遗传调控机制

白念珠菌是人体重要的条件性致病真菌。形态的多样性和可塑性是白念珠菌典型的生物学特征,这与它的致病性、宿主适应能力以及有性生殖过程密切相关。白念珠菌生物被膜(Biofilm)是由不同形态细胞(包括酵母型、菌丝和假菌丝)以及胞外基质组成的致密结构,也是毒性和耐药性形成的重要因子。生物被膜对抗真菌药物、宿主免疫系统和环境胁迫因子等都表现出较强的抵抗力和耐受性,是临床上病原真菌感染防治的重大挑战。随着基因表达谱和遗传操作技术的发展,白念珠菌生物被膜的形成及其耐药性的获得所依赖的遗传调控通路和分子调控机制越来越清楚。主要包括MAPK和cAMP介导的信号途径以及Bcr1和Tec1等因子介导的转录调控。此外,白念珠菌生物被膜的形成与形态转换和有性生殖之间存在密切的联系。文中综述了白念珠菌生物被膜形成的遗传调控机制,重点介绍了细胞壁相关蛋白、转录因子和交配型对该过程的调控以及生物被膜的耐药机制。     

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.     

Candida albicans VAC8 is required for vacuolar inheritance and normal hyphal branching

Hyphal growth is prevalent during most Candida albicans infections. Current cell division models, which are based on cytological analyses of C. albicans, predict that hyphal branching is intimately linked with vacuolar inheritance in this fungus. Here we report the molecular validation of this model, showing that a specific mutation that disrupts vacuolar inheritance also affects hyphal division. The armadillo repeat-containing protein Vac8p plays an important role in vacuolar inheritance in Saccharomyces cerevisiae. The VAC8 gene was identified in the C. albicans genome sequence and was resequenced. Homozygous C. albicans vac8Delta deletion mutants were generated, and their phenotypes were examined. Mutant vac8Delta cells contained fragmented vacuoles, and minimal vacuolar material was inherited by daughter cells in hyphal or budding forms. Normal rates of growth and hyphal extension were observed for the mutant hyphae on solid serum-containing medium. However, branching frequencies were significantly increased in the mutant hyphae. These observations are consistent with a causal relationship between vacuolar inheritance and the cell division cycle in the subapical compartments of C. albicans hyphae. The data support the hypothesis that cytoplasmic volume, rather than cell size, is critical for progression through G1.     

NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus

In Lotus japonicus, seven genetic loci have been identified thus far as components of a common symbiosis (Sym) pathway shared by rhizobia and arbuscular mycorrhizal fungi. We characterized the nup85 mutants (nup85-1, -2, and -3) required for both symbioses and cloned the corresponding gene. When inoculated with Glomus intraradices, the hyphae managed to enter between epidermal cells, but they were unable to penetrate the cortical cell layer. The nup85-2 mutation conferred a weak and temperature-sensitive symbiotic phenotype, which resulted in low arbuscule formation at 22 degrees C but allowed significantly higher arbuscule formation in plant cortical cells at 18 degrees C. On the other hand, the nup85 mutants either did not form nodules or formed few nodules. When treated with Nod factor of Mesorhizobium loti, nup85 roots showed a high degree of root hair branching but failed to induce calcium spiking. In seedlings grown under uninoculated conditions supplied with nitrate, nup85 did not arrest plant growth but significantly reduced seed production. NUP85 encodes a putative nucleoporin with extensive similarity to vertebrate NUP85. Together with symbiotic nucleoporin NUP133, L. japonicus NUP85 might be part of a specific nuclear pore subcomplex that is crucial for fungal and rhizobial colonization and seed production.     

Candida albicans Ecm33p is important for normal cell wall architecture and interactions with host cells

Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33Delta mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33Delta mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.     

N-cadherin mediates endocytosis of Candida albicans by endothelial cells

Candida albicans is the most common cause of fungal bloodstream infections. To invade the deep tissues, blood-borne organisms must cross the endothelial cell lining of the vasculature. We have found previously that C. albicans hyphae, but not blastospores, invade endothelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the endothelial cell receptor that mediates the endocytosis of C. albicans. We determined that endocytosis of C. albicans was not mediated by bridging molecules in the serum and that it was partially dependent on the presence of extracellular calcium. Using an affinity purification procedure, we discovered that endothelial cell N-cadherin bound to C. albicans hyphae but not blastospores. N-cadherin also co-localized with C. albicans hyphae that were being endocytosed by endothelial cells. Chinese hamster ovary (CHO) cells expressing human N-cadherin endocytosed significantly more C. albicans hyphae than did CHO cells expressing either human VE-cadherin or no human cadherins. The expression of N-cadherin by the CHO cells resulted in enhanced endocytosis of hyphae, but not blastospores, indicating the selectivity of the N-cadherin-mediated endocytosis. Down-regulation of endothelial cell N-cadherin expression with small interfering RNA significantly inhibited the endocytosis of C. albicans hyphae. Therefore, a novel function of N-cadherin is that it serves as an endothelial cell receptor, which mediates the endocytosis of C. albicans.     

Candida albicans biofilm development, modeling a host-pathogen interaction

Medical device-associated infections involve the attachment of cells to a surface, production of an extracellular matrix and development of a mature biofilm. Many Candida albicans disease states involve biofilm growth. These infections have great impact on public health because organisms in biofilms exhibit dramatically reduced susceptibility to antifungal therapy. Progression to a mature biofilm is dependent on cell adhesion, extracellular matrix production and the yeast-to-hyphae transition. Numerous in vitro biofilm model systems have been successfully used to examine biofilm architecture, development, cell phenotypes and drug resistance. Although these studies have included a number of experimental variables to mimic infections in patients, it is difficult to accurately account for the multitude of host and infection-site variables that are probably important in humans. Recent studies have begun to explore C. albicans biofilms using animal biofilm infection models in order to more completely reflect the complexity of this host-fungal interaction.     

Evaluation of the role of Candida albicans agglutinin-like sequence (Als) proteins in human oral epithelial cell interactions

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.     

Alterations of protein expression in macrophages in response to Candida albicans infection

Although macrophages are an important first line of cellular defense, they are unable to effectively kill phagocytosed C. albicans. To determine the physiological basis of this inability, we investigated the alterations of macrophage proteins caused by C. albicans infection. Since the formation of C. albicans hyphae caused cell death, proteins were prepared 3 h after infection and examined by two-dimensional gel electrophoresis (2-DE). The most prominent changes were in glycolytic enzymes, which could have caused energy depletion of the infected cells. Also changed were proteins involved in maintenance of cellular integrity and NO production. Treatment of the macrophages with either cytochalasin D or taxol did not alter their inability to kill C. albicans. Our results indicate that multiple factors contribute to cell death as the pathogenic form of C. albicans becomes fully active inside macrophage cells.     

Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development

Tyrosol and farnesol are quorum-sensing molecules produced by Candida albicans which accelerate and block, respectively, the morphological transition from yeasts to hyphae. In this study, we have investigated the secretion of tyrosol by C. albicans and explored its likely role in biofilm development. Both planktonic (suspended) cells and biofilms of four C. albicans strains, including three mutants with defined defects in the Efg 1 and Cph 1 morphogenetic signaling pathways, synthesized extracellular tyrosol during growth at 37°C. There was a correlation between tyrosol production and biomass for both cell types. However, biofilm cells secreted at least 50% more tyrosol than did planktonic cells when tyrosol production was related to cell dry weight. The addition of exogenous farnesol to a wild-type strain inhibited biofilm formation by up to 33% after 48 h. Exogenous tyrosol appeared to have no effect, but scanning electron microscopy revealed that tyrosol stimulated hypha production during the early stages (1 to 6 h) of biofilm development. Experiments involving the simultaneous addition of tyrosol and farnesol at different concentrations suggested that the action of farnesol was dominant, and 48-h biofilms formed in the presence of both compounds consisted almost entirely of yeast cells. When biofilm supernatants were tested for their abilities to inhibit or enhance germ tube formation by planktonic cells, the results indicated that tyrosol activity exceeds that of farnesol after 14 h, but not after 24 h, and that farnesol activity increases significantly during the later stages (48 to 72 h) of biofilm development. Overall, our results support the conclusion that tyrosol acts as a quorum-sensing molecule for biofilms as well as for planktonic cells and that its action is most significant during the early and intermediate stages of biofilm formation.     

Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.