Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.     

Up-regulation of the cell integrity pathway in saccharomyces cerevisiae suppresses temperature sensitivity of the pgs1Delta mutant

We have previously shown that mutants in the cardiolipin (CL) pathway exhibit temperature-sensitive growth defects that are not associated with mitochondrial dysfunction. The pgs1Delta mutant, lacking the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase (Pgs1p), has a defective cell wall due to decreased beta-1,3-glucan (Zhong, Q., Gvozdenovic-Jeremic, J., Webster, P., Zhou, J., and Greenberg, M. L. (2005) Mol. Biol. Cell 16, 665-675). Disruption of KRE5, a gene involved in cell wall biogenesis, restores beta-1,3-glucan synthesis and suppresses pgs1Delta temperature sensitivity. To gain insight into the mechanisms underlying the cell wall defect in pgs1Delta, we show in the current report that pgs1Delta cells have reduced glucan synthase activity and diminished levels of Fks1p, the glucan synthase catalytic subunit. In addition, activation of Slt2p, the downstream effector of the protein kinase C (PKC)-activated cell integrity pathway, was defective in pgs1Delta. The kre5W1166X suppressor restored Slt2p activation and dramatically increased (>10-fold) mRNA levels of FKS2, the alternate catalytic subunit of glucan synthase, partially restoring glucan synthase activity. Consistent with these results, up-regulation of PKC-Slt2 signaling and overexpression of FKS1 or FKS2 alleviated sensitivity of pgs1Delta to cell wall-perturbing agents and restored growth at elevated temperature. These findings demonstrate that functional Pgs1p is essential for cell wall biogenesis and activation of the PKC-Slt2 signaling pathway.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

A 55 kDa plasma membrane-associated polypeptide is involved in beta-1,3-glucan synthase activity in pea tissue

By glycerol gradient centrifugation of a detergent-solubilized plasma membrane fraction from pea tissue, we find a polypeptide of 55 kDa that copurifies with beta-1,3-glucan synthase activity. An antiserum against this polypeptide adsorbs glucan synthase activity and the 55 kDa polypeptide from digitonin-solubilized plasma membrane. These results indicate that the 55 kDa polypeptide is involved in pea beta-1,3-glucan synthase activity.     

Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan.

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.     

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.     

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

GAS2 and GAS4, a pair of developmentally regulated genes required for spore wall assembly in Saccharomyces cerevisiae

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.     

A Highlights from MBoC Selection: Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data

The cell wall of budding yeast is a rigid structure composed of multiple components. To thoroughly understand its involvement in morphogenesis, we used the image analysis software CalMorph to quantitatively analyze cell morphology after treatment with drugs that inhibit different processes during cell wall synthesis. Cells treated with cell wall–affecting drugs exhibited broader necks and increased morphological variation. Tunicamycin, which inhibits the initial step of N-glycosylation of cell wall mannoproteins, induced morphologies similar to those of strains defective in α-mannosylation. The chitin synthase inhibitor nikkomycin Z induced morphological changes similar to those of mutants defective in chitin transglycosylase, possibly due to the critical role of chitin in anchoring the β-glucan network. To define the mode of action of echinocandin B, a 1,3-β-glucan synthase inhibitor, we compared the morphology it induced with mutants of Fks1 that contains the catalytic domain for 1,3-β-glucan synthesis. Echinocandin B exerted morphological effects similar to those observed in some fks1 mutants, with defects in cell polarity and reduced glucan synthesis activity, suggesting that echinocandin B affects not only 1,3-β-glucan synthesis, but also another functional domain. Thus our multivariate analyses reveal discrete functions of cell wall components and increase our understanding of the pharmacology of antifungal drugs.     

A putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers

A novel plant gene CFL1 was cloned from cotton (Gossypium hirsutum L.) fibers by expressed sequence tag (EST) database searching and 5'-RACE (rapid amplification of cDNA ends). This gene shows sequence homology with FKS1 which has been identified as the putative catalytic subunit of the yeast beta-1,3-glucan synthase. It encodes a protein (CFL1p) of 219 kDa with 13 deduced transmembrane helices and 2 large hydrophilic domains, one of which is at the N-terminus and the other in the internal region of the polypeptide. CFL1 displays 21% identity and 41% similarity to FKS1 at the amino acid level over its entire length, with 31% identity and 52% similarity for the hydrophilic central domain. Using RNA and protein blot analysis, CFL1 was found to be expressed at higher levels in cotton fibers during primary wall development. CFL1 also had a strong expression in young roots. Using a calmodulin (CaM)-gel overlay assay, the hydrophilic N-terminal domain of CFL1p was shown to bind to CaM, while the hydrophilic central domain did not. A putative CaM-binding domain, 16 amino acids long, was predicted in the hydrophilic N-terminal domain. Moreover, a product-entrapment assay demonstrated that a protein associated with an in vitro-synthesized callose pellet could be labeled by anti-CFL1 antibodies. Our finding suggests that CFL1 is a putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 and could be involved in callose synthesis.     

α1,3 glucans are dispensable in Aspergillus fumigatus

A triple α1,3 glucan synthase mutant of Aspergillus fumigatus obtained by successive deletions of the three α1,3 glucan synthase genes (AGS1, AGS2, and AGS3) has a cell wall devoid of α1,3 glucans. The lack of α1,3 glucans affects neither conidial germination nor mycelial vegetative growth and is compensated by an increase in β1,3 glucan and/or chitin content.     

<Emphasis Type="Italic">Neurospora crassa</Emphasis> FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate,UDP-Glucose

The essential fungal cell-wall polymer (1,3)β-glucan is synthesized by the enzyme (1,3)β-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)β-glucan synthase. We have purified (1,3)β-glucan synthase from Neurospora crassa ∼400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[β-³²P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H⁺-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)β-glucan synthase. Received: 31 May 2002 / Accepted: 27 July 2002     

Aspergillus fumigatus devoid of cell wall β‐1,3‐glucan is viable,massively sheds galactomannan and is killed by septum formation inhibitors

Echinocandins inhibit β‐1,3‐glucan synthesis and are one of the few antimycotic drug classes effective against Aspergillus spp. In this study, we characterized the β‐1,3‐glucan synthase Fks1 of Aspergillus fumigatus, the putative target of echinocandins. Data obtained with a conditional mutant suggest that fks1 is not essential. In agreement, we successfully constructed a viable Δfks1 deletion mutant. Lack of Fks1 results in characteristic growth phenotypes similar to wild type treated with echinocandins and an increased susceptibility to calcofluor white and sodium dodecyl sulfate. In agreement with Fks1 being the only β‐1,3‐glucan synthase in A. fumigatus, the cell wall is devoid of β‐1,3‐glucan. This is accompanied by a compensatory increase of chitin and galactosaminogalactan and a significant decrease in cell wall galactomannan due to a massively enhanced galactomannan shedding. Our data furthermore suggest that inhibition of hyphal septation can overcome the limitations of echinocandin therapy. Compounds inhibiting septum formation boosted the antifungal activity of caspofungin. Thus, development of clinically applicable inhibitors of septum formation is a promising strategy to improve existing antifungal therapy.