β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

13 páginas, 7 figuras, 2 tablas.[Background]: The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of b-glucans. The b(1,3)-glucan synthase complex synthesizes linear b(1,3)- glucans, which remain unorganized until they are cross-linked to other b(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding b(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. [Methodology/Principal Findings]: Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1D mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display b(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. [Conclusions/Significance]: We conclude that b(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth.This research was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (BFU2004-00778) and Junta de Castilla y Leon (GR231) to C.R.V-A and from the European Community (LSHB-CT-2004-511952) to C.R.V-A. and J.P.L. M.M-R. held a fellowship from the Ministerio de Educacion y Ciencia.Peer reviewe

Dinámica de septinas y regulación de la separación celular durante el crecimiento hifal de Candida albicans ¿una diana de antifúngicos?

Resumen del trabajo presentado al "IV Congreso GEBMP: Simposio Hongos Patógenos" celebrado en Badajoz el 6 de julio del 2012.Peer Reviewe

A membrane-associated form of the transcription factor Ace2 controls septin ring dynamics during Candida albicans hyphal growth

Resumen del trabajo presentado a la Vth International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis celebrada en Primosten (Croacia) del 6 al 9 de Junio de 2012.Peer Reviewe

The β-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombe

17 pages, 8 figures.-- PMID: 18410286 [PubMed].-- Printed version published Jun 2008.-- et al.Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the β-1,3-glucan synthase bgs2p synthesizes linear β-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between β-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with β-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4+, a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4Δ diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4+ is strongly induced during sporulation and a yellow fluorescent protein (YFP)–gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.This research was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (BFU2004-00778 and BFU2007-60390BMC) to C.V.-A. and from the European Community (LSHB-CT-2004-511952) to C.V.-A. and J.P.L. The author M.M. held a fellowship from the Ministerio de Educación y Ciencia.Peer reviewe

Plasticity of septin filament assembly during hyphal development in Candida albicans

Resumen del trabajo presentado a la 10ª Reunión de la Red Española de Levaduras, celebrada en El Escorial (Madrid) del 16 al 18 de diciembre de 2015.Candida albicans is a major fungal pathogen in immunologically compromised patients. A key virulence trait is its ability to switch between the yeast and hyphal forms. Whereas yeast cells are required for dissemination, the filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth among dimorphic fungi is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is developmentally regulated. Previously, we have shown that the inhibition of cell separation in hyphae requires a modification of the dynamic properties of septins, a conserved family of GTPases that normally form a ring at the site of cytokinesis. Septins are GTP‐binding proteins that assemble into hetero‐oligomers and filaments and are important elements in morphogenesis in almost all eukaryotic organisms with the exception of plants. In C. albicans yeasts, septins form a ring at the bud emergence site that persists until cytokinesis, when it splits into two rings and dissasembles. The basic polymerization unit is a hetero‐tetramer, and two of them join into a nonpolar octamer (Cdc11‐Cdc12‐Cdc3‐Cdc10‐Cdc10‐Cdc3‐Cdc12‐Cdc11). Sep7, a fifth septin, can substitute Cdc11 at the terminal position. In hyphae septin rings are assembled inside the germ tube, split into two rings before cytokinesis and they are maintained after cytokinesis, resulting in multiple rings along the length of the hypha. Inhibition of hyphal cell separation intrinsic to hyphal development is dependent on the Sep7 septin and on its phosphorylation by the hyphaspecific cyclin‐CDK complex Hgc1‐Cdc28. sep7Δ mutants form normal hyphae, but the compartments separate after cytokinesis. In this work, we have analyzed the contribution of Sep7 and Cdc11 to the inhibition of hyphal cell separation. The results indicate that the proportion of Sep7/Cdc11 in the filaments is essential to maintain the hyphal compartments attached, since a sep7+/‐ heterozygous mutant has an intermediate phenotype between the WT and the sep7‐/‐ mutant, and this defect is corrected when the Cdc11 dosage is reduced in the sep7+/‐ cdc11+/‐ heterozygous. We have also analyzed the different domains of Sep7 required for normal hyphal development and the putative phosphorylation sites that might contribute to this regulation.MICINN/European Union FEDER (BFU2010-15884) y Ayudas de la Universidad de Salamanca.Peer Reviewe

β-glucanase Eng2 is required for ascus wall endolysis after sporulation in the fission yeast Schizosaccharomyces pombe

Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. WhenSchizosaccharomyces pombe diploid cells undergo meiosis, they differentiate into asci containing four haploid ascospores that are highly resistant to environmental stress. The formation of the ascospore wall requires the activity of several enzymes involved in the biosynthesis and modification of its components, such as α-and β-glucan synthases. Once the spores are completely mature, the wall of the ascus undergoes an endolytic process that results in the release of ascospores from the ascus, allowing their dispersal into the environment. This process requires the activity of the endo-α;-1,3-glucanase Agn2. Here, we focus on the characterization of the endo-β-1,3-glucanase Eng2, which is also required for ascospore release from the ascus. Although Eng2 is present during the mitotic cycle, the protein accumulates after meiosis II. The expression ofeng2+ is required for the efficient release of ascospores, as shown by placingeng2+ under the control of a repressible promoter. Furthermore, a point mutation that destroys the catalytic activity of the protein results in a phenotype similar to that of the mutant strain. Finally, we demonstrate that exogenous addition of purified Eng2 releases the ascospores from asci generated by aneng2δ mutant. We propose that Eng2 would act together with Agn2 to completely hydrolyze the ascus wall, thereby assisting in the release of ascospores in S. pombe. Copyright © 2009, American Society for Microbiology. All Rights Reserved.This work was supported by grants from Ministerio de Ciencia y Tecnología (BFU2004-00778 and BFU2007-60390).Peer Reviewe

Regulation of cell separation during Candida hyphal growth by the Ace2 transcription factor and septins

Resumen del trabajo presentado a la Vth International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis celebrada en Primosten (Croacia) del 6 al 9 de Junio de 2012.-- Tambien presentado por Carlos R. Vázquez de Aldana al "International Symposium: Biology and Communications" celebrado en Madrid del 26 al 27 de marzo de 2012.Peer Reviewe

The Int1 anillin and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans

Trabajo presentado en la 11º Reunión de la Red Española de Levaduras, celebrada en El Escorial (España), del 13 al 15 de diciembre de 2017Ploidy plasticity is a feature of the opportunistic fungal human pathogen Candida albicans that may promote adaptation to stress conditions such as exposure to antifungal drugs. Indeed, variation in cell ploidy is common in C. albicans clinical isolates resistant to the antifungal drug fluconazole. However, the molecular mechanisms by which cellular ploidy is controlled are poorly understood in this fungus. In budding yeasts, multiprotein complexes at the cleavage plane are important to ensure genome stability. Septins, a conserved filament-forming GTPases, are important in cytokinesis and host-pathogen interactions. In this study, we found that the Sep7 septin, independently of other septin subunits, collaborates with the anillin-related protein Int1 in the maintenance of cellular ploidy. Int1 is the ortholog of S. cerevisiae Bud4, and it is required for virulence and interacts and localizes with the septin ring along the cell cycle. Loss of Int1 gives rise to a rapid disassembly of duplicated septin rings from the bud neck at the onset of actomyosin ring contraction. Strikingly, the absence of split rings has no major impact on cytokinesis and septum formation. Thus, neither Int1 nor duplicated septin rings are essential for concentrating diffusible cytokinesis factors at the division site. However, Int1 genetically interacts with the Sep7 septin, maintaining the diffusion barrier function at the bud neck and guarantying a faithful nuclear segregation. Indeed, int1¿¿ sep7¿¿ mutant cells, in contrast to int1¿¿ cdc10¿¿, gave rise to a large proportion of cells with more than one nucleus (44%). We found that this was due to a premature activation of mitotic exit prior to the alignment of the mitotic spindle with the division axis, producing large multinucleated cells. Some of these multinucleated cells arise from trimeras similar to those observed upon fluconazole exposure. The defects in nuclear segregation could be in part due to the inability to maintain the Lte1 mitotic exit activator at the cortex of the daughter cell. These results suggest that Int1 and Sep7 play a role in maintaining genome stability by acting as a diffusion barrier for Lte1, and suggest that cortical cytoskeletal proteins located at the bud neck are essential for preserving genome stability in C. albicans.Peer reviewe

The anillin-related Int1 protein and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans

Variation in cell ploidy is a common feature of Candida albicans clinical isolates that are resistant to the antifungal drug fluconazole. Here, we report that the anillin-related protein Int1 interacts with septins for coupling cytokinesis with nuclear segregation. Loss of Int1 results in a rapid disassembly of duplicated septin rings from the bud neck at the onset of actomyosin ring contraction. Strikingly, this has no major impact on cytokinesis and septum formation. However, Int1 genetically interacts with the Sep7 septin, maintaining the diffusion barrier at the bud neck and guarantying a faithful nuclear segregation. Indeed, int1ΔΔ sep7ΔΔ mutant cells, in contrast to int1ΔΔ cdc10ΔΔ, undergo a premature activation of mitotic exit prior to the alignment of the mitotic spindle with the division axis, producing large multinucleated cells. Some of these multinucleated cells arise from trimeras similar to those observed upon fluconazole exposure. Finally, the defects in nuclear segregation could be in part due to the inability to maintain the Lte1 mitotic exit activator at the cortex of the daughter cell. These results suggest that Int1 and Sep7 play a role in maintaining genome stability by acting as a diffusion barrier for Lte1.This work was supported by grants from the Spanish Ministry of Science and Innovation to CRV (BFU2010-15884 and BIO2015-70195-C2-1-R) and to JC-B (BFU2012-39910 and BIO2015-70195-C2-2R) and from the Junta de Extremadura to JC-B (GR15008). All Spanish funding is co-sponsored by the European Union FEDER programme. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. SOM was supported by pre-doctoral fellowships (FPI program) from the Spanish Government. The support and help of the Bioscience Applied Techniques Facility (Servicio de Técnicas Aplicadas a las Biociencias) of the University of Extremadura is greatly acknowledged.Peer reviewe

A single nucleotide polymorphism uncovers a novel function for the transcription factor Ace2 during Candida albicans hyphal development

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al.Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages.This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO) (BFU2010-15884 to CRVdA and BFU2012-39910 to JCB), co-sponsored by the European Union FEDER program and from the Junta de Extremadura (GRU10-008) to JCB. DMCN and SOM were supported by pre-doctoral fellowships (FPI program) from the Spanish Government. CdE has received funding from the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence ‘‘Integrative Biology of Emerging Infectious Diseases’’ (Grant #ANR-10-LABX-62-IBEID). This work was also supported by NIH grant R01 AI077737 to GS.Peer Reviewe