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Semilethal effects of the double inactivation ofCHS3 and GAS1 genes. (A) Representative tetratype tetrads from LF-3 (chs3Δ gas1Δ heterozygous diploid) 8 days after dissection. (B) Cell viability assay. Stationary-phase cells from the first inoculum of spores in YNB−glucose at 30°C were concentrated to a value of 8A450. Then, 5 μl of this suspension and four subsequent 10-fold serial dilutions were spotted onto YEPDAT plates. (C) CF staining of four representative spores. Magnifications: upper panels and lower left panel, ×3,200; lower right panel, ×2,000. (D) Effects of CHS3 deletion on chitin levels.
Source publication
The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of
chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested
chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas...
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Citations
... These glycoproteins likely have related functional roles in regulating chitin biosynthesis and maintaining cell wall architecture. For example, loss of the S. cerevisiae GPI-anchored Gas1 associated with cell wall polymer assembly increases chitin levels and cell wall thickness and severely reduces growth rate and cell viability (53). This suggests that similar inhibitory effects can also be achieved via modulating the activity of other cell surface glycoproteins. ...
Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth.
... Ecm33p is a known activator of the CWI pathway, and its deletion starts a compensatory mechanism by increasing chitin deposition (Pardo et al. 2004, Arias et al. 2011. The chitin synthase CSIII was subsequently found to be the enzyme responsible for increasing cell wall chitin levels (Valdivieso et al. 2000, de Groot et al. 2001, Lagorce et al. 2003. Likewise, the disruption of CWP2, a gene encoding one of the most abundant CWP, was also shown to improve the secretion of heterologous cellulase (Li et al. 2020). ...
The cell wall is a dynamic organelle that determines the shape and provides the cell with mechanical strength. This study investigated whether modulation of cell wall composition can influence the production or secretion of small metabolites by yeast cell factories. We deleted and upregulated several cell wall-related genes KRE2, CWP1, CWP2, ECM33, PUN1, and LAS21 in yeast Saccharomyces cerevisiae engineered for p-coumaric acid or β-carotene production.
Deletions of las21∆ and ecm33∆ impaired the yeast growth on medium with cell wall stressors, calcofluor white, and caffeine. Both overexpression and deletion of ECM33 significantly improved the specific yield of p-coumaric acid and β-carotene. We observed no change in secretion in any cell wall altered mutants, suggesting the cell wall is not a limiting factor for small molecule secretion at the current production levels. We evaluated the cell wall morphology of the ECM33 mutant strains using transmission electron microscopy. The ecm33∆ mutants had an increased chitin deposition and a less structured cell wall, while the opposite was observed in ECM33-overexpressing strains. Our results point at the cell wall-related gene ECM33 as a potential target for improving production in engineered yeast cell factories.
... Cell wall stress results in activation of a compensatory mechanism and increased chitin deposition in a CHS3-dependent manner (Popolo et al. 1997;Garcı'a-Rodriguez et al. 2000;Valdivieso et al. 2000). Similarly, the expression of FKS2/GSC2 is also known to increase specifically upon cell wall stress (Zhao et al. 1998). ...
... Increase in chitin content has been observed in various mutants defective in cell wall biosynthesis (Popolo et al. 1997;Garcı'a-Rodriguez et al. 2000;Valdivieso et al. 2000). Cell wall defects can be assessed by testing sensitivity toward cell wall stress-inducing agents. ...
... An active CWI signaling pathway in cohesin mutants suggests that there could be a defect in cell wall biosynthesis in these mutants. Mutation in two genes, FKS1 (codes for one of the two catalytic subunits of b-1,3-glucan synthase) and GAS1 (a 1,3beta-glucanosyltransferase, involved in b-1,3-glucan chain elongation) lead to zymolyase resistance, increased chitin content in the cell wall and sensitivity to various cell wall stress-inducing agents (Popolo et al. 1997;Ram et al. 1998;Garcı'a-Rodriguez et al. 2000;Valdivieso et al. 2000). All these phenotypes are also shared by cohesin mutants, indicating that there may be a defect in b-1,3-glucan synthesis and/or elongation, in these mutants. ...
Cohesin is a conserved chromatin-binding multisubunit protein complex involved in diverse chromosomal transactions such as sister-chromatid cohesion, chromosome condensation, regulation of gene expression, DNA replication, and repair. While working with a budding yeast temperature-sensitive mutant, mcd1-1, defective in a cohesin subunit, we observed that it was resistant to zymolyase, indicating an altered cell wall organization. The budding yeast cell wall is a strong but elastic structure essential for maintenance of cell shape and protection from extreme environmental challenges. Here, we show that the cohesin complex plays an important role in cell wall maintenance. Cohesin mutants showed high chitin content in the cell wall and sensitivity to multiple cell wall stress-inducing agents. Interestingly, temperature-dependent lethality of cohesin mutants was osmoremedial, in a HOG1-MAPK pathway-dependent manner, suggesting that the temperature sensitivity of these mutants may arise partially from cell wall defects. Moreover, Mpk1 hyper-phosphorylation indicated activation of the cell wall integrity (CWI) signaling pathway in cohesin mutants. Genetic interaction analysis revealed that the CWI pathway is essential for survival of mcd1-1 upon additional cell wall stress. The cell wall defect was independent of the cohesion function and accompanied by misregulation of expression of several genes having cell wall-related functions. Our findings reveal a requirement of cohesin in maintenance of CWI that is independent of the CWI pathway, and that may arise from cohesin’s role in regulating the expression of multiple genes encoding proteins involved in cell wall organization and biosynthesis.
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(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.
... Several S. cerevisiae cell wall mutants have been reported to exhibit increased levels of chitin as a compensatory mechanism to reestablish the cell wall integrity (Bulik et al., 2003;Kapteyn et al., 1999a,b;Popolo et al., 1997;Ram et al., 1998). Because the absence of Chs3 causes a reduction in the content of chitin in some of these cell wall mutants (Bulik et al., 2003;Carotti et al., 2002;García-Rodríguez et al., 2000;Valdivieso et al., 2000), it has been proposed that Chs3 is responsible for the compensatory increase of chitin levels in response to cell wall damage (Popolo et al., 2001). Similarly, C. albicans cells treated with echinocandin display high levels of cell wall chitin . ...
In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is absent in host mammalian cells, it stands as an important target when developing selective compounds for the treatment of fungal infections. Consequently, treatment with echinocandins, a family of antifungal agents that specifically inhibits the biosynthesis of cell wall (1-3)β-D-glucan, has been established as an alternative and effective antifungal therapy. However, the existence of many pathogenic fungi resistant to single or multiple antifungal families, together with the limited arsenal of available antifungal compounds, critically affects the effectiveness of treatments against these life-threatening infections. Thus, new antifungal therapies are required. Here we review the fungal cell wall and its relevance in biotechnology as a target for the development of new antifungal compounds, disclosing the most promising cell wall inhibitors that are currently in experimental or clinical development for the treatment of some invasive mycoses.
... Alternatively, the TM-resistant mutants may have acquired extra gene dosages to decrease TM import into the cell (37). Accumulation of chitin, a structural component of the cell wall, has been associated with increased resistance to multiple stresses (38,39) and may be able to explain decreased permeability of the cell wall to TM. However, we found that aneuploid mutants did not show increased resistance to cell wall stress, suggesting that ER stress resistance in aneuploid mutants is unlikely due to decreased permeability of the cells to TM. ...
Significance
In eukaryotes, about one-third of all proteins are folded in the endoplasmic reticulum (ER). When protein folding demand exceeds capacity of the ER, the unfolded protein response (UPR) is activated to counteract ER stress. However, the mechanisms of adaptation to ER stress are not restricted to the UPR. Here, we report that gain of chromosome II in yeast allows adaptation of cells to protein misfolding. We narrowed down the effect of chromosome II duplication to three genes, which promote ER stress resistance independently of the UPR. Overexpression of these genes was sufficient to protect cells from ER stress and led to the elimination of aneuploidy. Thus, aneuploidy can serve as an important genome adaptation protecting cells against ER stress.
... IRC8 has unknown function, and its product localizes in bud tips during cell division (25). An increase in chitin deposition has been observed as a compensation mechanism that mutants defective in cell wall synthesis or assembly activate to maintain cell integrity (26). ...
Protein haze formation in bottled wines is a significant concern for the global wine industry, and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, the addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels, indicating differences in haze-protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli-produced green fluorescent protein (GFP)-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities that also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and we propose a strategy for optimizing wine yeast strains to improve wine clarification.
IMPORTANCE
In this study, we establish a new mechanism by which wine yeast strains can impact the protein haze formation of wines, and we demonstrate that yeast cell wall chitin binds grape chitinase in a chitin concentration-dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also investigate how yeast cell wall chitin levels are affected by environmental conditions.
... In comparison to WT, 1Δ2Δ3tyΔ were much more sensitive to CFW, as well as to the β1,3glucan synthase inhibitor caspofungin, and the inhibitor of N-glycosylation tunicamycin, but, when placed on sorbitol, they remained only sensitive to CFW (Fig 3C). This argues that sorbitol is efficiently combating the cell wall deficiency of 1Δ2Δ3tyΔ, but does not eliminate it completely since CFW hypersensitivity signals an elevated chitin content of the cell wall, suggesting that chitin synthesis remains elevated since the cells continue to sense a cell wall problem [32][33][34]. ...
While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data.
... The recruitment of other CHS isoforms during HWS reveals a difference between C. albicans and S. cerevisiae. In S. cerevisiae the absence of Chs3p does not lead to chitin deposition by other isoforms, namely the Class I chitin synthase Chs1p (equivalent to Chs2p and Chs8p) despite the fact that ScCHS1 is always up-regulated by cell wall stress [57,64]. ...
Background
The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, β(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH. We exploited the pH-conditional nature of a PHR1 null mutant to analyze the impact of glucan remodeling on the hyphal transcriptional program and the role of chitin synthases in the hyphal wall stress (HWS) response.
Results
In hyphal growth inducing conditions, phr1Δ germ tubes are defective in elongation, accumulate chitin, and constitutively activate the signaling pathways mediated by the MAP kinases Mkc1p, Cek1p and Hog1p. The transcriptional profiles revealed an increase of transcript levels for genes involved in cell wall formation (CHS2 and CHS8, CRH11, PGA23, orf19.750, RBR1, RBT4, ECM331, PGA6, PGA13), protein N-glycosylation and sorting in the ER (CWH8 and CHS7), signaling (CPP1, SSK2), ion transport (FLC2, YVC1), stress response and metabolism and a reduced expression of adhesins. A transient up-regulation of DNA replication genes associated with entry into S-phase occurred whereas cell-cycle regulating genes (PCL1, PCL2, CCN1, GIN4, DUN1, CDC28) were persistently up-regulated. To test the physiological relevance of altered CHS gene expression, phr1Δ chsxΔ (x = 2,3,8) mutant phenotypes were analyzed during the Y-H transition. PHR1 deletion was synthetic lethal with CHS3 loss on solid M199 medium-pH 7.5 and with CHS8 deletion on solid M199-pH 8. On Spider medium, PHR1 was synthetic lethal with CHS3 or CHS8 at pH 8.
Conclusions
The absence of Phr1p triggers an adaptive response aimed to reinforce the hyphal cell wall and restore homeostasis. Chs3p is essential in preserving phr1Δ cell integrity during the Y-H transition. Our findings also unveiled an unanticipated essential role of Chs8p during filamentation on solid media. These results highlight the flexibility of fungal cells in maintaining cell wall integrity and contribute to assessments of glucan remodeling as a target for therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2853-5) contains supplementary material, which is available to authorized users.
