Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 921-928 ◽  
Author(s):  
Serena Selvaggini ◽  
Carol A. Munro ◽  
Serge Paschoud ◽  
Dominique Sanglard ◽  
Neil A. R. Gow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
De Novo ◽  
Gene Families ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Growing Cell ◽  
Chitinase Genes

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

scholarly journals Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

2010 ◽  
Vol 9 (9) ◽  
pp. 1329-1342 ◽  
Author(s):  
Claire A. Walker ◽  
Beatriz L. Gómez ◽  
Héctor M. Mora-Montes ◽  
Kevin S. Mackenzie ◽  
Carol A. Munro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Cell Walls ◽  
Chitin Synthesis ◽  
Melanin Production ◽  
Content Type ◽  
Encoding Gene ◽  
Chitinase Genes

ABSTRACT The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

scholarly journals CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis.

1992 ◽  
Vol 12 (4) ◽  
pp. 1764-1776 ◽  
Author(s):  
C E Bulawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xenopus Laevis ◽  
Chitin Synthase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Significant Similarity ◽  
Rhizobium Species

In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein of Xenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.

Effect of cerulenin and sodium butyrate on chitin synthesis in Candida albicans

1987 ◽  
Vol 33 (6) ◽  
pp. 546-550 ◽  
Author(s):  
Phyllis C. Braun ◽  
Richard F. Hector ◽  
Michael E. Kamark ◽  
John T. Hart ◽  
Ronald L. Cihlar
Keyword(s):  
Candida Albicans ◽  
Sodium Butyrate ◽  
Marked Decrease ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Time Intervals ◽  
Whole Cells ◽  
Insoluble Material ◽  
Membrane Bound

Experiments were conducted to gain insight concerning the mechanism(s) whereby cerulenin and sodium butyrate affect chitin synthesis in Candida albicans. In vitro studies with isolated membrane-bound chitin synthase from C. albicans, strain 4918, showed that neither agent affected the level of either unactivated or trypsin-activated enzyme activity. Subsequent studies utilizing protoplasts revealed that early in the cell wall regeneration process, cells treated with cerulenin or butyrate synthesized chitin at a rate equal to untreated controls, as measured by the incorporation of [3H]-N-acetylglucosamine (GlcNAc) into acid–alkali insoluble material. However, after 40 min of incubation, the incorporation of [3H]GlcNAc into chitin is reduced in cells treated with either agent. On the other hand, samples taken during the same time intervals and analyzed by flow cytometry suggested that the amount of chitin synthesis in treated and untreated cells was identical. A marked decrease in fluorescence was observed in similar experiments using polyoxin D, a direct inhibitor of chitin synthase activity. Experiments that measured uptake of [3H]GlcNAc into both whole cells and protoplasts demonstrated that cerulenin and butyrate had no effect on the transport of the chitin precursor.

CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis

1992 ◽  
Vol 12 (4) ◽  
pp. 1764-1776
Author(s):  
C E Bulawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xenopus Laevis ◽  
Chitin Synthase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Significant Similarity ◽  
Rhizobium Species

In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein of Xenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.

scholarly journals Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

1989 ◽  
Vol 108 (5) ◽  
pp. 1665-1672 ◽  
Author(s):  
E Cabib ◽  
A Sburlati ◽  
B Bowers ◽  
S J Silverman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Acidic Ph ◽  
Chitin Synthesis ◽  
Ph Optimum ◽  
Repair Synthesis ◽  
Septum Formation ◽  
Nuclear Separation

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Potent Chitin Synthase Inhibitors from Plants

2020 ◽  
Vol 16 (1) ◽  
pp. 58-63
Author(s):  
Amrutha Vijayakumar ◽  
Ajith Madhavan ◽  
Chinchu Bose ◽  
Pandurangan Nanjan ◽  
Sindhu S. Kokkal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Caenorhabditis Elegans ◽  
Aspergillus Niger ◽  
Small Molecules ◽  
Tip Growth ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Hyphal Tip Growth ◽  
Hyphal Tip

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

scholarly journals Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

1996 ◽  
Vol 7 (12) ◽  
pp. 1909-1919 ◽  
Author(s):  
M Ziman ◽  
J S Chuang ◽  
R W Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Endocytic Pathway ◽  
Chitin Synthase ◽  
Cell Fractionation ◽  
Cell Wall Polysaccharide ◽  
Chitin Synthesis ◽  
A Cell ◽  
Steady State Levels ◽  
Membrane Bound

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

scholarly journals N-Acetylglucosamine Utilization by Saccharomyces cerevisiae Based on Expression of Candida albicans NAG Genes

2009 ◽  
Vol 75 (18) ◽  
pp. 5840-5845 ◽  
Author(s):  
Jürgen Wendland ◽  
Yvonne Schaub ◽  
Andrea Walther
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
De Novo ◽  
Biofuel Production ◽  
Bioethanol Production ◽  
Catabolic Pathway ◽  
Cellular Functions ◽  
Kinase A

ABSTRACT Synthesis of chitin de novo from glucose involves a linear pathway in Saccharomyces cerevisiae. Several of the pathway genes, including GNA1, are essential. Genes for chitin catabolism are absent in S. cerevisiae. Therefore, S. cerevisiae cannot use chitin as a carbon source. Chitin is the second most abundant polysaccharide after cellulose and consists of N-acetylglucosamine (GlcNAc) moieties. Here, we have generated S. cerevisiae strains that are able to use GlcNAc as a carbon source by expressing four Candida albicans genes (NAG3 or its NAG4 paralog, NAG5, NAG2, and NAG1) encoding a GlcNAc permease, a GlcNAc kinase, a GlcNAc-6-phosphate deacetylase, and a glucosamine-6-phosphate deaminase, respectively. Expression of NAG3 and NAG5 or NAG4 and NAG5 in S. cerevisiae resulted in strains in which the otherwise-essential ScGNA1 could be deleted. These strains required the presence of GlcNAc in the medium, indicating that uptake of GlcNAc and its phosphorylation were achieved. Expression of all four NAG genes produced strains that could use GlcNAc as the sole carbon source for growth. Utilization of a GlcNAc catabolic pathway for bioethanol production using these strains was tested. However, fermentation was slow and yielded only minor amounts of ethanol (approximately 3.0 g/liter), suggesting that fructose-6-phosphate produced from GlcNAc under these conditions is largely consumed to maintain cellular functions and promote growth. Our results present the first step toward tapping a novel, renewable carbon source for biofuel production.

Sign in / Sign up

Export Citation Format

Share Document