Chitin synthase III activity,but not the chitin ring,is required for remedial septa formation in budding yeast

Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall. In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential. However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa. We examined which of two CSIII functions, the formation of a chitin ring at bud emergence or of chitin in the remedial septa, was required for viability. By using cell cycle synchronization in combination with nikkomycin Z, a specific inhibitor of CSIII, we inhibited chitin synthesis in a chs2 mutant, during formation of either the ring or the remedial septa. The results show that only synthesis of the chitin during aberrant septa formation is essential for viability. Thus, the unique function of the chitin ring seems to be maintenance of the integrity of the mother-bud neck, as we recently found, and the importance of chitin in septum closure, both in normal and abnormal situations, is underlined.     

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.     

Analysis of the landmark protein Bud3 of Ashbya gossypii reveals a novel role in septum construction

Cell division in fungal cells requires the coordination of three different processes: determination of the site of division, actomyosin ring formation, and the concomitant contraction of this ring together with chitin deposition at septal sites. This report describes the isolation of the AgBUD3 homologue and the characterization of Bud3 protein function in Ashbya gossypii. Bud3 fused to green fluorescent protein was shown to localize transiently either as a single ring to multiple sites of future septation or as a double ring to newly established septa. Deletion of AgBUD3 leads to a striking change in actin ring localization involving the mislocalization of AgCyk1, which is required for actin ring assembly. Aberrant chitin accumulation occurs subsequently, generating delocalized septa. Thus, in A. gossypii, Bud3 acts as a landmark, tagging future septal sites, and is involved in the positioning of the contractile ring, whereas it does not direct lateral branching.     

Effect of Polyoxin D on Chitin Synthesis and Septum Formation in Saccharomyces cerevisiae

The normal sequence of cell separation in Saccharomyces cerevisiae begins with the formation of a primary septum, presumably consisting of chitin, on which secondary septa are later deposited. In the presence of the antibiotic polyoxin D, a potent inhibitor of chitin synthetase, pairs of abnormal cells of two different types were observed by phase-contrast microscopy: the "exploded pair," consisting of two lysed cells from which the cytoplasm had been extruded at the cell junction, and the "refringent pair," consisting of two highly refractile cells joined by a thin bridge. Thus, in both cases the septal region appears to be affected. Observations with the electron microscope showed that the primary chitin septum was not formed in either of these cell types, and as a consequence secondary septa of varying thicknesses were laid down in an abnormal pattern. With [(3)H]glucose as carbon source the incorporation of tritium into the chitin of abnormal cells was inhibited about 90%, whereas the labeling of mannan was normal and that of glucan somewhat reduced. The effective concentrations of polyoxin D (0.1 to 1 mg/ml) were much greater than those required to inhibit chitin synthesis in vitro. Dimethylsulfoxide and amphotericin B, both known to increase cell permeability, enhanced the action of the antibiotic.     

A preliminary report on complex fibrillar aggregates in the gill epithelium of Panulirus interruptus (Randall)

Complex structures have been found in gill epithelium of the spiny lobster, Panulirus interruptus. These fibrillar structures are located at the base of gill filaments and at the ends of longitudinal septa which divide the gill filaments into channels for directional blood flow. The complex fibrillar aggregates compare favorably with secretory granules seen in crustacean integument and are believed to play a role in chitin metabolism.     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.     

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinA^rigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.     

The structure and development of septa inNeurospora crassa

Summary Electron microscopy of septa ofNeurospora crassa has shown that microfibrillar material is a major structural component. These microfibrils are arranged in a predominantly tangential orientation and in young (24 hours) mycelium can often be seen in unextracted septal preparations. In older (5 days) mycelium they are overlain with amorphous material, which can be removed by treatment with Pronase, or by chemical extractions. The structure of septa is described and compared with that of the lateral hyphal wall.Developing septa are typically surrounded by vesicles, and sometimes by alignments of cytoplasmic tubules. Electron and light microscopic autoradiography have shown that N-acetylglucosamine is quickly and specifically incorporated into developing septa, and there is no evidence for its prior incorporation into any cytoplasmic site. Glucose is also incorporated into septa, but less specifically. From the autoradiographic results and from experiments with chemical and enzymic extractions it is suggested that chitin is a major component of the septum. A model of the septum is constructed from these data.     

几丁质合酶在人类致病真菌中的研究进展

抑制真菌细胞壁的合成常作为防治真菌感染的安全有效手段。几丁质是真菌细胞壁及隔膜的重要结构成分，几丁质合酶是催化几丁质合成的关键酶。真菌细胞中几丁质合酶家族的不同成员在调控几丁质的合成中存在着差异，因此产生不同的生物学效应。本文通过综述几丁质合酶在人体三大条件致病真菌白色念珠菌、烟曲霉、新生隐球菌中的研究进展，分析了几丁质合酶对真菌致病性影响的机制，总结了几丁质合酶调控真菌细胞增殖、形态转换、病原菌与宿主的相互作用和细胞壁损伤诱导的补偿效应，展望了抗真菌感染的新策略及关于真菌几丁质合酶的未来研究方向。     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.     

Hyphal Wall Synthesis in Aspergillus nidulans: Effect of Protein Synthesis Inhibition and Osmotic Shock on Chitin Insertion and Morphogenesis

Pulse-labeling with N-[acetyl-(3)H] glucosamine and radioautography were used to follow the sites of chitin incorporation in hyphae of an Aspergillus nidulans mutant blocked in amino sugar synthesis. Growing hyphae incorporated N-acetylglucosamine almost exclusively at the tip. Cycloheximide addition greatly increased the label in subapical regions of the hyphae and reduced that at the tip. This effect of cycloheximide was immediate, could be reversed by removing the inhibitor, and did not appear to be due to chitin turnover. A similar change from apical to subapical N-acetylglucosamine incorporation occurred after hyphae were subjected to an osmotic shock which did not inhibit protein synthesis. The two treatments induced morphogenetic changes in the hyphae which produced abnormally large numbers of branches and septa.     

Induction of Chitin-Binding Proteins during the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

Previous work has shown that attachment of Vibrio harveyi to chitin is specific and involves at least two chitin-binding peptides. However, the roles and regulation of these chitin-binding peptides in attachment are still unclear. Here we show that preincubation with the oligomeric sugars composing chitin stimulated chitinase activity, cellular attachment to chitin, and production of chitin-binding peptides. One of these peptides, a 53-kDa peptide, is produced constitutively and appears to mediate initial attachment to chitin. Synthesis of another peptide, a 150-kDa chitin-binding peptide, is induced by chitin and thus may be involved in time-dependent attachment. Coordinated regulation of attachment and degradation of chitin may give bacteria like V. harveyi a selective advantage over other bacteria in nutrient-poor aquatic environments.     

Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.Chitin, a polymer of β-1,4-linked N-acetylglucosmine, is one of the major structural components of the fungal cell wall. Its metabolism, including synthesis, degradation, assembly, and cross-linking to other cell wall components, is thought to be very important for many fungi (5, 22, 24, 36, 45). Fungal chitin synthases have been classified into seven groups, classes I to VII, depending on the structures of their conserved regions (6). The genes encoding the synthases belonging to classes III, V, VI, and VII are only found in fungi with high chitin contents in their cell walls. We have identified six chitin synthase genes from Aspergillus nidulans and designated them chsA, chsB, chsC, chsD, csmA, and csmB; these gene products belong to classes II, III, I, IV, V, and VI, respectively (9, 13, 30, 31, 44, 52). The chsB deletion mutant grew very slowly and formed small colonies with highly branched hyphae, suggesting its important role in hyphal tip growth (3, 52). Repression of chsB expression in the deletion mutant of chsA, chsC, or chsD exaggerated the defects in the formation of aerial hyphae, the production of cell mass, or the growth under high-osmolarity conditions, respectively, compared to each single mutant. These results indicate that chsB functions at various stages of development (15, 16).The deletion of class III chitin synthase-encoding genes leads to severe defects in most of the filamentous fungi thus far investigated. However, their detailed functions are currently unknown. In Neurospora crassa, inactivation of the gene encoding Chs-1, a class III chitin synthase with 63% identity to A. nidulans ChsB, leads to slow growth, aberrant hyphal morphology, and a decrease in chitin synthase activity. The mutant of chs-1 became sensitive to Nikkomycin Z, a chitin synthase inhibitor (53). In Aspergillus fumigatus, two genes encoding class III chitin synthases, chsC and chsG, have been identified. Their gene products showed 66 and 89% identity, respectively, to A. nidulans ChsB. The chsG deletion mutant showed slow growth and defects in conidiation, and its hyphae were highly branched. chsC deletion did not cause any phenotypic change. The chsC chsG double deletion mutant showed almost the same phenotype as the chsG single deletion mutant (28). Class III chitin synthases have been reported to be involved in the virulence of some pathogens. Deletion of Bcchs3a in the phytopathogenic fungus Botrytis cinerea and double deletion of WdCHS3 and class I chitin synthase WdCHS2 in the human pathogen Wangiella dermatitidis both caused a reduction of virulence (40, 48). On the other hand, the deletion mutant of a class III chitin synthase-encoding gene, CgChsIII, of the maize pathogen Colletotrichum graminicola did not exhibit the significant phenotypic difference from the wild-type strain (50). Deletion of a gene, chs1, encoding a class III chitin synthase of the maize pathogenic dimorphic fungi Ustilago maydis caused minor defects in the growth of haploid yeastlike cells and conjugation tube formation (49). These results indicate that the functions of class III chitin synthases has evolutionally diverged.In the present study, we characterized the cytological defects of the A. nidulans chsB deletion mutant and investigated the localization of ChsB using FLAG- or enhanced green fluorescent protein (EGFP)-tagged ChsB. We reveal that the deletion mutant formed hyphae with aberrant cell wall structures and that ChsB tagged with EGFP primarily localized at polarized growth sites during germination, hyphal growth, septation, and conidiation. These findings suggest that ChsB functions at the polarized growth sites and forming septa during the hyphal growth and conidia development.     

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.