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.     

The characterization of a chitin-associated D-glucan from the cell walls of Aspergillus niger

Exhaustive extraction of the cell walls of Aspergillus niger with 10% NaOH solution leaves an alkali-resistant residue containing chitin and glucan as the major components. The glucan in this residue comprises 58.7% of the total cell wall glucan and was characterized by permethylation, and identification of the resulting $\text{O-}\text{methyl-}\text{D}\text{-glucoses}$ obtained after hydrolysis by gas-liquid chromagtography and mass spectrometry of the derived partially acetylated, partially methylated, [1-²H]alditols. The glucan was separated from the chitin by acetylation of the alkali-resistance material, a procedure which separates a large portion of the total glucan as a chloroformsoluble acetate, abd by treatment of the alkali-insoluble residue with nitrous acid, a procedure which was found to render the complex soluble in dimethylsulfoxide and amenable, therefore, to permethylation. The data collected suggests that the preparation is an essentially linear glucan containing 85–95% 1 → 3 linkages and 10–15% 1 → 4 linkages. An analysis of the glycosidic linkages using NMR spectroscopy indicate that both α and β linkages are present in the ratio of 4:1. An identical glucan appears to be present in the cell walls of Penicillium chrysogenum as well as the spore cell walls of both organisms, as evidenced by methylation studies.     

Functional stratification of the Spitzenkörper of Neurospora crassa

GS‐1 (ncu04189) is a protein required for the synthesis of β‐1,3‐glucan in Neurospora crassa. As chitin, β‐1,3‐glucan is a morphogenetically relevant component of the fungal cell wall. Previously, we showed that chitin synthases are delivered to the growing hyphal tip of N. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the Spitzenkörper (Spk). Tagged with the green fluorescent protein (GFP), GS‐1 accumulated in the hyphal apex forming a dynamic and pleomorphic ring‐like structure (‘Spitzenring’) that corresponded to the Spk outer macrovesicular stratum and surrounded the inner core of chitin synthase‐containing microvesicles. TIRF microscopy revealed that GS‐1‐GFP reached the hyphal apex as a population of heterogeneous‐size particles that moved along defined paths. On sucrose density gradients, GS‐1‐associated particles mainly sedimented in a high density range 1.1272–1.2124 g ml^?1. Clearly, GS‐1 and chitin synthases of N. crassa are contained in two different types of secretory vesicles that accumulate in different strata of the Spk, a differentiation presumably related to the spatial control of cell‐wall synthesis.     

Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique, crh1Δ crh2Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Δ crh2Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.The fungal cell wall protects the cell against internal turgor pressure and external mechanical injury. To fulfill these functions, it must be endowed with a resilient structure. Presumably, the cell wall strength is largely due to the cross-links that bind together its components, mainly polysaccharides, giving rise to a tightly knit mesh (6, 11-13). Interestingly, the cross-links must be created outside the plasma membrane, because most of the polysaccharides are extruded as they are synthesized at the membrane; therefore, they do not exist inside the cell. This posits a thermodynamic problem, because there are no obvious sources of energy in the periplasmic space. About 20 years ago we proposed that the free energy may come from existing bonds in the polysaccharide chains and that the new cross-links may be originated by transglycosylation, thus creating a new linkage for each one that is broken (5).Ascertaining the mechanism of cross-link formation seemed a worthwhile endeavor, both because of the theoretical implications and because the cell wall is a proven target for antifungal compounds; therefore, more knowledge about its synthesis can be of practical interest. For this type of investigation to proceed, it was necessary to devise some method for the quantitative analysis of cell wall cross-links. We developed such a procedure for the evaluation of the proportion of cell wall chitin that is free or bound to β(1-3)- or β(1-6)glucan (4). In this methodology, chitin was specifically labeled in vivo with [¹⁴C]glucosamine; cell walls were isolated, and their proteins were eliminated by alkali treatment. The insoluble residue was solubilized by carboxymethylation and analyzed by size fractionation chromatography. By treating the cell walls with different glucanases before carboxymethylation and comparing the chromatographic profiles, we were able to determine the amount of chitin bound to the different glucans, as well as the fraction that was free (4). Armed with this procedure, we could now analyze the cell wall of different mutants that appeared to be candidates for cross-links defects. In this way we found that the two putative transglycosylases Crh1p and Crh2p were redundantly required for the formation of the chitin-β(1-6)glucan linkage. A double mutant crh1Δ crh2Δ had no chitin attached to β(1-6)glucan, although it still contained apparently normal amounts of chitin-β(1-3)glucan complex (7). Further work supported the notion that Crh1p and Crh2p function as transglycosylases, transferring portions of chitin chains to glucan (8). This confirmed our earlier hypothesis.With the initial intention of finding easier and faster methods, I devised two novel procedures for cell wall analysis. One is based on the affinity between β(1-3)glucan chains, the other on the conversion of chitin in situ into its deacetylated product, chitosan, followed by extraction of the chitosan with acetic acid before or after treatment with specific glucanases. With a wild-type strain, both procedures gave similar results to those of the carboxymethylation-chromatography technique. However, in the double mutant crh1Δ crh2Δ all of the chitin appeared to be free with both new methods. Further investigation showed that the older procedure led to erroneous results for the double mutant, because of the presence of a small amount of chitinase in the β(1-3)glucanase preparation used. After reconciling the results, I conclude that Crh1p and Crh2p are necessary for the formation of cross-links between chitin and either β(1-6) or β(1-3)glucan.     

Cell wall assembly ofNeurospora crassa: Isolation and analysis of cell wall-less mutants

Cell-wall-less mutants ofNeurospora crassa were isolated by mutagenizing cells of the temperature-sensitive, protoplast-formingosmotic-1 mutant and screening for cultures that did not regenerate cell wall. Of the 24 strains isolated, 22 were found to have significantly reduced levels of (1–3)-glucan synthase activity; none was found to be defective in chitin synthase activity. Genetic analysis by standard transmission genetics revealed that the glucan synthase defect was ascospore lethal. Complementation analysis by forced heterokaryosis revealed four complementation groups. In each case in which complementation of cell wall formation was observed, there was concomitant restoration of (1–3)-glucan synthase activity. Our results indicate that glucan synthase activity is necessary, but not sufficient, for cell wall assembly and resulting morphogenesis.     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a β-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology. Received: 1 April 1996 / Accepted: 2 September 1996     

On the cell wall composition of the apiculate yeasts

Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae. The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine. The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls. It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.     

Vascular bundle sheath and mesophyll cells modulate leaf water balance in response to chitin

Plants can detect pathogen invasion by sensing microbe‐associated molecular patterns (MAMPs). This sensing process leads to the induction of defense responses. Numerous MAMP mechanisms of action have been described in and outside the guard cells. Here, we describe the effects of chitin, a MAMP found in fungal cell walls and insects, on the cellular osmotic water permeability (P_f) of the leaf vascular bundle‐sheath (BS) and mesophyll cells (MCs), and its subsequent effect on leaf hydraulic conductance (K_leaf). BS is a parenchymatic tissue that tightly encases the vascular system. BS cells (BSCs) have been shown to influence K_leaf through changes in their P_f, for example, after sensing the abiotic stress response‐regulating hormone abscisic acid. It was recently reported that, in Arabidopsis, the chitin receptors‐like kinases, chitin elicitor receptor kinase 1 (CERK1) and LYSINE MOTIF RECEPTOR KINASE 5 (LYK5) are highly expressed in the BS as well as the neighboring mesophyll. Therefore, we studied the possible impact of chitin on these cells. Our results revealed that BSCs and MCs exhibit a sharp decrease in P_f in response to chitin treatment. In addition, xylem‐fed chitin decreased K_leaf and led to stomatal closure. However, Atlyk5 mutant showed none of these responses. Complementing AtLYK5 in the BSCs (using the SCARECROW promoter) resulted in the response to chitin that was similar to that observed in the wild‐type. These results suggest that BS play a role in the perception of apoplastic chitin and in initiating chitin‐triggered immunity.     

Alterations in the cell wall ofSaccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases inSaccharomyces cerevisiae after arrest of acdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it representedde novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and thecdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.Abbreviations GlcNAc N-acetyl glucosamine - UDPGIcNAc uridine-diphosphate-N-acetyl glucosamine - UDPGlc uridine-diphosphate glucose - TCA trichloroacetic acid - EDTA ethylene diamino tetraacetate - TAME tosyl-L-arginyl methyl ester - GTP guanosine triphosphate - WGA wheat germ agglutinin     

Chitin and β(1–3) glucan synthases in the protoplastic entomophthorales

(1–3) glucan and chitin synthases were studied in spontaneously produced protoplasts and in the mycelium (hyphal body) of the entomopathogenic Entomophthorale species Entomophaga aulicae, Conidiobolus obscurus and Entomophthora muscae. The absence of wall in protoplasts was correlated to an absence of chitin synthase and to a very low (1–3) glucan synthase activity, whereas these two polysaccharide synthases were present and active in the walled hyphal bodies. Physicochemical properties of chitin and (1–3) glucan synthases such as localization, optimum pH and temperature, activation by disaccharides and proteases were similar to those found in other fungi unable to spontaneously produce protoplasts and could not be related to the ability for protoplastic Entomophthorale species to produce and proliferate under a protoplast form. The absence or the low chitin and glucan synthase activites in Entomophthorale protoplasts was not due to an absence of proteolytic activation of the enzyme. However, all protoplast fractions contained inhibitory substances of glucan and chitin synthase activities. These inhibitors were stable and specific of the protoplast stage. They were not glucanase nor chitinase. These results suggest that the absence of wall synthesis in Entomophthorale protoplasts is due to a continuous inhibition of (1–3) glucan and chitin synthase activities by intracellular compounds and also for glucan synthase by protoplast medium constituents such as NaCl and fetal calf serum.Abbreviations BSA bovine serum albumin - DFP diisopropylfluorophosphate - EDTA ethylenediamine tetraaoetic acid - FCS fetal calf serum - GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - 2 k pellet 2,000 g wall fraction - 140 k pellet 140,000 g particulate fraction - 140 k supernatant 140,000 g soluble fraction     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Chitinolytic and antifungal activity of a <Emphasis Type="Italic">Bacillus pumilus</Emphasis> chitinase expressed in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to beta(1-3)glucan or beta(1-6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [14C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan.