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     

Survival and differential development of Entomophaga maimaiga and Entomophaga aulicae (Zygomycetes: Entomophthorales) in Lymantria dispar hemolymph

The closely related entomophthoralean fungi Entomophaga aulicae and E. maimaiga are both host-specific pathogens of lepidopteran larvae. However, these fungi do not have the same host range. The first objective of this study was to compare the fate of E. aulicae in the nonpermissive host Lymantria dispar with the fate of the successful pathogen E. maimaiga over the same time period. In the hemolymph of L. dispar injected with E. maimaiga protoplasts, the number of hemocytes demonstrated a decreasing trend after the first day postinjection and hemocytes completely disappeared by day 5, with the majority of larvae dying in 5.6 +/- 0.1 days. In L. dispar larvae, E. maimaiga infections developed successfully, evidenced by increasing numbers of protoplasts and hyphal bodies prior to host mortality. In contrast, at day 5 hemocytes were readily visible in hemolymph of E. aulicae-injected larvae, but E. aulicae cells did not increase in numbers, although persisting in the hemolymph for at least 16 days postinjection. For both fungal species, when hemolymph samples from injected insects were introduced to culture media viable fungal cultures were always produced. Both E. aulicae and E. maimaiga occurred in hemolymph initially after injection as protoplasts. For E. maimaiga, after day 3, <50% of fungal cells were hyphal bodies until insect death when most cells regenerated cell walls. For E. aulicae, from day 2 equal numbers of fungal cells in the hemolymph occurred as protoplasts and hyphal bodies. To investigate the cause of fungistasis in E. aulicae-injected larvae, E. aulicae cell cultures exposed to partially purified protein fractions from hemolymph of larvae infected with either fungus displayed increased lysis and decreased viability at lower concentrations of protein fractions compared with E. maimaiga cell cultures. These studies demonstrate that E. aulicae does not increase in L. dispar hemolymph, although it persists and results suggest that proteinaceous factors induced within the hemolymph may limit the capacity of E. aulicae to develop successful infections.     

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening

Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC₅₀ values of 1.8 and 10 μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100 μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.     

Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm

Chitin formation depends on the activity of a family II glycosyltransferase known as chitin synthase, whose biochemical and structural properties are largely unknown. Previously, we have demonstrated that the chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS-2), one of two isoenzymes encoded by the Chs-1 and Chs-2 genes (also named Chs-A and Chs-B), and that CHS-2 is located at the apical tips of the brush border microvilli. Here we report the purification of the chitin synthase from the Manduca midgut as monitored by its activity and immuno-reactivity with antibodies to the chitin synthase. After gel permeation chromatography, the final step of the developed purification protocol, the active enzyme eluted in a fraction corresponding to a molecular mass between 440 and 670 kDa. Native PAGE revealed a single, immuno-reactive band of about 520 kDa, thrice the molecular mass of the chitin synthase monomer. SDS-PAGE and immunoblotting indicated finally that an active, oligomeric complex of the chitin synthase was purified. In summary, the chitin synthase from the midgut of Manduca may prove to be a good model for investigating the enzymes' mode of action.     

Swm1p, a subunit of the APC/cyclosome, is required to maintain cell wall integrity during growth at high temperature in Saccharomyces cerevisiae

Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant.     

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.     

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana. The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli. The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus, whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa, whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N-acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.     

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune.

In the presence of MgSo4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (alpha-1,3-glucan), R-glucan (beta-1,3, beta-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion of hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 mug/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

Phospholipid requirements of membrane-bound chitin synthase from Absidia glauca

Abstract Membrane-bound chitin synthase, a key enzyme in chitin biosynthesis, had a specific requirement for phospholipid. The activity of the enzyme was enhanced 2.7-fold by adding phosphatidylinositol from porcine liver but not by other phospholipids. Each of the constituents of phospholipids inhibited enzyme activity at concentrations over 0.05%. Sterols and glycolipids had little effect on chitin synthase activation. Moreover, investigation using define species of phosphatidylcholine revealed that 1-palmytoyl-2-arachidoyl and 1-stearoyl-2-arachidoyl phosphatidylcholine activated the enzyme. In contrast to the arachidoyl acyl chain, other species having unsaturated fatty acyl chains inhibited enzyme activity at a concentration of 0.01%.     

Alpha-proteobacteria cultivated from marine sponges display branching rod morphology

PCR amplification of two CHS gene fragments of the obligate biotroph Plasmopara viticola, the causal agent of downy mildew of grapevine, is described. While one fragment shows homology to fungal class IV chitin synthases, the other fragment groups with other oomycete chitin synthases to form a novel class of chitin synthases most closely related to class I-III. RT-PCR experiments indicate that PvCHS1 is constitutively expressed, whereas PvCHS2 is specifically transcribed in sporangiophores and sporangia. Analyses of wheat germ agglutinin labeling patterns by confocal laser scanning microscopy show that chitin is present on the surface of hyphal cell walls during in planta growth, and of sporangiophores and sporangia.     

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus

Abstract A chitin synthase-like gene ( chsD ) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other N acetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD⁻ mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD⁻ strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus .     

Chitin synthase homologs in three ectomycorrhizal truffles

Abstract Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii . A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber , and suggested a close relationship between T. magnatum and T. uncinatum .     

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.     

Construction of an RNAi expression vector and transformation into Penicillium chrysogenum

In this work, we constructed an RNAi vector for attenuation of the class III chitin synthase gene chs4, which plays a major role in hyphal growth and conidia formation. To achieve a high transformation frequency, factors affecting the preparation and regeneration of protoplasts were analyzed. The maximum numbers of protoplasts (1.41?×?10⁷ mL^?1) were released when mycelia cultured for 48 h were incubated at 30 °C for 5 h in a buffer containing 4 mg mL^?1 lysing enzyme. The maximum regeneration rate (33 %) was obtained when mycelia were digested for 4 h and plated on a regeneration medium containing 1 % overlaid agar. Quantitative real-time PCR was performed to validate the transformation efficiency, and it revealed knockdown of chs4 gene in randomly selected transformants at different levels. Dramatic reductions in the formation of conidia and the hyphal growth rate were observed in most of the transformants.     

Insect chitin synthases: a review

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP–N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes‘ catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.