Studies on the Mode of Action of Organophosphorus Fungicide,Kitazin

Studies were conducted on the influence of Kitazin analogues on the incorporation of ¹⁴C-glucosamine into the mycelial cell wall fraction of Pyricularia oryzae. Compounds of thiolates and phosphates, both having in vitro inhibitory activities toward the mycelial growth, inhibited the incorporation, whereas those of thionates and dithioates, either having no fungitoxicity, did not inhibit the incorporation. Mycelia of P. oryzae treated with Kitazin-P (S-benzyl O,O′-diisopropyl phosphrothioate; IBP) accumulated about twice as much an amino sugar derivative as untreated ones. Mycelia treated with thiono or dithio analogues, which have no fungitoxicity, showed no accumulation. The accumulated substance gave a identical spot with authentic UDP-N-acetyl glucosamine on paper chromatograms developed with four solvent systems.     

Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.     

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.     

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.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures

The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.Abbreviations FPP farnesyl diphosphate - HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase     

Chitin synthesis inhibitors prevent cyst formation by Entamoeba trophozoites

Cysts of Entamoeba invadens obtained under axenic culture conditions have been reported to be similar to cysts of the human intestinal parasite E histolytica both in morphology and chitin presence in their wails. Mature E. invadens cyst forms, isolated from cultures following discontinuous Percoll gradient sedimentation were resistant (>80%) to detergent treatment. Addition of chitin synthesis inhibitors such as Polyoxin D and Nikkomycin (50 μg/ml) to cultures in encystation media markedly inhibited (>85%) the formation of detergent resistant cysts and prevented the incorporation of radiolabeled chitin precursor N-acetyl[³H]glucosamine. These findings suggest that chitin synthesis inhibitors may serve as drugs which specifically block the life cycle of the Entamoeba parasite.     

Studies on the Biosynthesis of Kanamycins

Streptomyces kanamyceticus produces ¹⁴C-kanamycin and other labeled kanamycin-related compounds in the presence of ¹⁴C-glucose or ¹⁴C-glucosamine. One of the latter is paromamine. In order to find a condition to obtain high incorporation rate of radioactive precursors, the culture phase for the addition of labeled compounds was studied. The maximal incorporation rate was obtained by addition of ¹⁴C-glucosamine in the later phase of the culture and the specific activity of resultant kanamycin A was found to be much higher than in the case of the addition of ¹⁴C-glucose. In some experiments one third of radioactivity of the added ¹⁴C-glucosamine was taken into kanamycin, and the distribution was limited only in 6-amino-6-deoxy-D-glucose moiety of kanamycin A when ¹⁴C-glucosamine was added. In contrast, when ^1JC-glucose was added, approximately equal distribution of radioactivity was observed in each of three moieties of kanamycin A molecule.     

多氧霉素及其生物合成的研究进展

多氧霉素(Polyoxins)是高效广谱抗真菌核苷类抗生素,在农业上广泛用于防治植物真菌病害。本文综述了多氧霉素化学结构和理化性质,尤其是武汉大学组合生物合成与新药发现(教育部)重点实验室近年来在该抗生素生物合成基因簇的克隆、生物合成途径的阐明以及多氧霉素组合生物合成等多个方面的研究进展与成果,并对今后以多氧霉素为代表的核苷类抗生素的生物合成研究进行了展望。     

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。     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Existence of Hyphal Extension Factor and Hyphal Extension Inhibitor in the Hyphae of Rhizoctonia solani

Brefeldin A (BFA) is an antibiotic having diverse biological effects such as antifungal, antiviral and antitumor activities. The effect of BFA on biosynthesis of cellular components was examined to elucidate the mode of action of BFA using C. albicans IAM 4888.

When C. albicans was grown in the presence of BFA, cells became rounded and enlarged several times larger than the untreated control cells. Cell walls of the treated cells became irregular and a number of Sudan III-stainable lipid droplets was formed in the cytoplasm. Accompanying these morphological changes, a marked alteration occurred in the cellular lipid composition; neutral lipids increased whereas phospholipid decreased. [¹⁴C]Acetate incorporation into the lipid fraction proceeded in accordance with the growth in the presence of BFA. On the other hand, [³²P]orthophosphate incorporation into phospholipid was severely inhibited. Incorporation of radiolabeled precursors into DNA, RNA and protein was not affected on a cell weight basis.     

The effect of rifampicin onMycobacterium smegmatis

Rifampicin was found to inhibit the growth and incorporation of¹⁴C-adenine,¹⁴C-leucine and¹⁴C-glycine in exponentially growing cells ofM. smegmatis cultivated in Merrill’s synthetic medium. Increasing concentrations of the antibiotic inhibited respiration in resting cells, in the presence of glucose or 2-oxoglutarate as substrates in particular. In addition to the well-known interference of rifampicin with the biosynthesis of RNA, the effect on the energy metabolism should also be considered.     

Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea

The ascomycete Botrytis cinerea is a destructive and ubiquitous plant pathogen and represents a model organism for the study of necrotrophic fungal pathogens. Higher fungi possess a complex and dynamic multilayer cell wall involved in crucial aspects of fungal development, growth and pathogenicity. Plant resistance to microbial pathogens is determined often by the capacity of the plant to recognize molecular patterns associated with the surface of an interacting microbe. Here we report the chemical characterization of cell walls from B. cinerea during axenic growth. Neutral sugars and proteins constituted most of the mass of the B. cinerea cell walls, although chitin and uronic acids were detected. Glucose was the most abundant neutral sugar, but arabinose, galactose, xylose and mannose also were present. Changes in cell wall composition during culture were observed. As the culture developed, protein levels declined, while chitin and neutral sugars increased. Growth of B. cinerea was associated with a remarkable decline in the fraction of its cell wall material that was soluble in hot alkali. These results suggest that the cell wall of B. cinerea undergoes significant modifications during growth, possibly becoming more extensively covalently cross-linked, as a result of aging of mycelia or in response to decreasing nutrient supply or as a consequence of increasing culture density.     

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.     

Surface ��-1,3-Glucan Facilitates Fungal Stealth Infection by Interfering with Innate Immunity in Plants

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant''s defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants'' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.     

Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells

Soybean cells in suspension culture incorporate [³H]mannose into dolichyl-phosphoryl-mannose and into lipid-linked oligosaccharides as well as into extracellular and cell wall macromolecules. Tunicamycin completely inhibited the formation of lipid-linked oligosaccharides at a concentration of 5 to 10 micrograms per milliliter, but it had no effect on the formation of dolichyl-phosphoryl-mannose. Tunicamycin did inhibit the incorporation of [³H]mannose into cell wall components and extracellular macromolecules, but even at 20 micrograms per milliliter of antibiotic there was still about 30% incorporation of mannose. The radioactivity in these macromolecules was localized in mannose (70%), rhamnose (20%), galactose (8%), and fucose (2%) in the absence of antibiotic. But when tunicamycin was added, very little radioactive mannose was found in cell wall or extracellular components. The incorporation of [³H]leucine into membrane components and [¹⁴C]proline into cell wall components by these suspension cultures was unaffected by tunicamycin. However, tunicamycin did inhibit the appearance of leucine-labeled extracellular macromolecules, probably because it prevented their secretion.     

Effect of the Urtica dioica agglutinin on germination and cell wall formation of Phycomyces blakesleeanus Burgeff

The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [¹⁴C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.Abbreviations GlcNAc N-Acetylglucosamine - PDB potato dextrose broth - PMM Phycomyces minimal medium - UDA Urtica dioica agglutinin - TEA tri-ethyl-amine - DAB 1,4-diaminobutanone