Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

Stability and zymogenic nature of chitin synthase fromCandida albicans

Chitin synthase preparations from both yeast and mycelialCandida albicans were chiefly in a zymogenic form, activatable by trypsin treatment. This was especially marked for preparations that had been solubilized with digitonin treatment. Endogenous activation of chitin synthase zymogen was observed over many days in preparations stored in glycerol (33%, wt/v) at?12°C and over many hours in preparations stored at 30°C. Gel chromatography of enzyme preparations suggested that zymogen was preferentially retarded on the columm matrices in comparison with active enzyme.     

Chitin synthase in Candida albicans: comparison of digitonin-permeabilized cells and spheroplast membranes.

The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid); partially prevented by the protease inhibitors antipain, leupeptin, and N alpha-tosyl-L-lysyl chloromethyl ketone; and also partially prevented by EDTA. Thus, a clostripain-like protease may be involved in the endogenous activation phenomenon. The pH activity profile, cofactor requirements, and kinetic parameters of the endogenously activated chitin synthase were identical to those of the trypsin-activated enzyme in protoplast membranes.     

昆虫几丁质合成酶及其抑制剂

几丁质合成酶(CS)是几丁质合成的关键酶,它具有3个结构域:结构域A、结构域B和结构域C,其中结构域B是催化域。根据氨基酸序列的差异,几丁质合成酶分为两类:CS-A及CS-B,分别在表皮及围食膜基质中催化合成几丁质。关于几丁质合成有2种假想模型。有多种抑制剂可以抑制几丁质的合成,其中核苷肽抗生素类及核苷磷酸类作用于CS的催化部位,是竞争性抑制剂,其它抑制剂的作用机理仍不明确。     

A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.     

Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus Ustilago maydis

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

Umchs5,a Gene Coding for a Class IV Chitin Synthase inUstilago maydis

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungusUstilago maydiswas amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product ofUmchs5has highest similarity with class IV chitin synthases encoded by theCHS3genes fromSaccharomyces cerevisiaeandCandida albicans, chs-4fromNeurospora crassa,andchsEfromAspergillus nidulans. Umchs5null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase,Umchs6.It is suggested that multigenic control of chitin synthesis inU. maydisoperates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.     

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     

定虫隆对锈赤扁谷盗生长发育影响及其作用机理初探

定虫隆是一种昆虫生长调节抑制剂,研究其对锈赤扁谷盗的控制作用,将为锈赤扁谷盗的综合防治提供理论基础。本研究以锈赤扁谷盗幼虫为研究对象,用定虫隆拌粮进行饲喂,观察定虫隆对锈赤扁谷盗的亚致死效应,并利用实时荧光定量PCR(qPCR)技术,测定经定虫隆处理后锈赤扁谷盗幼虫几丁质合成酶1(CfCHS1)和几丁质合成酶2(CfCHS2)基因表达量的变化。结果表明:定虫隆对锈赤扁谷盗的毒力回归方程为y=7.514+1.81x(r=0.974),LD 10、LD 20、LD 30分别为0.008 mg/kg、0.014 mg/kg、0.021 mg/kg,亚致死剂量定虫隆可使锈赤扁谷盗幼虫发育历期明显延长(P<0.05),体重和几丁质含量明显降低(P<0.05),这说明了定虫隆对锈赤扁谷盗的生长发育有明显的抑制作用,可以有效控制锈赤扁谷盗的为害。qPCR结果显示,与对照组相比,LD 30处理后的锈赤扁谷盗幼虫的CfCHS 1基因表达量提高了74.7%,CfCHS 2基因的表达量提高了27.4%,均差异显著(P<0.05)。CfCHS 1和CfCHS2基因表达量的上调,可能是几丁质合成酶受到了抑制,锈赤扁谷盗为了维持机体的稳定,提高几丁质合成酶基因的表达量,从而形成一种代偿性增加的反馈机制,由此推测定虫隆可能作用于几丁质合成酶。     

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%.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

甜菜夜蛾cDNA文库的构建

以甜菜夜蛾Spodoptera exigua(Hübner)幼虫为材料建立了cDNA表达文库,经检测文库的初始滴度为1.1×105pfu/mL,重组率为97%,扩增后文库的滴度为5.4×107pfu/mL。用设计的2对引物筛选该文库,得到468 bp的1个片段,分析后证实是几丁质合成酶基因I保守区域的1个片段。该cDNA文库为进一步筛选甜菜夜蛾功能基因奠定了基础。     

DIETARY LUFENURON REDUCES EGG HATCH AND INFLUENCES PROTEIN EXPRESSION IN THE FRUIT FLY Bactrocera latifrons (HENDEL)

Lufenuron (LFN), a chitin synthase inhibitor, impacts the fertility of Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons. We posed the hypothesis that LFN curtails egg hatch in the solanaceous fruit fly, B. latifrons. In this study, newly emerged virgin adults were sexed and fed for 12 days with varying concentrations of LFN‐laced agar diets until sexual maturation. Eggs were collected from 12‐d‐old adults and the egg hatch was assessed. Egg hatch decreased in adults reared on LFN‐treated diets. LFN‐treated media did not influence fertility after one gender was reared on experimental and the other on control media before mating. Exposure to LFN‐treated medium after mating led to reduced egg hatch. We infer that LFN is not a permanent sterilant, and reduced egg hatch depends on continuous exposure to dietary LFN after mating. Proteomic analysis identified two differentially expressed proteins, a pheromone binding protein and a chitin binding protein, between adults maintained on LFN‐treated and control diets. Expression of two genes encoding chitin synthase 2, and chitin binding protein, was altered in adults exposed to dietary LFN. LFN treatments also led to increased expression of two odorant binding proteins one in females and one in males. We surmise these data support our hypothesis and provide insight into LFN actions.     

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。     

Evidence for a phospholipid requirement of chitin synthase inSchizophyllum commune

Evidence for a lipid dependence of membrane-associated chitin synthase inSchizophyllum commune is based on the following observations: Arrhenius plots of the temperature dependence of this enzyme showed deflections from linearity that are characteristic for lipid-affected membrane-bound enzymes. The activity of chitin synthase dissociated by digitonin decreased at increasing digitonin/protein ratios and could be restored by addition of egg lecithin. After further delipification by sucrose gradient centrifugation, enzyme activity progressively decreased, banded at higher densities, and was less effectively restored by lecithin. The activity of dissociated chitin synthase was also restored by soybean phosphatidylcholine and low concentrations of phosphatidylinositol and phosphatidylserine. At higher concentrations, phosphatidylinositol and phosphatidylserine were inhibitory. Lysophosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas no effect resulting from ergosterol was observed.