枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l^?1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l^?1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性。将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤, 分离得到脂肽类抗生素的纯品。经IC50实验和抗菌谱测定, 考察了脂肽类抗生素对多种植物病原菌的作用, 确定了脂肽类抗生素的抗菌谱。深入研究表明, 枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质, 能够抑制灰霉病菌孢子的萌发和菌丝的生长。脂肽类抗生素和挥发性抑菌物质的协同作用, 有助于提高枯草芽孢杆菌的生物防治效果。     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Effect of the microbial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C₁₃-lipopeptide<C₁₄-lipopepitde<C₁₅-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC₅₀ values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

In Silico Discovery of Novel Ligands for Antimicrobial Lipopeptides for Computer-Aided Drug Design

The increase in antibiotic-resistant strains of pathogens has created havoc worldwide. These antibiotic-resistant pathogens require potent drugs for their inhibition. Lipopeptides, which are produced as secondary metabolites by many microorganisms, have the ability to act as potent safe drugs. Lipopeptides are amphiphilic molecules containing a lipid chain bound to the peptide. They exhibit broad-spectrum activities against both bacteria and fungi. Other than their antimicrobial properties, they have displayed anti-cancer properties as well, but their mechanism of action is not understood. In silico drug design uses computer simulation to discover and develop new drugs. This technique reduces the need of expensive and tedious lab work and clinical trials, but this method becomes a challenge due to complex structures of lipopeptides. Specific agonists (ligands) must be identified to initiate a physiological response when combined with a receptor (lipopeptide). In silico drug design and homology modeling talks about the interaction between ligands and the binding sites. This review summarizes the mechanism of selected lipopeptides, their respective ligands, and in silico drug design.     

渤海湾仿刺参养殖圈内贝莱斯芽胞杆菌B-VE的分离鉴定及其产抗菌脂肽的研究

为分离筛选具有广谱抑菌作用的细菌并研究其抗菌物质,实验利用琼脂扩散法和牛津杯法,以溶壁微球菌、金黄色葡萄球菌、铜绿假单胞杆菌、大肠埃希菌为指示菌,从渤海湾大连海域仿刺参养殖圈中分离筛选出9株活性菌,2株具有广谱抑菌效果,其中1株抑菌效果最强.通过形态学和分子生物学检测分析,鉴定该菌株为贝莱斯芽胞杆菌(Bacillus ...     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

Vesicle Leakage Reflects the Target Selectivity of Antimicrobial Lipopeptides from Bacillus subtilis

Cyclic lipopeptides act against a variety of plant pathogens and are thus highly efficient crop-protection agents. Some pesticides contain Bacillus subtilis strains that produce lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). The antimicrobial activity of these peptides is mainly mediated by permeabilizing cellular membranes. We used a fluorescence-lifetime based leakage assay to examine the effect of individual lipid components in model membranes on lipopeptide activity. Leakage induction by FE was strongly inhibited by cholesterol (CHOL) as well as by phosphatidylethanolamine (PE) and -glycerol (PG) lipids. Already moderate amounts of CHOL increased the tolerable FE content in membranes by an order of magnitude to 0.5 FE per PC + CHOL. This indicates reduced FE-lipid demixing and aggregation, which is known to be required for membrane permeabilization and explains the strong inhibition by CHOL. Ergosterol (ERG) had a weak antagonistic effect. This confirms results of microbiological tests and agrees with the fungicidal activity and selectivity of FE. SF is known to be much less selective in its antimicrobial action. In line with this, liposome leakage by SF was little affected by sterols and PE. Interestingly, PG increased SF activity and changed its leakage mechanism toward all-or-none, suggesting more specific, larger, and/or longer-lived defect structures. This may be because of the reduced energetic cost of locally accumulating anionic SF in an anionic lipid matrix. IT was found largely inactive in our assays. B. subtilis QST713 produces the lipopeptides in a ratio of 6 mol SF: 37 mol FE: 57 mol IT. Leakage induced by this native mixture was inhibited by CHOL and PE, but unaffected by ERG and by PG in the absence of PE. Note that fungi contain anionic lipids, but little PE. Hence, our data explain the strong, fungicidal activity and selectivity of B. subtilis QST713 lipopeptides.     

元江仙人掌植物内生真菌的抑菌活性筛选研究

从云南元江野生仙人掌的肉质茎中分离出28株内生真菌,经形态学分类鉴定归于3个目,3个科,10个属。同时选择30种病原微生物作为指示菌进行抑菌试验,研究其抑菌活性。结果表明,3株元江仙人掌内生真菌分别对细菌、植物病原真菌和皮肤致病真菌多种病原微生物有较为明显的抑制生长作用。     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin

A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid- modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S- acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC- and -CXC(farnesyl)- OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC- motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S- acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl- motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC- occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment.     

Mycoviruses of filamentous fungi and their relevance to plant pathology

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal–virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.     

Are the short cationic lipopeptides bacterial membrane disruptors? Structure-Activity Relationship and molecular dynamic evaluation

Short cationic lipopeptides are amphiphilic molecules that exhibit antimicrobial activity mainly against Gram-positives. These compounds bind to bacterial membranes and disrupt their integrity. Here we examine the structure-activity relation (SAR) of lysine-based lipopeptides, with a prospect to rationally design more active compounds. The presented study aims to explain how antimicrobial activity of lipopeptides is affected by the charge of lipopeptide headgroup and the length of lipopeptide acyl chain. The obtained SAR models suggest that the lipophilicity of short synthetic cationic lipopeptides is the major factor that determines their antimicrobial activities. In order to link the differences in antimicrobial activity to the mechanism of action of lipopeptides containing one and two hydrophobic chains, we additionally performed molecular dynamic (MD) simulations. By using combined coarse-grained and all-atom simulations we also show that these compounds neither affect the organization of the membrane lipids nor aggregate to form separate phases. These results, along with the onset of antimicrobial activity of lipopeptides well below the critical micelle concentration (CMC), indicate that lipopeptides do not act in a simple detergent-like manner.     

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.     

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA₃ and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA₃ and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA₃ and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA₃ to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA₃ produces rapid changes in fungal membrane permeability. It also induces H₂O₂ production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.     

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.