致病性曲霉的耐药性研究进展

随着抗真菌药物在临床上的广泛使用,致病性真菌的耐药率越来越高,耐药曲霉对侵袭性曲霉病的诊治产生了重要影响。目前,致病性曲霉耐药性的确定主要依靠抗真菌药敏试验和分子诊断。在有关曲霉耐药机制的研究中,报道最多的是曲霉对唑类药物的耐药,其机制主要包括外排泵表达增加、靶酶Cyp51突变和表达水平增高、形成生物膜,以及热休克蛋白90（Hsp90）介导的信号通路参与而导致的耐药。本文就上述领域近年来的主要进展进行综述。     

条件致病真菌烟曲霉麦角甾醇合成通路遗传调控机制研究进展

环境中普遍存在的腐生性条件致病真菌——烟曲霉是引起人类侵袭性曲霉病的重要病原,因此,研究烟曲霉的致病机理,开发有效的治疗药物是全球关注的热点。麦角甾醇是真菌细胞膜的主要成分,参与细胞内许多生物学过程,麦角甾醇合成通路中的羊毛甾醇14-α-去甲基化酶Erg11A (Cyp51A同源蛋白)是抗曲霉病唑类药物的重要靶点,其受到转录因子Srb A与CCAAT结合复合物(CBC)的协同调控作用。文中阐述了主要的抗真菌药物以及抗真菌唑类药物的作用靶点-麦角甾醇及其合成途径的遗传调控机制的研究进展,同时分析了烟曲霉产生抗性的机制,期望为认识烟曲霉耐药产生和研发新型抗真菌药物提供帮助。     

米卡芬净对分离自中国的念珠菌和曲霉临床株体外抑菌活性的研究

目的了解新型抗真菌药物米卡芬净(micafungin,MFG)对分离自中国的念珠菌和曲霉临床株的体外抑菌活性。方法参照CLSI(Clinical and Laboratory Standards Institute,以前为NCCLS)制定的M27-A2和M38-A方案测定86株念珠菌和35株曲霉的最低抑菌浓度(MIC)或最低有效浓度(MEC)。结果MFG对大多数念珠菌属和曲霉属均有较好的抑菌作用。对念珠菌属的MIC90从高到低依次为:氟康唑(FLC)敏感的白念珠菌、热带念珠菌、光滑念珠菌为0.125μg/ml,FLC耐药和剂量依赖敏感株为0.25μg/ml,克柔念珠菌为0.5μg/ml,近平滑念珠菌8μg/ml,季也蒙念珠菌>16μg/ml。MFG对烟曲霉的MEC90为≤0.03μg/ml,对非烟曲霉的曲霉属MEC90为0.06μg/ml。MFG与唑类药物、两性霉素B(AMB)不存在交叉耐药,对FLC耐药的念珠菌、伊曲康唑耐药的曲霉、AMB不敏感的曲霉均有好的抑菌活性。结论MFG对多数念珠菌属和曲霉属(包括对唑类耐药和AMB不敏感的菌株)有较好的体外抑菌作用。     

外排转运蛋白介导的抗真菌药物耐药研究进展

近年来,随着广谱抗生素,免疫抑制剂,抗肿瘤化疗药物的广泛应用,器官移植的普遍开展以及AIDS患者的逐年增加,各系统侵袭性真菌感染日益增多。抗真菌药物的大量应用使得真菌耐药现象日渐严重。大量研究表明,耐药真菌细胞膜上外排转运蛋白的过量表达对抗真菌药物耐药形成起到重要作用。ATP结合盒式蛋白(ABC转运体)和易化扩散载体超家族蛋白(MFS转运体)便是其中最重要的两种。本文从ABC及MFS转运体的结构和功能出发,分析其在抗真菌药物耐药形成中的作用,并对相关研究进展进行综述。     

念珠菌耐药机制的研究进展

近年来,真菌感染尤其是机会性真菌感染的发生率和病死率呈不断上升的趋势,这主要与真菌耐药和抗真菌药物品种有限有关。念珠菌是人体内的重要条件致病真菌,其常见的耐药机制包括靶酶的改变、靶位的缺乏、真菌细胞膜对药物的通透性下降、多药耐药蛋白表达增高、固醇合成旁路下游的改变、生物膜的形成等,最新文献报道分泌型天冬氨酸蛋白酶在念珠菌的耐药形成中存在一定作用。该文就念珠菌感染的流行病学和念珠菌的耐药机制两个方面进行综述。     

抗真菌药物及其耐药机制研究进展

侵袭性真菌感染,由于其高发病率和死亡率,对人类健康造成严重威胁。针对侵袭性真菌自身生长繁殖的生物学过程,人们开发了不同的抗真菌药物对其进行阻断、干扰,从而达到杀菌抑菌的目的。我们简要介绍临床常见抗真菌药物的种类、作用机制、耐药机制以及应对耐药的方法。     

抗真菌抗生素179M产生菌的分离鉴定和生理特性研究

从大亚湾海底沉积物中分离出一株具广谱抗真菌作用的曲霉（编号为179）,经初步鉴定为黄柄曲霉(Aspergillus flavipes),该曲霉最适生长温度为36℃,最适生长pH为6。其抗真菌代谢产物179M对酵母类真菌的最小抑菌浓度为0.78-12.5μg/mL,对皮肤感染真菌石膏样小孢子菌(Microsporum gypseum)的最小抑菌浓度为1.56μg/mL,179M还能抑制多种植物病原真菌的生长。     

解淀粉芽胞杆菌抗真菌活性研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有很强的抑制植物病原真菌的能力。其菌体细胞能产生多种酶类、脂肽类抗生素、生物表面活性素、聚酮类化合物和抑菌蛋白,同时具有诱导植物产生系统抗性(ISR)的能力,因此在工农业、种植业、养殖业、食品加工业、果蔬的采后保鲜和饲料业等行业具有重要价值。本文对解淀粉芽胞杆菌抗真菌作用、抗真菌能力提高策略、抗菌化合物合成调节、抑制真菌机制及其引发的ISR等问题进行了深入探讨和综述。     

病原性曲霉三唑类药物耐药机制的研究进展

近年来随着免疫抑制人群的不断增加,侵袭性曲霉病的发病率不断增高。然而随着三唑类药物在临床上的广泛使用,病原性曲霉对三唑类药物的耐药率逐渐增加,是临床治疗重大挑战。文中综述了病原性曲霉对三唑类药物耐药机制的研究进展。曲霉对三唑类药物的耐药机制主要包括cyp51的突变与过表达、药物外排泵的过表达、应激适应通路的激活、生物膜的形成,以及脂质合成相关基因参与而导致的耐药。     

基于丙酮酸累积的真菌抗逆机制

环境中的真菌经常会遇到多种非生物逆境胁迫,对真菌耐受逆境胁迫的机制已开展大量研究。在多种不同的真菌中发现热胁迫会诱发丙酮酸快速累积。在罗伯茨绿僵菌中,累积的丙酮酸能及时消除热胁迫诱导产生的活性氧自由基(ROS),降低ROS所引起的蛋白质羰基化修饰水平,并抑制线粒体膜电位崩溃,从而提高真菌的耐热能力。高渗、氧化胁迫和紫外线辐射也会诱发丙酮酸累积,并消除产生的ROS,这是丙酮酸累积参与多逆境交叉保护的一个机制。通过基因工程技术改造丙酮酸代谢途径,提高了在最适温度下分生孢子中丙酮酸的含量。遗传改良的孢子中丙酮酸能够快速有效地消除由热处理诱发产生的ROS,降低了ROS对分生孢子的损伤,进而提高了孢子对热胁迫的耐受性,为创建高效的真菌杀虫剂提供了菌株。     

Update on Antifungal Resistance and its Clinical Impact

Candida and Aspergillus species are important causes of opportunistic infection in an ever-growing number of vulnerable patients, and these infections are associated with high mortality. This has partly been attributed to the emerging resistance of pathogenic fungi to antifungal therapy, which potentially compromises the management of infected patients. Multi-azole resistance of Aspergillus fumigatus is a current health problem, as well as is the co-resistance of Candida glabrata to both azoles and echinocandins. In most cases, negative clinical consequences of reduced in vitro fungal susceptibility to azoles and/or echinocandins can be traced to acquisition of particular resistance mechanisms. While strategies using antifungal combinations or adjunctive agents that maximize the efficacy of existing antifungals may limit treatment failures, new therapeutic approaches, including antifungal agents with novel mechanisms of action, are urgent. In the meantime, more efforts should be devoted to close monitoring of antifungal resistance and its evolution in the clinical setting.     

Tolerance to drug-induced cell death favours the acquisition of multidrug resistance in Leishmania

The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions.     

Proteome analysis of the farnesol-induced stress response in Aspergillus nidulans--The role of a putative dehydrin

The isoprenoid alcohol farnesol represents a quorum-sensing molecule in pathogenic yeasts, but was also shown to inhibit the growth of many filamentous fungi. In order to gain a deeper insight into the antifungal activity of farnesol, we performed 2D-differential gel electrophoretic analysis (2D-DIGE) of Aspergillus nidulans exposed to farnesol. We observed an increased abundance of antioxidative enzymes and proteins involved in protein folding and the ubiquitin-mediated protein degradation. A striking finding was the strong up-regulation of a dehydrin-like protein (DlpA). Expression analyses suggested the involvement of DlpA in the cellular response to oxidative, osmotic and cold stress. In line with these data, we demonstrated that dlpA expression was regulated by the MAP kinase SakA/HogA. The generation of both a dlpA Tet(on) antisense RNA-producing A. nidulans strain (dlpA-inv) and a ΔdlpA deletion mutant indicated a role of DlpA in conidiation and stress resistance of dormant conidia against heat and ROS. Furthermore, the production of the secondary metabolite sterigmatocystin was absent in both strains dlpA-inv and ΔdlpA. Our results demonstrate the complexity of the farnesol-mediated stress response in A. nidulans and describe a farnesol-inducible dehydrin-like protein that contributes to the high tolerance of resting conidia against oxidative and heat stress.     

Augmenting the activity of antifungal agents against aspergilli using structural analogues of benzoic acid as chemosensitizing agents

A number of benzoic acid analogues showed antifungal activity against strains of Aspergillus flavus, Aspergillus fumigatus and Aspergillus terreus, causative agents of human aspergillosis, in in vitro bioassays. Structure-activity analysis revealed that antifungal activities of benzoic and gallic acids were increased by addition of a methyl, methoxyl or chloro group at position 4 of the aromatic ring, or by esterification of the carboxylic acid with an alkyl group, respectively. Thymol, a natural phenolic compound, was a potent chemosensitizing agent when co-applied with the antifungal azole drugs fluconazole and ketoconazole. The thymol-azole drug combination demonstrated complete inhibition of fungal growth at dosages far lower than the drugs alone. Co-application of thymol with amphotericin B had an additive effect on all strains of aspergilli tested with the exception of two of three strains of A. terreus, where there was an antagonistic effect. Use of two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus, sakAΔ and mpkCΔ, having gene deletions in the oxidative stress response pathway, indicated antifungal and/or chemosensitization activity of the benzo analogues was by disruption of the oxidative stress response system. Results showed that both these genes play overlapping roles in the MAPK system in this fungus. The potential of safe, natural compounds or analogues to serve as chemosensitizing agents to enhance efficacy of commercial antifungal agents is discussed.     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 μg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.     

Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast

The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.     

Antifungal agents: mechanisms of action

Clinical needs for novel antifungal agents have altered steadily with the rise and fall of AIDS-related mycoses, and the change in spectrum of fatal disseminated fungal infections that has accompanied changes in therapeutic immunosuppressive therapies. The search for new molecular targets for antifungals has generated considerable research using modern genomic approaches, so far without generating new agents for clinical use. Meanwhile, six new antifungal agents have just reached, or are approaching, the clinic. Three are new triazoles, with extremely broad antifungal spectra, and three are echinocandins, which inhibit synthesis of fungal cell wall polysaccharides--a new mode of action. In addition, the sordarins represent a novel class of agents that inhibit fungal protein synthesis. This review describes the targets and mechanisms of action of all classes of antifungal agents in clinical use or with clinical potential.     

First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus

A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.     

Aspergillus terreus Causing Probable Invasive Aspergillosis in a Patient with Cystic Fibrosis

Mycopathologia - Aspergillus terreus may colonize the airways of patients with cystic fibrosis (CF). Whether this merits antifungal treatment is still unclear due to heterogeneous reports regarding...