Molecular genetics, biochemistry and therapy of pathogenic fungi

病原真菌的分子遗传学、生物化学和治疗

• 批准号: 6431622
• 负责人: John E Bennett
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431622
• 关键词: Candida albicans; Cryptococcus neoformans; Saccharomyces cerevisiae; candidiasis; cerebrospinal fluid; dihydroxyphenylalanine; enzyme activity; fungal genetics; gene deletion mutation; genetic strain; hydrogen peroxide; laboratory mouse; laboratory rabbit; melanins; microorganism metabolism; monophenol monooxygenase; mutant; neutrophil; nitrogen oxides; phenols; polymerase chain reaction; site directed mutagenesis; virulence

Candidiasis remains the most common deep mycosis, being the fourth most common cause of nosocomial bloodstream infections and occurring commonly in patients with neutropenia, prematurity and complicated abdominal surgery. Mucosal candidiasis is also the most common mycosis in AIDS patients. Broad use of fluconazole, particularly for prophylaxis and empiric therapy, has slowly increased resistance to fluconazole and other azole antifungals. Fluconazole resistance appears to rise rapidly in Candida glabrata, a species accounting for 15% of candidemias. In a series of 9 bone marrow transplant recipients who were persistently colonized by C. glabrata, minimum inhibitory concentrations of fluconazole rose in five patients from 16-32 mcg/ml to at least 128 mcg/ml during fluconazole prophylaxis. In all five patients, azole resistance arose in their original strain, rather than being due to acquisition of a resistant strain, as judged by computer- assisted analysis of macrodigested CHEF patterns. The major mechanism of fluconazole resistance in this species appears to be activation of drug efflux pumps. We have used Northern blotting with phosphoimager analysis to examine transcription of the ABC transporter gene, PDH1. Although disruption of this gene does not change fluconazole susceptibility, transcript abundance correlates with azole susceptibility, indicating coregulation with other transporters, at least one of which transports fluconazole. A wide range of apparently unrelated toxic substances increases abundance of the PDH1 transcript, including oligomycin, cycloheximide and rhodamine 6G. We extended our studies on the disruption of PDH1 to a second azole susceptible strain, confirming increased toxicity of oligomycin, cycloheximide, and cadmium, as well as increased intracellular concentrations of rhodamine. Increased estradiol uptake was also noted in this pdh1 mutant. No change in any of these parameters was observed when PDH1 was disrupted in one markedly azole resistant strain, probably because other transporters with overlapping specificity were activated and compensated for loss of pdh1p transport activity. Overlapping function of multidrug resistance genes and their transcriptional regulation may account for this species? rapid ability to overcome growth inhibition by azole antifungals.We have continued our study of azole resistant Candida albicans strains. The importance of the Y132H and I471T mutations in the gene coding for the azole target, C14demeythylase (ERG11) was confirmed by site directed mutagenesis. One copy of ERG11 gene in an azole susceptible strain of C. albicans was replaced with a C. albicans ERG11 gene that these mutations. Resistance was increased markedly by either mutation but was greatest with both mutations. To assist in identifying transmission of azole resistant strains, we compared three different molecular typing techniques and found that the macrodigested CHEF had the appropriate discimination index for our purpose. Both the CA3 and CARE2 probes of EcoR1 digested genomic DNA gave patterns that were so discriminating that microevolution within a single strain could not be distinguished from acquisition of a new strain. Cryptococcal meningitis in previously normal patients can respond poorly to all available antifungal agents. Cytokine therapy has not been evaluated clinically, largely because of concern that increased inflammation might worsen cerebral edema. In order to begin to understand how immunity may be altered favorably, we have begun study of experimental cryptococcosis. In IL12 knockout mice in a Balb/c background, death occurred much earlier than in Balb/c controls, confirming the importance of the Th1 arm of the immune system. In a clinical study, neurosurgical placement of a shunt in ten patients with hydrocephalus due to cryptococcal meningitis was found to restore neurologic function and not impair response to antifungal therapy by acting as a foreign body.Pneumonia due to respiratory syncytial virus (RSV) causes death in approximately 5% of allogeneic bone marrow transplant recipients. We have begun a study of early diagnosis and immunoprophylaxis of RSV in the NIH bone marrow transplant unit. - azole, antifungal, resistance, Candida, demethylase, transporter - Human Subjects

念珠菌病仍然是最常见的深部真菌病，是医院内血流感染的第四大常见原因，常见于中性粒细胞减少症、早产和复杂腹部手术患者。粘念珠菌病也是艾滋病患者最常见的真菌病。氟康唑的广泛使用，特别是用于预防和经验性治疗，使氟康唑和其他唑类抗真菌药的耐药性缓慢增加。氟康唑耐药性似乎在光滑念珠菌中迅速上升，光滑念珠菌占念珠菌的15%。在9例骨髓移植受者中，C. glabrata，在氟康唑预防期间，5名患者的氟康唑最低抑菌浓度从16-32 mcg/ml升高至至少128 mcg/ml。在所有5名患者中，唑类耐药性均出现在其原始菌株中，而不是由于获得耐药菌株，如通过计算机辅助分析大量消化的CHEF模式所判断的。氟康唑耐药的主要机制似乎是药物外排泵的激活。我们使用北方印迹与磷光成像分析，以检查ABC转运蛋白基因，PDH 1的转录。虽然该基因的破坏不会改变氟康唑的敏感性，转录丰度与唑类药物的敏感性，表明与其他转运蛋白，其中至少一个运输氟康唑的协同调节。一系列明显不相关的有毒物质增加了PDH 1转录物的丰度，包括寡霉素、放线菌酮和罗丹明6 G。我们扩展了我们的研究对破坏的PDH 1的第二唑敏感菌株，确认寡霉素，放线菌酮，镉的毒性增加，以及罗丹明的细胞内浓度增加。在pdh 1突变体中也观察到雌二醇摄取增加。当PDH 1在一个明显的唑类耐药菌株中被破坏时，没有观察到任何这些参数的变化，这可能是因为其他具有重叠特异性的转运蛋白被激活并补偿了pdh 1 p转运活性的损失。多药耐药基因的重叠功能及其转录调控可能是导致该物种耐药的原因之一。快速克服唑类抗真菌药生长抑制的能力。我们继续研究唑类耐药白色念珠菌菌株。通过定点突变证实了编码唑靶点C14脱甲基酶（ERG 11）的基因中Y132 H和I471 T突变的重要性。在唑类敏感的C.白色念珠菌替换为C.白念珠菌ERG 11基因存在这些突变。任何一种突变都能显著增加耐药性，但两种突变都是最大的。为了帮助确定唑类耐药菌株的传播，我们比较了三种不同的分子分型技术，发现粗消化的CHEF具有适合我们目的的区分指数。EcoR 1消化的基因组DNA的CA 3和CARE 2探针都给出了如此有区别的模式，以至于单个菌株内的微进化不能与新菌株的获得区分开来。隐球菌性脑膜炎在以前正常的病人可以响应差，所有可用的抗真菌药物。细胞因子治疗尚未进行临床评估，主要是因为担心炎症增加可能会加重脑水肿。为了开始了解免疫力如何被有利地改变，我们已经开始了实验性隐球菌病的研究。在Balb/c背景下的IL 12敲除小鼠中，死亡发生时间比Balb/c对照组早得多，证实了免疫系统Th 1臂的重要性。在一项临床研究中，在10例隐球菌脑膜炎引起脑积水的患者中，神经外科手术放置分流管，发现神经功能恢复，并且不会因异物作用而损害抗真菌治疗的反应。呼吸道合胞病毒（RSV）引起的肺炎导致约5%的异基因骨髓移植受者死亡。我们已经开始在NIH骨髓移植单位进行RSV的早期诊断和免疫预防的研究。- 唑类，抗真菌，耐药性，念珠菌，脱甲基酶，转运蛋白-人类受试者