Molecular Genetics And Therapy Of Pathogenic Fungi

病原真菌的分子遗传学和治疗

• 批准号: 8946298
• 负责人: John E Bennett
• 金额: $ 6.75万
• 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/8946298
• 关键词: ATP-Binding Cassette Transporters; Adult; Affect; Antifungal Agents; Aspartic Endopeptidases; Autophagocytosis; Azole resistance; Azoles; Benomyl; CYP2C19 gene; CYP2C9 gene; Candida; Candida glabrata; Carbohydrates; Cell Wall; Cell physiology; Cells; Chemistry; Cholesterol; Clinical; Development; Dose; Drug Binding Site; Drug toxicity; Energy-Generating Resources; Environment; Ergosterol; Evaluation; Future; Gene Expression; Genes; Genetic Transcription; Genotype; Gluconeogenesis; Glyoxylates; Growth; Hallucinations; Hepatotoxicity; Human; In Vitro; Incubated; Knockout Mice; Metabolism; Microarray Analysis; Minor; Molecular Genetics; Mus; Nitrogen; Nutrient; Oligomycins; Oral; Oxides; Oximes; Pathway interactions; Patients; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Photosensitivity; Predisposition; Prospective Studies; Proteins; Reporting; Resistance; Resources; Sepsis; Serum; Specificity; Spleen; Sterols; Therapeutic; Time; Tissues; Toxic effect; Up-Regulation; Visual; Voriconazole; Yeasts; absorption; deprivation; fatty acid metabolism; fungus; glyoxylate; in vivo; milbemycin; neutrophil; novel; prophylactic; prospective; response; rhodamine 6G

1. Milbemycin Azole resistance in Candida glabrata, a pathogenic yeast, has prompted studies of compounds that have therapeutic potential by reversing azole resistance. Milbemycin A4 oxime blocked azole efflux and enhanced azole susceptibility fourfold in 28 clinical isolates of C. glabrata. Specificity of the milbemycin A4 oxime effect depended on the drug transporter and the substrate being effluxed. The major effect of milbemycin A4 oxime was inhibition of azole and rhodamine 6G efflux by the ATP-binding cassette (ABC) transporters CgCDR1 and PDH1. Milbemycin A4 oxime effect did not extend to oligomycin, transported by the ABC transporter YOR1 or to benomyl, transported by the major facilitator superfamily transporter, CgFLR1. Milbemycin A4 oxime did not suppress transcription of CgCDR1 but increased CgCDR1 expression 126-fold. Selectivity of the effect is compatible with the concept that milbemycin A4 oxime may interact directly with one or more drug-binding sites of the major azole transporters. 2. Voriconazole Prospective evaluation of the antifungal drug, voriconazole, is needed to determine whether drug toxicity correlates with CYP2C19 genotype or serum concentrations of voriconazole or its metabolites. We conducted a prospective study of 95 patients to determine voriconazole toxicity and its relationship to genotype and serum levels of voriconazole and its two metabolites. Efficacy was not evaluated because, in most cases, the drug was given for empirical or prophylactic therapy. Hallucinations occurred in 16 patients (16.8%), visual changes in 17 (17.9%), photosensitivity in 10 (10.5%), and hepatotoxicity in 6 (6.3%). There was no correlation between photosensitivity or hepatotoxicity and levels of voriconazole or metabolites. Patients with hallucinations had higher average voriconazole levels (4.5 vs 2.5 μg/mL) but with extensive overlap. The recommended oral dose of 200 mg did not provide consistently detectable serum voriconazole levels in adults. CYP2C19 and CYP2C9 genotypes had a minor influence over levels, though the 4 patients homozygous for the 2C19*2 genotype had higher average levels for voriconazole (4.3 vs 2.5 μg/mL) and lower N-oxide levels (1.6 vs 2.5 μg/mL). We concluded that CYP2C19 and 2C9 genotypes were not major determinants of voriconazole metabolism. No toxic serum level of voriconazole or its metabolites could be identified. 3. Gene expression of Candida glabrata in vivo. Expression microarray analysis of Candida glabrata following phagocytosis by human neutrophils was performed, and results were compared with those from C. glabrata incubated under conditions of carbohydrate or nitrogen deprivation. Twenty genes were selected to represent the major cell processes altered by phagocytosis or nutrient deprivation. Quantitative real-time PCR (qRT-PCR) with TaqMan chemistry was used to assess expression of the same genes in spleens of mice infected intravenously with Candida glabrata. The results in spleen closely paralleled gene expression in neutrophils or following carbohydrate deprivation. Fungal cells responded by upregulating alternative energy sources through gluconeogenesis, glyoxylate cycle, and longchain fatty acid metabolism. Autophagy was likely employed to conserve intracellular resources. Aspartyl protease upregulation occurred and may represent defense against attacks on cell wall integrity. Downregulated genes were in the pathways of protein and ergosterol synthesis. Upregulation of the sterol transport gene AUS1 suggested that murine cholesterol may have been used to replace ergosterol, as has been reported in vitro. C. glabrata isolates in spleens of gp91phox/knockout mice with reduced oxidative phagocyte defenses were grossly similar although with a reduced level of response. These results are consistent with reported results of other fungi responding to phagocytosis, indicating that a rapid shift in metabolism is required for growth in a carbohydrate-limited intracellular environment.

1.米尔倍霉素 光滑念珠菌（一种致病性酵母）的唑类抗性促使人们研究通过逆转唑类抗性而具有治疗潜力的化合物。在 28 个光滑念珠菌临床分离株中，米尔倍霉素 A4 肟可阻断唑类外流，并使唑类敏感性增强四倍。米尔倍霉素 A4 肟作用的特异性取决于药物转运蛋白和外排底物。米尔倍霉素 A4 肟的主要作用是通过 ATP 结合盒 (ABC) 转运蛋白 CgCDR1 和 PDH1 抑制唑类和罗丹明 6G 外流。米尔倍霉素 A4 肟效应并未延伸至寡霉素（由 ABC 转运蛋白 YOR1 转运）或苯菌灵（由主要易化子超家族转运蛋白 CgFLR1 转运）。 Milbemycin A4 oxime 不会抑制 CgCDR1 的转录，但会使 CgCDR1 的表达增加 126 倍。该效应的选择性与米尔贝霉素 A4 肟可能与主要唑类转运蛋白的一个或多个药物结合位点直接相互作用的概念相一致。 2.伏立康唑 需要对抗真菌药物伏立康唑进行前瞻性评估，以确定药物毒性是否与 CYP2C19 基因型或伏立康唑或其代谢物的血清浓度相关。我们对 95 名患者进行了一项前瞻性研究，以确定伏立康唑毒性及其与伏立康唑及其两种代谢物基因型和血清水平的关系。未评估疗效，因为在大多数情况下，该药物用于经验性或预防性治疗。 16 名患者（16.8%）出现幻觉，17 名患者（17.9%）出现视力变化，10 名患者（10.5%）出现光过敏，6 名患者（6.3%）出现肝毒性。光敏性或肝毒性与伏立康唑或代谢物的水平之间没有相关性。有幻觉的患者伏立康唑平均水平较高（4.5 vs 2.5 μg/mL），但有广泛的重叠。推荐口服剂量 200 mg 并不能在成人中提供一致可检测的血清伏立康唑水平。 CYP2C19 和 CYP2C9 基因型对水平的影响较小，尽管 4 名 2C19*2 基因型纯合患者的伏立康唑平均水平较高（4.3 vs 2.5 μg/mL），N-氧化物水平较低（1.6 vs 2.5 μg/mL）。我们得出结论，CYP2C19 和 2C9 基因型不是伏立康唑代谢的主要决定因素。未发现伏立康唑或其代谢物的毒性血清浓度。 3.光滑念珠菌体内基因表达。 对被人中性粒细胞吞噬后的光滑念珠菌进行表达微阵列分析，并将结果与​​在碳水化合物或缺氮条件下孵育的光滑念珠菌的结果进行比较。选择了二十个基因来代表因吞噬作用或营养剥夺而改变的主要细胞过程。实时定量PCR (qRT-PCR) 和 TaqMan 化学方法用于评估静脉内感染光滑念珠菌的小鼠脾脏中相同基因的表达。脾脏中的结果与中性粒细胞中或碳水化合物剥夺后的基因表达密切相关。真菌细胞通过糖异生、乙醛酸循环和长链脂肪酸代谢上调替代能源来做出反应。自噬可能被用来保存细胞内资源。天冬氨酰蛋白酶发生上调，可能代表对细胞壁完整性攻击的防御。下调的基因位于蛋白质和麦角甾醇合成途径中。正如体外报道的那样，甾醇转运基因 AUS1 的上调表明小鼠胆固醇可能已被用来替代麦角甾醇。氧化吞噬细胞防御能力降低的 gp91phox/基因敲除小鼠脾脏中分离的光滑念珠菌非常相似，尽管反应水平降低。这些结果与其他真菌响应吞噬作用的报道结果一致，表明在碳水化合物有限的细胞内环境中生长需要新陈代谢的快速转变。

项目成果

The changing face of febrile neutropenia-from monotherapy to moulds to mucositis. Management of mycoses in neutropenic patients: a brief history, 1960-2008.

发热性中性粒细胞减少症的变化——从单一疗法到霉菌再到粘膜炎。

• DOI: 10.1093/jac/dkp079
• 发表时间: 2009
• 期刊: The Journal of antimicrobial chemotherapy
• 作者: Bennett,JohnE

Is real-time polymerase chain reaction ready for real use in detecting candidemia?

实时聚合酶链反应是否准备好真正用于检测念珠菌血症？

• DOI: 10.1086/528692
• 发表时间: 2008
• 期刊: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
• 作者: Bennett,John

Milbemycin A4 oxime as a probe of azole transport in Candida glabrata.

米尔倍霉素 A4 肟作为光滑念珠菌中唑类转运的探针。

• DOI: 10.1111/1567-1364.12164
• 发表时间: 2014
• 期刊: FEMS yeast research
• 影响因子: 3.2
• 作者: Walker,Bryan;Izumikawa,Koichi;Tsai,Huie-Fung;Bennett,JohnE
• 通讯作者: Bennett,JohnE

Disseminated microsporidiosis in an immunosuppressed patient.

• DOI: 10.3201/eid1807.120047
• 发表时间: 2012-07
• 期刊: Emerging infectious diseases
• 影响因子: 11.8
• 作者: Meissner EG;Bennett JE;Qvarnstrom Y;da Silva A;Chu EY;Tsokos M;Gea-Banacloche J
• 通讯作者: Gea-Banacloche J

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment.

• DOI: 10.1186/1471-2199-13-22
• 发表时间: 2012-06-29
• 期刊: BMC molecular biology
• 作者: Li QQ;Skinner J;Bennett JE
• 通讯作者: Bennett JE