Stress Management of Cryptococcal Pathogens

隐球菌病原体的压力管理

• 批准号: 9786414
• 负责人: Kyung Kwon-Chung
• 金额: $ 51.33万
• 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/9786414
• 关键词: ABCB1 gene; ATP-Binding Cassette Transporters; Acquired Immunodeficiency Syndrome; Aerobic Bacteria; Aneuploidy; Antifungal Agents; Azole resistance; Azoles; Behavior; Binding Proteins; Brain; Bypass; Carbon Dioxide; Categories; Cell Nucleus; Cells; Cellular Stress; Chemical Agents; Chromosomes; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 4; Complement; Cryptococcal Meningitis; Cryptococcosis; Cryptococcus gattii; Cryptococcus neoformans; DNA Sequence Rearrangement; Endoplasmic Reticulum; Environment; Ergosterol; Etiology; Fluconazole; Fluconazole resistance; Flucytosine; Frequencies; Gene Mutation; Gene Targeting; Genes; Goals; Gold; Growth; HIV Seropositivity; Habitats; Histones; Human; Hypersensitivity; Hypoxia; In Vitro; Individual; Infection; Intermediate resistance; Libraries; Link; Maintenance Therapy; Meningoencephalitis; Molecular; Monitor; Mus; Mutation; Neoadjuvant Therapy; Nuclear; Nuclear Fusion; Organism; Oxygen; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Pump; Reactive Nitrogen Species; Reactive Oxygen Species; Regulatory Element; Resistance; Role; Serotyping; Signal Pathway; Sister; Sterol Biosynthesis Pathway; Sterols; Stress; Substrate Specificity; System; T-Lymphocyte; Time; Treatment Failure; Virulence; cell behavior; fungus; gene function; man; mutant; neurotropic; pathogen; planetary Atmosphere; resistance mechanism; stress management

Cryptococcus neoformans is a neurotropic pathogen that,unless treated,causes fatal meningoencephalitis primarily in individuals with T-cell deficiency such as the AIDS patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. C. neoformans is an obligate aerobe which is commonly found in the human environment world-wide. The fungus requires atmospheric concentrations of oxygen for optimum growth in vitro and the growth is drastically retarded at oxygen levels lower than that of the atmosphere. In the past three years, we have constructed an insertional mutant library composed of 30,000 mutant clones and screened them for their ability to grow under hypoxic conditions in 1% oxygen and 5% carbon dioxide. We identified numerous clones that were unable to grow under hypoxic conditions. These mutants belonged to two categories, those that had mutations in genes that function in SREBP (sterol regulatory element binding protein termed as Sre1) pathway and the others that belonged to cyptcococcus specific-gene mutations which are not known to be associated with the oxygen sensing system. The genes identified in these two categories were deleted in the serotype D reference strain, B-3501. Deletants expressing the expected phenotype (unable to grow under low oxygen) were complemented with their respective wild type genes. During 2008-2009, we charcterized mutants with genetic defects in 6 genes that share homology to genes that function in the mammalian SREBP pathway. Three of these genes, SFB2, KAP123, and GSK3, are not reported to be involved in the SREBP pathway of other fungi. In addition, C. neoformans contains an extra gene, DAM1, which functions in the SREBP pathway but has not been described previously in other organisms. Mutants associated with the steps prior to formation of the nuclear SREBP form dramatically reduced the accumulation of the nuclear form. Concurrently, two of these mutants, scp1 and stp1, and the previously isolated sre1 mutants showed a reduction in ergosterol levels, hypersensitivity to several chemical agents including azole antifungals, CoCl2, compounds producing reactive oxygen species or reactive nitrogen species and most importantly, showed reduced virulence in mice. Mutations in genes involved in the later steps of the Sre1 pathway, such as those required for the import and phosphorylation of proteins in nucleus, showed less compelling phenotypes. These findings suggest that the SREBP pathway is highly conserved in C. neoformans and it serves as an important link between sterol biosynthesis, oxygen sensing, CoCl2 sensitivity and virulence in C. neoformans.During 2008-2009, we also characterized the levels of azole heteroresistance in C. neoformans strains that had been isolated prior to the advent of azole antifungals as well as those isolated from AIDS patients undergoing azole maintenance therapy and found that heteroresistance to fluconazole is universal in C. neoformans. During 2009-2010, we focused on the mechanism of heteroresistance in C. neoformans as well as the status of intrinsic heteroresistance to fluconazole in C. gattii, the sister species of C. neoformans that causes cryptococcosis. We found that C. neoformans strains possess an innate ability to produce subpopulations that can form disomic chromosomes when the cells are stressed by high concentration of fluconazole and the number of these disomic chromosomes was proportional to the concentration of the drug. For example, at 32 ug/ml fluconazole, the surviving clones were disomic for chromosome 1 while those that survived at 64 ug/ml fluconazole contained disomies of chromosomes one and four. Chromosome one was observed to be disomic in every clone that survived drug concentrations higher than the strains minimal inhibitory concentration of the drug. Chromosome one was found to contain ERG11, the target gene of azoles, and AFR1, an efflux pumpknown for azoles. These results suggest that complete duplication of chromosome 1 is a major mechanism by which C. neoformans overcomes the stress of azole drugs. C. gattii was found to be innately heteroresistant to fluconazole as is the case with C. neoformans but with higher levels of heteroresistance to fluconazole. During 2010-2011, we identified the genes on chromosome 4 that are relevant for azole resistance and duplication of the chromosome under high concentration of fluconazole.During 2011-2012, we have characterized the importance of the endoplasmic reticulum (ER) integrity in C. neoformans to resist azole stress by forming aneuploidy for chromosomes 1 and 4. During 2013-2014, we have confirmed that chromosome duplication occure in the C. neoformans fluconazole resistant clones that emerge during long term therapy with fluconazole.During 2014-2015, we initiated the molecular characterization of cryptococcal resistance to 5-fluorocytosine since the drug is used widely during an induction therapy of cryptococcosis but with very little understanding in the mechanism of resistance. During 2015-2016,we generated a strain of C. neoformans containing GFP tagged histone to monitor the nuclear behavior in growing cells under fluconazole stress. The majority of cells stopped growth after a few division while less than 1 % of cells continued growth and formed colony. Nuclear behavior of the cells monitored for 3 days by time laps micrographs. Our observation indicated the possibility of chromosome endoduplication rather than more common mechanism of aneuploidy formation via nuclear fusion followed by chromosome missegregation. We also discovered the additive role of AFR2, a secondary ABC transporter for effluxing azoles. During 2016-2017, we investigated the substrate specificity of three ABC transporters in two different etiologic agents of cryptococcosis and found that in both species, AFR1 is the major drug pump and the two other pumps, AFR2 and MDR1 show differences in substrate specificity between the two cryptococcosis agents.2017-2018, we have found the evidence that the extra copy of chromosome 1 formed under the azole stress was the result of endogenous duplication. During 2018-2019, we discovered that C. neoformans undergo genomic rearrangement and produce extraordinary number of clones resistant to 5-fluorocytosine, which is an essential part of gold-standard induction therapy for cryptococcal meningitis

隐型新羊角菌是一种神经性病原体，除非接受过治疗，否则会导致致命脑膜脑炎，主要是在T细胞缺乏症（例如AIDS患者）中引起的致命脑膜炎。但是，真菌还会在低频中引起正常患者的感染。 C. neoformans是一种强制性的可氧化锅，在全球人类环境中通常发现。该真菌需要大气浓度的氧气才能在体外最佳生长，并且在氧气水平低于大气水平的情况下，生长急剧延伸。在过去的三年中，我们构建了一个由30,000个突变克隆组成的插入突变库，并筛选了它们在1％氧和5％二氧化碳在低氧条件下生长的能力。我们确定了许多在低氧条件下无法生长的克隆。这些突变体属于两个类别，在SREBP（固醇调节元件结合蛋白称为SRE1）途径的基因中具有突变的突变体，而其他属于Cyptcocococcus特异性基因突变的基因（固醇调节元件结合蛋白）中的突变，这些突变与氧气传感系统相关。在这两个类别中鉴定的基因在血清型D参考菌株B-3501中被删除。表达预期表型（无法在低氧气下生长）的缺失与其各自的野生型基因相辅相成。在2008 - 2009年期间，我们在6个基因中具有遗传缺陷的突变体，这些突变体与在哺乳动物SREBP途径中起作用的基因具有同源性。据报道，这些基因中的三个，SFB2，KAP123和GSK3没有涉及其他真菌的SREBP途径。此外，新梭菌包含一个额外的基因DAM1，该基因在SREBP途径中起作用，但在其他生物体中尚未描述。与核SREBP形成之前的相关步骤相关的突变体大大降低了核形式的积累。同时，这些突变体SCP1和STP1中的两个，并且先前分离的SRE1突变体显示出麦角固醇水平的降低，对包括硫唑抗真菌剂，COCL2，COCL2，化合物，产生活性氧或反应性氮种的高敏性，以及最重要的毒素，在小鼠中显示出毒素的降低。 SRE1途径后期步骤中涉及的基因的突变，例如核中蛋白质的进口和磷酸化所需的突变表现出较不引人注目的表型。这些发现表明，SREBP途径在Neoformans中高度保守，它是固醇生物合成，氧气感应，COCL2敏感性和毒力之间的重要联系。从接受硫唑维持疗法的艾滋病患者中，发现氟康唑的异质症在新梭菌中是普遍的。在2009年至2010年期间，我们重点关注Neoformans杂质的机理，以及在Gattii中对氟康唑的内在杂质的状态，C. gattii是新生梭菌的姐妹种类，导致了加密co虫病。我们发现，当细胞受到高浓度的氟康唑压力时，新生梭菌菌株具有生产亚群的先天能力，可以形成疾病的染色体，并且这些疾病染色体的数量与该药物的浓度成正比。例如，在32 ug/ml氟康唑时，幸存的克隆是1染色体的疾病，而在64 ug/ml氟康唑中存活的克隆含有染色体的疾病和第四个。在每个克隆中，染色体一染色体都是在药物浓度高的药物浓度中生存的每个克隆中是缺陷的。发现一染色体包含Azole的靶基因和AFR1的ERG11，AFR1是Azoles的外排泵。这些结果表明，染色体1的完全重复是Neoformans C. Neoformans克服硫唑药物的应力的主要机制。发现gattii与氟康唑一样天生具有异性抗性，就像Neoformans一样，但具有较高水平的氟康唑的杂质。在2010-2011期间，我们确定了4号染色体上的基因，这些基因与高浓度的氟康唑在高浓度的氟康唑之下与染色体的抗性和重复相关。Ding2011-2012，我们表征了内质网（ER）在C. Neooformans中以抗azole premose在Aneofors中的cros and and anneplems and anneeplom and anneepleom的重要性。 2013 - 2014年，我们已经证实，在长期用氟康唑治疗期间出现的C. Neoformans抗甲唑烷耐药克隆发生染色体重复。抗性机制。 在2015 - 2016年期间，我们产生了一组含有GFP标记组蛋白的Neoformans菌株，以监测氟康唑应激下生长细胞的核行为。大多数细胞在几次分裂后停止了生长，而不到1％的细胞持续生长并形成菌落。按时间圈显微照片监测的细胞的核行为。我们的观察结果表明，通过核融合，随后是染色体错误分析的非整倍性形成的染色体内二拟补的可能性。我们还发现了AFR2的加性作用，AFR2是一种用于排出叠唑的次级ABC转运蛋白。在2016年至2017年期间，我们调查了三个ABC转运蛋白在两个不同的隐孢子虫病病学剂中的底物特异性，并且发现这两个物种中，AFR1是主要的药物泵，而其他两个泵均显示出AFR2和MDR1在两个副作用中的底物特异性差异，该副作用的副作用是跨越杂物。压力是内源性重复的结果。在2018-2019期间，我们发现Neoformans经历了基因组重排并产生非凡的克隆对5-氟环胞嘧啶的耐药性，这是加密赛脑膜炎的金标准诱导疗法的重要组成部分