Molecular Genetics And Therapy Of Pathogenic Fungi

病原真菌的分子遗传学和治疗

• 批准号: 8336091
• 负责人: John E Bennett
• 金额: $ 70.24万
• 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/8336091
• 关键词: ATP-Binding Cassette Transporters; Adult; American; Amino Acids; Anabolism; Antifungal Agents; Azole resistance; Azoles; Binding; CDR1 gene; Caffeine; Candida; Candida glabrata; Carbohydrates; Carboxylic Acids; Carrier Proteins; Cell Membrane Proteins; Cells; Cholesterol; Code; Communicable Diseases; Complement; Complex; Cyclophilins; Data; Drug Efflux; EWS/FLI 1 Type 1 antisense oligonucleotide; Environment; Ergosterol; Evaluation; Fluconazole; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic Transcription; Glucose; Growth; Homologous Gene; Host Defense; Hour; Housekeeping Gene; Human; Hydrogen Peroxide; Infection; Internal Medicine; Knockout Mice; Mediating; Messenger RNA; Metabolism; Methionine; Methods; Molecular Genetics; Mouse Strains; Multidrug Resistance Gene; Mus; N-terminal; Nitrogen; Nutrient; Open Reading Frames; Oxidative Stress; Phagocytosis; Pharmaceutical Preparations; Physicians; Process; Protein Biosynthesis; Qualifying; RNA; RNA, ribosomal, 25S; RPL10 gene; Relative (related person); Reporting; Resistance; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal RNA; Role; Saccharomyces cerevisiae; Sepsis; Source; Spleen; Sterols; Stimulus; Stress; System; TFRC gene; Testing; Time; Training Programs; Transcription Repressor/Corepressor; Transforming Protein ERG; Tubulin; UBE2D2 gene; Up-Regulation; Validation; Yeasts; catalase; deprivation; fungus; gene repression; graduate medical education; human UBE2G2 protein; in vivo; interest; internal control; killings; mRNA Expression; microorganism; mutant; neutrophil; novel; overexpression; pathogen; research study; response; transcription factor

1)STB5, a transcriptional repressor in Candida glabrata The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals. Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata the ORF designated CaGl0I02552g, is syntenic with STB5 (YHR178w) in S. cerevisiae and has 43.4% amino acid identity with STB5, including an N-terminal Zn2Cys6 binuclear cluster domain, causing us to test for homologous function and a possible role in azole resistance. CaGl0I02552g was able to restore vegetative growth rate, complemented increased sensitivity to cold, hydrogen peroxide and caffeine in a Δstb5 mutant, and was designated CgSTB5.Overexpression of Cg STB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 increased resistance, both by a mechanism independent of CgPDR1.Real time PCR analysis showed that overexpression of CgSTB5 repressed transcription of the transporter genes CDR1, PDH1 and YOR1. Transcriptional repression by CgSTB5 was not found to be due to competition with PDR1 for binding to the Pleomorphic Drug Response Element (PDRE), TCCGCCCA, but may be acting through binding to the transcriptional repressor complex, SIN3, as the S. cerevisiae homologue is conjectured to do. 2) Global transcriptional profiling of Candida glabrata during phagocytosis by neutrophils and in the infected mouse spleen Neutrophils provide an important host defense against deep C. glabrata infections but the effect neutrophils or the host environment have on the fungus transcriptome is unknown. We used microarray to study the transcriptome of C. glabrata cells after 30 and 60 minutes incubation with human neutrophils under conditions in which more than 80% of the cells were phagocytosed. A total of 519 genes were up-regulated and 360 down-regulated at 30 min incubation. Several highly up-regulated genes were involved in methionine biosynthesis (MET28, STR3), related to nitrogen depletion. We also found induction of processes involved in carboxylic acid transport (SFC1, MUP1, GAP1, ADY2), cellular carbohydrate metabolic processes (FBP1, PCK1, ICL1) and transcriptional factors which regulate gene expression for utilization of alternative carbohydrate sources instead of glucose (SIP4, CAT8, ADR1). This induction was strongly related to carbohydrate depletion . For our interest, multidrug resistance genes were also upregulated (PDR1, PDH1) , also seen as a response to carbohydrate depletion. Many of the down-regulated genes were involved in protein synthesis (RPL12A, GCD10) and ergosterol biosynthetic process (ERG 10, ERG9). These results suggested that C. glabrata cells were decreasing synthesis of proteins and cell membrane sterols because of nutrient depletion. We confirmed this by performing microarray on C. glabrata exposed to nitrogen or glucose deprivation. We hypothesized that C. glabrata in mouse spleen might be exposed to the same environmental conditions as in human neurophils. Therefore, we selected 20 target genes from our neutrophil experiments and tested expression in spleen 24 hours after infection of C57BL/6J mice, using quantitative real-time PCR. Our results indicated that expression of 15 genes was similar in neutrophils and mouse spleen, whereas 3 genes (AUS1, PCK1, CDC19) were significantly upregulated in C57BL/6J mouse spleen compared to neutrophils. These results suggested Cg cells are exposed to even more severe nutrient condition in mouse spleen compared with that in neutrophils. The 6-fold upregulation of AUS1 may indicate a need for importing exogenous sterol, such as cholesterol, in response to decreased sterol synthesis. As we have reported, C. glabrata can utilize cholesterol when ergosterol synthesis is impaired. To the extent that the cell can utilise cholesterol, azoles ability to block growth by blocking ergosterol synthesis is nullified. To clarify the effect of oxidative stress against C. glabrata cells in vivo, we also compared gene expression of C. glabrata in spleens of C57BL/6J and gp91phox- knock-out mice. The latter mice have reduced oxidative capacity for neutrophil killing of microorganisms. Expression of the 20 genes was similar in both mouse strains, including expression of CTA1, which codes for catalase and is upregulated under oxidative stress. This suggests that oxidative stress may not be the major mechanism by which the neutrophil inhibits growth. Response of C. glabrata to neutrophil phagocytosis reflects the lack of essential carbohydrate and nitrogen sources. The gene expression of C glabrata cells in mouse spleens showed the fungus was exposed to at least as severe nutrient depletion. Up-regulation of multidrug resistance genes, such as PDH1, in neutrophils and mouse spleens even in the absence of drugs is an integral part of C. glabrata response and this, as well as increased sterol transport, may contributes to azole resistance in vivo. 3)Evaluation of housekeeping genes for RT-PCR analysis of gene expression in Candida glabrata responding to azole stress Selection of stable and suitable reference genes for quantitative real-time PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions and environmental stimuli. The present study aimed at identifying and selecting housekeeping genes as internal controls for gene expression studies by RT-qPCR in fluconazole-stimulated Candida glabrata. The expression stability of 20 housekeeping genes was evaluated using the 2-∆∆CT Method. Our data revealed that the mRNA expression levels of the three ribosomal RNAs 5.8S rRNA, 18S rRNA, and 25S rRNA remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNA were relatively stable with only approximately 2.3-, 2.3-, and 1.9-fold induction, respectively. By contrast, mRNA levels of the other 14 housekeeping genes (ACT1, cyclophilin, EF-1α, GAPDH, RPL2A, RPL10, RPL13B, RPP2B, SDHA, TBP, TFRC, α-tubulin, β-tubulin, and UBC4) tested were significantly increased in C. glabrata following drug treatment, with changes ranging from 3.5 to 25 fold. We then validated the suitability of 6 candidate reference genes (5.8S rRNA, 18S rRNA, ACT1, PGK1, UBC7, and UBC13) as endogenous controls for 10 target genes in this system using the ∆∆CT Method. We chose to assess 5 ATP-binding cassette transporter genes (CDR1, PDH1, PDR1, SNQ2, and YOR1) and 5 sterol biosynthetic genes (ERG2, ERG3, ERG4, ERG10, and ERG11) as the targets in this study because these genes have been shown to be implicated in azole resistance in C. glabrata. Our validation experiments passed for all the 6 control genes analyzed except for 18S rRNA, where the absolute value of the slope of log input RNA vs. ∆CT of the targets was >0.1. Finally, we demonstrated that the relative quantification of the target gene expression varied according to the internal control used, thus highlighting the importance of the choice of endogenous controls in such experiments. 4)Infectious Disease Training Program This training program continues to attract 4-5 highly qualified physicians each year and remains fully accredited by the Adult Committee for Graduate Medical Education. The pass rate of graduates on the American Board of Internal Medicines Infectious Disease certifying exam remains 100% for the decade and 95% since inception of the examination in 1972.

1)STB5，光滑念珠菌中的转录抑制因子 机会性酵母病原体光滑念珠菌因其在唑类抗真菌药物的长期治疗过程中获得耐药性的能力而受到认可。 对唑类药物的耐药性主要由转录因子 PDR1 介导，导致 ATP 结合盒 (ABC) 转运蛋白和药物外流上调。 对相关酵母酿酒酵母的研究表明，Pdr1p 与另一个转录因子 Stb5p 形成异二聚体。 在光滑 C. glabrata 中，指定为 CaGl0I02552g 的 ORF 与酿酒酵母中的 STB5 (YHR178w) 同线，并且与 STB5 具有 43.4% 的氨基酸同一性，包括 N 端 Zn2Cys6 双核簇结构域，使我们能够测试同源功能和在唑类抗性中的可能作用。 CaGl0I02552g能够恢复营养生长速率，补充Δstb5突变体中对寒冷、过氧化氢和咖啡因的敏感性增加，并被命名为CgSTB5。光滑念珠菌中Cg STB5的过表达抑制了唑类抗性，而CgSTB5的缺失增加了抗性，两者都是通过独立于CgPDR1的机制实现的。实时PCR 分析表明，CgSTB5 的过表达抑制了转运蛋白基因 CDR1、PDH1 和 YOR1 的转录。未发现 CgSTB5 的转录抑制是由于与 PDR1 竞争与多形性药物反应元件 (PDRE)、TCCGCCCA 的结合，而是可能通过与转录抑制复合物 SIN3 的结合来发挥作用，正如酿酒酵母同源物所推测的那样。 2) 光滑念珠菌在中性粒细胞吞噬过程中和感染小鼠脾脏中的整体转录谱 中性粒细胞为宿主抵御深部光滑念珠菌感染提供了重要的防御，但中性粒细胞或宿主环境对真菌转录组的影响尚不清楚。我们使用微阵列研究了光滑 C. glabrata 细胞与人中性粒细胞孵育 30 和 60 分钟后的转录组，其中超过 80% 的细胞被吞噬。孵育 30 分钟时，共有 519 个基因上调，360 个基因下调。几个高度上调的基因参与蛋氨酸生物合成（MET28、STR3），与氮消耗相关。我们还发现了涉及羧酸转运（SFC1、MUP1、GAP1、ADY2）、细胞碳水化合物代谢过程（FBP1、PCK1、ICL1）和调节基因表达以利用替代碳水化合物来源而不是葡萄糖的转录因子（SIP4、CAT8、ADR1）的诱导过程。这种诱导与碳水化合物消耗密切相关。出于我们的兴趣，多药耐药基因也被上调（PDR1、PDH1），也被视为对碳水化合物消耗的反应。许多下调的基因涉及蛋白质合成（RPL12A、GCD10）和麦角甾醇生物合成过程（ERG 10、ERG9）。 这些结果表明，光滑念珠菌细胞由于营养耗尽而减少了蛋白质和细胞膜甾醇的合成。 我们通过对暴露于氮或葡萄糖剥夺的光滑 C. glabrata 进行微阵列证实了这一点。 我们假设小鼠脾脏中的光滑念珠菌可能暴露于与人类神经细胞相同的环境条件下。因此，我们从中性粒细胞实验中选择了 20 个目标基因，并使用定量实时 PCR 测试了感染 C57BL/6J 小鼠 24 小时后脾脏中的表达。我们的结果表明，中性粒细胞和小鼠脾脏中 15 个基因的表达相似，而与中性粒细胞相比，C57BL/6J 小鼠脾脏中 3 个基因（AUS1、PCK1、CDC19）显着上调。这些结果表明，与中性粒细胞相比，小鼠脾脏中的 Cg 细胞面临着更严重的营养条件。 AUS1 的 6 倍上调可能表明需要输入外源性甾醇，例如胆固醇，以应对甾醇合成的减少。正如我们所报道的，当麦角甾醇合成受损时，光滑念珠菌可以利用胆固醇。在细胞可以利用胆固醇的情况下，唑类通过阻断麦角甾醇合成来阻断生长的能力被抵消。为了阐明体内氧化应激对光滑念珠菌细胞的影响，我们还比较了 C57BL/6J 和 gp91phox 敲除小鼠脾脏中光滑念珠菌的基因表达。后者小鼠的中性粒细胞杀死微生物的氧化能力降低。两种小鼠品系中 20 个基因的表达相似，包括 CTA1 的表达，CTA1 编码过氧化氢酶，并在氧化应激下上调。这表明氧化应激可能不是中性粒细胞抑制生长的主要机制。 光滑 C. glabrata 对中性粒细胞吞噬作用的反应反映了必需碳水化合物和氮源的缺乏。小鼠脾脏中光滑念珠菌细胞的基因表达表明，真菌至少遭受了同样严重的营养消耗。即使在没有药物的情况下，中性粒细胞和小鼠脾脏中的多药耐药基因（例如 PDH1）的上调也是光滑念珠菌反应的一个组成部分，并且这以及甾醇转运的增加可能有助于体内唑类耐药。 3)评估管家基因，用于RT-PCR分析光滑念珠菌响应唑类胁迫的基因表达 选择稳定且合适的内参基因进行实时定量PCR（RT-qPCR）是在不同实验条件和环境刺激下进行可靠的基因表达分析的关键前提。本研究旨在通过 RT-qPCR 在氟康唑刺激的光滑念珠菌中鉴定和选择管家基因作为基因表达研究的内部对照。使用2-ΔΔCT方法评估20个管家基因的表达稳定性。我们的数据显示，三种核糖体 RNA 5.8S rRNA、18S rRNA 和 25S rRNA 的 mRNA 表达水平对氟康唑的反应保持稳定，而 PGK1、UBC7 和 UBC13 mRNA 相对稳定，分别仅诱导约 2.3、2.3 和 1.9 倍。相比之下，药物治疗后光滑念珠菌中其他 14 个管家基因（ACT1、亲环蛋白、EF-1α、GAPDH、RPL2A、RPL10、RPL13B、RPP2B、SDHA、TBP、TFRC、α-微管蛋白、β-微管蛋白和 UBC4）的 mRNA 水平显着增加，变化范围为 3.5 至 25 倍。然后，我们使用 ΔΔCT 方法验证了 6 个候选参考基因（5.8S rRNA、18S rRNA、ACT1、PGK1、UBC7 和 UBC13）作为该系统中 10 个目标基因的内源对照的适用性。我们选择评估 5 个 ATP 结合盒转运蛋白基因（CDR1、PDH1、PDR1、SNQ2 和 YOR1）和 5 个甾醇生物合成基因（ERG2、ERG3、ERG4、ERG10 和 ERG11）作为本研究的靶标，因为这些基因已被证明与光滑念珠菌的唑类抗性有关。我们的验证实验通过了所有分析的 6 个对照基因（18S rRNA 除外），其中对数输入 RNA 与目标 ΔCT 的斜率绝对值 >0.1。最后，我们证明了靶基因表达的相对定量根据所使用的内部对照而变化，从而强调了在此类实验中选择内源对照的重要性。 4）传染病培训项目 该培训计划每年继续吸引 4-5 名高素质医生，并得到研究生医学教育成人委员会的充分认可。美国内科医学委员会传染病认证考试的毕业生通过率十年来一直保持在 100%，自 1972 年考试开始以来，通过率高达 95%。