白念珠菌耐药性产生的“线粒体氧化呼吸抑制”机制
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摘要
白念珠菌易于感染、难以防治的主要原因在于其具有“高适应性”的特点,主要表现为高致病性和高耐药性。近年来,白念珠菌适应氧化刺激的机制受到日益关注。本课题前期研究结果提示,白念珠菌线粒体氧化呼吸功能的改变在其适应性耐药性形成过程中可能也发挥着重要作用,即白念珠菌耐药子代的线粒体氧化呼吸功能减弱,代偿性加强细胞质内糖酵解、乙醛酸循环等代谢途径以供能,从而使氟康唑不能有效促进其内源性活性氧升高,使其对氟康唑的敏感性下降。因此,深入研究白念珠菌线粒体呼吸功能与其药物敏感性的关系对进一步阐明白念珠菌耐药机制具有重要意义。
目的从多方面、多层次地阐明我们所提出的“线粒体氧化呼吸抑制”假说:一方面,使用线粒体呼吸链抑制剂阻断或抑制白念珠菌线粒体氧化呼吸,考察菌株对唑类药物的敏感性是否降低。另一方面,通过抑制糖酵解的酶活性,阻断糖酵解代谢通路,或者使用氧化磷酸化解偶联剂,进而增强线粒体氧化呼吸,考察白念珠菌对唑类药物的敏感性是否升高。同时,通过靶向敲除和原位高表达线粒体醛脱氢酶ALD5基因,考察该基因对于白念珠菌线粒体呼吸、对唑类药物敏感性的影响,从而间接证明线粒体呼吸功能在白念珠菌耐药性形成中的作用。此外,考察白念珠菌ERG3、ERG11、TAC1基因耐药相关特异性突变位点;考察白念珠菌新基因ORF19.1510、ORF19.3216、ORF19.5518的耐药相关功能。
方法(一)通过点板实验和抑菌圈实验,分别考察氰化钾和水杨基氧肟酸作用下,白念珠菌对唑类药物敏感性的变化。通过抑菌圈实验,考察白念珠菌交替氧化酶AOX基因缺失菌分别在氰化钾和水杨基氧肟酸作用下,对唑类药物敏感性的变化。通过荧光染色,检测白念珠菌交替氧化酶AOX基因缺失菌在咪康唑或苯菌灵作用下的细胞内活性氧水平。通过棋盘式微量液基稀释法检测氟康唑与水杨基氧肟酸联合用药的抑菌效果。通过抑菌圈实验,分别考察氧化磷酸化解偶联剂、7%乙醇、6-磷酸海藻糖作用下,白念珠菌对唑类药物敏感性的变化。(二)采用Ura-blast策略,构建含有目的基因上、下游同源重组片段的URA3-HisG-URA3敲除盒,醋酸锂法转染白念珠菌,通过两次同源重组,使亲本菌的ALD5基因被敲除盒同源重组,从而构建ALD5基因缺失菌,并通过基因组DNA的套式PCR和southern杂交验证。构建ALD5基因高表达质粒,将ALD5基因完整开放阅读框置于pCaEXP高表达载体中MET3启动子的控制下;醋酸锂法转染白念珠菌ALD5基因缺失菌,PCR鉴定ALD5基因的原位整合,实时定量PCR筛选ALD5基因表达量明显升高的菌株。利用微量液基稀释法和点板法考察ALD5基因缺失菌在不同碳源培养基上对唑类药物的敏感性是否改变;通过流式细胞仪检测ALD5基因敲除菌的细胞周期;通过荧光显微镜检测ALD5基因敲除菌的细胞膜、细胞核形态;分别使用不同培养条件,考察ALD5基因敲除菌的菌丝形成能力和生物膜形成能力;与哺乳动物巨噬细胞共孵育,考察ALD5基因敲除菌对巨噬细胞的敏感性;通过荧光染料检测ALD5基因敲除菌的线粒体膜电位和内源性活性氧水平。(三)以白念珠菌敏感亲本y0109S和耐药子代y0109R基因组DNA为模板,PCR扩增ERG3、ERG11、TAC1基因全长开放阅读框,并与白念珠菌基因组数据库比对,分析基因突变位点。(四)分别构建含有目的基因ORF19.1510上、下游同源重组片段的HIS1、LEU2敲除盒,醋酸锂法转染白念珠菌,通过两次同源重组,使亲本菌的ORF19.1510基因被敲除盒同源重组,从而构建ORF19.1510基因插入失活缺失菌,并通过基因组DNA套式PCR验证。按照同样方法,分别构建ORF19.3216、ORF19.5518基因插入失活缺失菌。通过荧光显微镜分别检测ORF19.1510、ORF19.3216、ORF19.5518基因敲除菌的细胞膜、细胞核形态;分别使用不同培养条件,考察ORF19.1510基因敲除菌的菌丝形成能力;利用微量液基稀释法和点板法分别考察ORF19.1510、ORF19.3216、ORF19.5518基因缺失菌对唑类药物、盐刺激、渗透刺激、过氧化氢刺激、DNA抑制剂的敏感性是否改变;通过流式细胞仪分别检测ORF19.1510、ORF19.3216、ORF19.5518基因敲除菌的细胞周期。
结果(一)氰化钾作用下,含唑类药物培养基上存活菌落数量增加、含唑类药物纸片周围抑菌圈明显缩小,即菌株对唑类药物的敏感性降低。水杨基氧肟酸作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。白念珠菌交替氧化酶AOX1b基因缺失菌在氰化钾作用下,含唑类药物纸片周围抑菌圈缩小,但是缩小程度弱于亲本菌和AOX1a基因缺失菌。咪康唑或苯菌灵作用下,AOX基因缺失菌的细胞内活性氧升高程度明显高于亲本菌,而AOX基因高表达菌的细胞内活性氧升高程度明显低于亲本菌。水杨基氧肟酸与氟康唑体外联合使用,能明显降低氟康唑的最低抑菌浓度,对白念珠菌特别是耐药菌株,产生明显协同抑菌作用。氧化磷酸化解偶联剂作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。7%乙醇作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。6-磷酸海藻糖作用下,含唑类药物纸片周围抑菌圈无明显变化,即菌株对唑类药物的敏感性未改变。(二)分别通过PCR扩增、酶切、测序验证ALD5基因敲除盒构建正确;通过基因组DNA套式PCR和Southern杂交验证ALD5基因一条等位基因敲除菌、两条等位基因敲除菌构建正确。通过酶切和测序验证ALD5基因高表达质粒构建正确;通过PCR验证ALD5基因高表达菌株构建正确,并通过实时定量PCR筛选得到一株ALD5基因表达量相对较高的菌株。在非发酵碳源的培养基内,ALD5基因敲除菌的生长倍增时间延长;ALD5基因敲除菌呈椭圆球形,以一端或两端极化出芽生长;在液体和固体培养条件下,ALD5基因敲除菌的菌丝形成能力与亲本菌相当,生物膜形成能力略增强;ALD5基因敲除菌在巨噬细胞存在时的存活率与亲本菌相当;ALD5基因敲除菌的线粒体膜电位水平与亲本菌无明显差别;在含有过氧化氢、氯化钠、山梨醇的培养基上,ALD5基因敲除菌的存活菌落数量与亲本菌相当。随着孵育时间的延长,ALD5基因敲除菌对唑类药物的敏感性明显降低;在乙醇、乙酸等非发酵碳源培养基上,ALD5基因敲除菌对唑类药物的敏感性略降低;咪康唑作用下,ALD5基因敲除菌细胞内活性氧升高程度明显低于亲本菌。(三)敏感亲本y0109S和耐药子代y0109R的ERG11基因均无点突变;耐药子代y0109R的ERG3基因存在D19E突变;耐药子代y0109R的TAC1基因存在L47K和N977K突变。(四)通过融合PCR扩增得到含有目的基因ORF19.1510上下游同源重组片段和标记HIS1、LEU2的敲除盒,通过基因组DNA套式PCR验证ORF19.1510基因一条等位基因敲除菌、两条等位基因敲除菌构建正确。按照同样方法和原理,成功构建ORF19.3216、ORF19.5518基因敲除菌。与亲本菌相比,ORF19.1510基因缺失菌在完全培养基和极限培养基中对数生长期的倍增时间无明显变化;ORF19.1510基因缺失菌单个细胞增大,可从细胞表面多个方位不规则出芽生长;ORF19.1510基因缺失菌在各种诱导条件下菌丝形成能力明显降低;ORF19.1510基因缺失菌对唑类药物、氯化钠的敏感性明显升高;ORF19.1510基因缺失菌对过氧化氢、山梨醇的敏感性无明显变化;ORF19.1510基因缺失菌对DNA单链抑制剂甲磺酸甲酯、羟基脲的敏感性明显降低,对DNA双链抑制剂腐草霉素的敏感性升高;ORF19.1510基因缺失菌的细胞周期在G2/S期转变发生阻滞。与亲本菌相比,ORF19.3216、ORF19.5518基因缺失菌形态、菌丝形成能力、细胞周期、对唑类药物、盐刺激、过氧化氢刺激、渗透刺激的敏感性等表型均无明显变化。
结论(一)白念珠菌经典氧化呼吸通路(细胞色素途径)受到抑制或阻断,转而使用交替氧化为主要电子传递通路时,可明显降低菌株对唑类药物的敏感性;使用氧肟酸化合物抑制白念珠菌交替氧化呼吸通路,可明显升高菌株对唑类药物的敏感性。白念珠菌在唑类药物作用下,其交替氧化酶可被药物作用后升高的内源性活性氧所诱导高表达,从而发挥电子分流作用,在一定程度上减少真菌细胞内活性氧水平,削弱药物的抑菌作用,进而降低菌株对唑类药物的敏感性。这是白念珠菌耐药性产生的新机制之一。白念珠菌根据生存环境变化及时调节电子在细胞色素途径和交替呼吸途径的流量,并减少活性氧产生,是其高适应性的表现之一。
(二)白念珠菌醛脱氢酶Ald5p主要参与细胞内乙醛代谢,不改变细胞周期进展,有助于维持真菌细胞在非发酵碳源环境中的生存。CaAld5p有助于维持白念珠菌细胞呈圆球形形态,略降低菌株的生物被膜形成能力;但是不改变细胞以一端或两端为主的极化出芽生长方式,不改变酵母态细胞向菌丝态细胞的转变,不改变菌株对哺乳动物巨噬细胞的敏感性,也不改变白念珠菌对外源性氧化刺激、盐刺激、渗透刺激的敏感性。白念珠菌Ald5p不影响其线粒体膜电位水平,但是,可以增加将线粒体内乙醛转变为乙酸时产生的还原态NADPH,从而增加细胞内活性氧水平,增加菌株对唑类药物的敏感性。
(三)白念珠菌TAC1基因可因N977K突变而使转录因子Tac1p活化,并调控其靶基因CDR1、CDR2、PDR17等高表达,进而表现出对唑类药物敏感性降低。白念珠菌ERG3基因可因D19E突变而使Erg3p功能受到抑制,并改变白念珠菌甾醇代谢通路,麦角甾醇含量减少,中间代谢产物和麦角甾醇替代物含量增加,进而表现出对唑类药物敏感性降低。
(四)白念珠菌新基因ORF19.1510有助于维持菌株的细胞周期G2/S期转变,有助于白念珠菌由酵母态向菌丝态的转变;可明显降低菌株对唑类药物、盐刺激、DNA双链抑制剂的敏感性,明显升高菌株对DNA单链抑制剂的敏感性。白念珠菌新基因ORF19.1510、ORF19.3216、ORF19.5518的功能有待进一步研究。
More recently, it has been widely accepted that Candida albicans can exploit several cellular responses to facilitate tolerance of antifungal agents and can further acquire resistance by multiple mechanisms. The response to oxidative stress is considered to contribute significantly to modulation of fluconazole (FLC) tolerance in C. albicans in response to antifungal stress. We previously presented evidence that a protective metabolic shift due to mitochondrial respiration deficiency may enable the FLC-resistant C. albicans strain to survive drug challenge partly due to a reduction in generation of intracellular reactive oxygen species (ROS). As a result, it is expected that mitochondrial functions that regulate metabolic behaviour are likely to contribute to fitness and flexibility of C. albicans strains in response to external challenges.
Goal To explore the antifungal susceptibility of C. albicans under conditions of mitochondrial respiration blockages and mitochondrial respiration enhancers. To investigate the function of Ald5 in C. albicans, a potential mitochondrial-function-related protein probably encoding an aldehyde dehydrogenase. To identify the potential point mutations in ERG3, ERG11, and TAC1 genes in the matched pair of FLC-resistant and FLC-sensitive C. albicans strains. To characterize and identify the unigenes of ORF19.1510, ORF19.3216, and ORF19.5518 in C. albicans.
Methods (Ⅰ) In order to investigate whether cyanide or SHAM would affect drug susceptibility in C. albicans, we used spot and filter disk assays to test C. albicans both clinical and laboratory strains in the presence of cyanide or SHAM. To evaluate possible differences in azole tolerance conferred by Aox1a and Aox1b activity, we compared azole sensitivities of four strains - the WT strain, the isogenetic aox1a/aox1a mutant strain, the aox1b/aox1b mutant strain, and the aox1a/aox1a aox1b/aox1b double mutant strain - by the filter disk method with the treatment of cyanide or SHAM. Alteration of ROS production as measured through DCF fluorescence over time was measured for these four strains. We explored the in vitro combination use of FLC and SHAM against clinical C. albicans isolates by using filter disk and microtiter plates methods. (Ⅱ) To investigate the function of ALD5, we constructed the ald5 null mutant using wild-type strain CAI4. Replacements of the ALD5 alleles with linear disruption fragments were monitored by PCR and southern-blotting with genomic DNA. Then, the susceptibility testing, endogenous ROS production, hyphae induction, biofilm induction, and the interaction with macrophage cell assays were investigated with the wild-type and ald5 null mutant. (Ⅲ) Point mutations in ERG3, ERG11, and TAC1 genes were further examined in the matched pair of C. albicans strains, y0109S and y0109R. (Ⅳ) We performed gene disruption of ORF19.1510, ORF19.3216 and ORF19.5518 in C. albicans using fusion PCR and heterologous markers. First, two pairs of primers were used to amplify genomic DNA on the 5’side and 3’side of the target gene separately. Another pair of primers was used to amplify the selectable markers C. dubliniensis HIS1 and C. maltose LEU2 separately. The first round of gene disruption was carried out with C. dubliniensis HIS1, and the second round was carried out with C. maltose LEU2. After selection of transformants on the appropriate single or double amino acid dropout medium, gene disruption candidates were screened by PCR for expected 5’and 3’junctions as well as the size of the disrupted gene. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. Then, morphosis, the susceptibility testing, and the cell cycle analysis were investigated with the wild-type and the target genes null mutants.
Results (Ⅰ) The addition of 1 mM cyanide to the culture medium resulted in decreased susceptibility to FLC, itroconazole (ITC), ketoconazole (KTC), and miconazole (MCZ) for both FLC-resistant and -sensitive C. albicans strains. The drug sensitivity was further confirmed for these four strains by the filter disk method, in which the diameters of the inhibition zones of FLC, ITC, KTC, and MCZ disks were significantly diminished in the presence of 1 mM cyanide. Meanwhile, all four strains were strikingly susceptible to the four azoles tested in the presence of 5 mM SHAM, with larger inhibition zones than those in the absence of SHAM. Furthermore, with the addition of 1 mM cyanide, the wild-type and the isogenetic aox1a/aox1a mutant strain strains with the induction of the Aox1b showed hyper-tolerance to all azoles tested, whereas the degree of reduced sensitivity to azole antifungals for aox1b/aox1b mutant strain with the Aox1a induction was less evident. With the treatment of the ROS-inducing agents, MCZ and benomyl, ROS generation was augmented in the aox1a/aox1a mutant, aox1b/aox1b mutant, and aox1a-aox1b/aox1a-aox1b mutant, to a greater degree than in the wild-type strain. The addition of SHAM remarkably reduced the MIC80s of FLC, as was evident from the checkboard analysis and filter disk assay. FLC MIC80s against resistant isolates decreased more than 128 fold, and the MIC80s against susceptible isolates decreased from 16 to 25 fold. Among the 20 clinical isolates, synergy between FLC and SHAM was observed in 17 (85%), and indifference was observed in 3 (15%) susceptible isolates. (Ⅱ) We successfully constructed the ald5 null mutant in C. albicans. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in cells. The results showed that the ald5 null mutant has no significant difference in forming hyphae in comparison to the wild-type strain. The susceptibility of the ald5 null mutant to macrophage cells, hydrogen peroxide, salt, and osmotic stresses was similar to it of the wild-type strain. In the non-fermentation medium, the ald5 null mutant grew slowly, while had no cell cycle arrest. The ald5 null mutant showed decreased susceptibility to azoles in a certain degree in the non-fermentation medium. (Ⅲ) For the strains y0109S and y0109R, the only one missense mutation in ERG3 gene was the single amino acid substitution D19E. Meanwhile, both strains harbored one nucleotide change at base position 304 (ACC) in ERG3 which had no effect in the amino acid sequence. There were no mutations in TAC1 in the strain y0109S. In contrast, TAC1 gene from the strain y0109R was showed a total of two missense mutations (L47K and N977K) in comparison with the published sequence dataset. In addition, there was no amino acid difference in Erg11 in these two strains. (Ⅳ) We performed the disruption of ORF19.1510, ORF19.3216, and ORF19.5518 using a two-step fusion PCR and heterologous markers. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. The orf19.1510 null mutant showed a changed budding pattern: in the middle-exponential phase, a small proportion of the orf19.1510 null mutant was unusually enlarged, and with random budding scars; in the stationary phase, most of the orf19.1510 null mutant cells were the same size as the wild-type strain, but there were still a proportion of cells that were enlarged, round, and with random budding scars. The orf19.1510 null mutant had a small effect on germ tube formation in YPD containing 10% FBS liquid culture at 37℃, and no hyphal growth was seen in spider or Lee’s medium, either on solid or in liquid culture at 37℃. The orf19.1510 null mutant was sensitive to azoles, sodium chloride, and double-strand DNA damage agent, phleomycin (PHL), but was resistant to single-strand DNA damage agents, methyl methane sulfonate (MMS), and hydroxyurea (HU). The orf19.1510 null mutant has the same sensitivity to peroxide oxygen and sorbitol. The DNA contents of many more single cells of the orf19.1510 null mutant were in 4N (G2) phase in the early-, middle-log growth phase. The DNA contents of a majority of single cells of the orf19.1510 null mutant in the stationary phase were in between the 2N (G1) and 4N (G2) phases. The ORF19.3216 and ORF19.5518 null mutants showed no difference in the phenotypes in our study.
Conclusion (Ⅰ) Our present study indicated that alternative, cyanide-insensitive, respiration in C. albicans leads to azole tolerance through the reduction of intracellular ROS production. This is a novel mechanism contributing to decreased FLC susceptibility and increased survival of C. albicans. We showed in this study that cyanide treatment, with the resulting induction of the alternative pathway in C. albicans, caused reduced susceptibility to antifungal azoles, and that the inhibition of alternative respiration by SHAM resulted in enhanced susceptibility to azoles. By testing the aox mutant C. albicans strains, we identified that Aox1b played a major role in conferring azole tolerance. Our analysis of intracellular ROS formation further proved that the Aox in C. albicans lowered mitochondrial ROS levels upon antifungal drug treatment. These findings provide a novel mechanistic insight into FLC tolerance in C. albicans and explain how the alternative oxidative pathway might regulate electron flux and mitochondrial respiration and thereby generate a survival advantage during antifungal stress.
(Ⅱ) We successfully constructed the ald5 null mutant in C. albicans and showed that the deletion of ald5 decreased susceptibility of C. albicans to azoles in a certain degree in the non-fermentation medium. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in C. albicans cells.
(Ⅲ) We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hyperactivity of Tac1 could up-regulate CDR1, CDR2, and PDR17 in C. albicans and result in resistance to azole. A single amino acid difference (D19E) in ERG3 that led to inactivation of Erg3 could accumulate sterol precursors along with changes in expression of ergosterol biosynthesis genes, which could contribute to azoles resistance in C. albicans.
(Ⅳ) We successfully constructed the orf19.1510 null mutant, orf19.3216 null mutant, and orf19.5518 null mutant of C. albicans. The results showed that the deletion of ORF19.1510 significantly increased susceptibility of C. albicans to azoles, sodium chloride, and double-strand DNA damage agent (PHL), but decreased sensitivity to single-strand DNA damage agents (MMS and HU). The deletion of ORF19.1510 was deficient in the yeast to hyphal switch. We proposed that the primary effect of ORF19.1510 depletion is accumulation of unrepaired DNA damage and the subsequent activation of the DNA-damage checkpoint. One of the activated molecules triggers the phenotypes. The deletion of ORF19.3216 and ORF19.5518 had no effect in the phenotypes in current study. Further experimental investigations are needed.
目的从多方面、多层次地阐明我们所提出的“线粒体氧化呼吸抑制”假说:一方面,使用线粒体呼吸链抑制剂阻断或抑制白念珠菌线粒体氧化呼吸,考察菌株对唑类药物的敏感性是否降低。另一方面,通过抑制糖酵解的酶活性,阻断糖酵解代谢通路,或者使用氧化磷酸化解偶联剂,进而增强线粒体氧化呼吸,考察白念珠菌对唑类药物的敏感性是否升高。同时,通过靶向敲除和原位高表达线粒体醛脱氢酶ALD5基因,考察该基因对于白念珠菌线粒体呼吸、对唑类药物敏感性的影响,从而间接证明线粒体呼吸功能在白念珠菌耐药性形成中的作用。此外,考察白念珠菌ERG3、ERG11、TAC1基因耐药相关特异性突变位点;考察白念珠菌新基因ORF19.1510、ORF19.3216、ORF19.5518的耐药相关功能。
方法(一)通过点板实验和抑菌圈实验,分别考察氰化钾和水杨基氧肟酸作用下,白念珠菌对唑类药物敏感性的变化。通过抑菌圈实验,考察白念珠菌交替氧化酶AOX基因缺失菌分别在氰化钾和水杨基氧肟酸作用下,对唑类药物敏感性的变化。通过荧光染色,检测白念珠菌交替氧化酶AOX基因缺失菌在咪康唑或苯菌灵作用下的细胞内活性氧水平。通过棋盘式微量液基稀释法检测氟康唑与水杨基氧肟酸联合用药的抑菌效果。通过抑菌圈实验,分别考察氧化磷酸化解偶联剂、7%乙醇、6-磷酸海藻糖作用下,白念珠菌对唑类药物敏感性的变化。(二)采用Ura-blast策略,构建含有目的基因上、下游同源重组片段的URA3-HisG-URA3敲除盒,醋酸锂法转染白念珠菌,通过两次同源重组,使亲本菌的ALD5基因被敲除盒同源重组,从而构建ALD5基因缺失菌,并通过基因组DNA的套式PCR和southern杂交验证。构建ALD5基因高表达质粒,将ALD5基因完整开放阅读框置于pCaEXP高表达载体中MET3启动子的控制下;醋酸锂法转染白念珠菌ALD5基因缺失菌,PCR鉴定ALD5基因的原位整合,实时定量PCR筛选ALD5基因表达量明显升高的菌株。利用微量液基稀释法和点板法考察ALD5基因缺失菌在不同碳源培养基上对唑类药物的敏感性是否改变;通过流式细胞仪检测ALD5基因敲除菌的细胞周期;通过荧光显微镜检测ALD5基因敲除菌的细胞膜、细胞核形态;分别使用不同培养条件,考察ALD5基因敲除菌的菌丝形成能力和生物膜形成能力;与哺乳动物巨噬细胞共孵育,考察ALD5基因敲除菌对巨噬细胞的敏感性;通过荧光染料检测ALD5基因敲除菌的线粒体膜电位和内源性活性氧水平。(三)以白念珠菌敏感亲本y0109S和耐药子代y0109R基因组DNA为模板,PCR扩增ERG3、ERG11、TAC1基因全长开放阅读框,并与白念珠菌基因组数据库比对,分析基因突变位点。(四)分别构建含有目的基因ORF19.1510上、下游同源重组片段的HIS1、LEU2敲除盒,醋酸锂法转染白念珠菌,通过两次同源重组,使亲本菌的ORF19.1510基因被敲除盒同源重组,从而构建ORF19.1510基因插入失活缺失菌,并通过基因组DNA套式PCR验证。按照同样方法,分别构建ORF19.3216、ORF19.5518基因插入失活缺失菌。通过荧光显微镜分别检测ORF19.1510、ORF19.3216、ORF19.5518基因敲除菌的细胞膜、细胞核形态;分别使用不同培养条件,考察ORF19.1510基因敲除菌的菌丝形成能力;利用微量液基稀释法和点板法分别考察ORF19.1510、ORF19.3216、ORF19.5518基因缺失菌对唑类药物、盐刺激、渗透刺激、过氧化氢刺激、DNA抑制剂的敏感性是否改变;通过流式细胞仪分别检测ORF19.1510、ORF19.3216、ORF19.5518基因敲除菌的细胞周期。
结果(一)氰化钾作用下,含唑类药物培养基上存活菌落数量增加、含唑类药物纸片周围抑菌圈明显缩小,即菌株对唑类药物的敏感性降低。水杨基氧肟酸作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。白念珠菌交替氧化酶AOX1b基因缺失菌在氰化钾作用下,含唑类药物纸片周围抑菌圈缩小,但是缩小程度弱于亲本菌和AOX1a基因缺失菌。咪康唑或苯菌灵作用下,AOX基因缺失菌的细胞内活性氧升高程度明显高于亲本菌,而AOX基因高表达菌的细胞内活性氧升高程度明显低于亲本菌。水杨基氧肟酸与氟康唑体外联合使用,能明显降低氟康唑的最低抑菌浓度,对白念珠菌特别是耐药菌株,产生明显协同抑菌作用。氧化磷酸化解偶联剂作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。7%乙醇作用下,含唑类药物纸片周围抑菌圈明显扩大,即菌株对唑类药物的敏感性升高。6-磷酸海藻糖作用下,含唑类药物纸片周围抑菌圈无明显变化,即菌株对唑类药物的敏感性未改变。(二)分别通过PCR扩增、酶切、测序验证ALD5基因敲除盒构建正确;通过基因组DNA套式PCR和Southern杂交验证ALD5基因一条等位基因敲除菌、两条等位基因敲除菌构建正确。通过酶切和测序验证ALD5基因高表达质粒构建正确;通过PCR验证ALD5基因高表达菌株构建正确,并通过实时定量PCR筛选得到一株ALD5基因表达量相对较高的菌株。在非发酵碳源的培养基内,ALD5基因敲除菌的生长倍增时间延长;ALD5基因敲除菌呈椭圆球形,以一端或两端极化出芽生长;在液体和固体培养条件下,ALD5基因敲除菌的菌丝形成能力与亲本菌相当,生物膜形成能力略增强;ALD5基因敲除菌在巨噬细胞存在时的存活率与亲本菌相当;ALD5基因敲除菌的线粒体膜电位水平与亲本菌无明显差别;在含有过氧化氢、氯化钠、山梨醇的培养基上,ALD5基因敲除菌的存活菌落数量与亲本菌相当。随着孵育时间的延长,ALD5基因敲除菌对唑类药物的敏感性明显降低;在乙醇、乙酸等非发酵碳源培养基上,ALD5基因敲除菌对唑类药物的敏感性略降低;咪康唑作用下,ALD5基因敲除菌细胞内活性氧升高程度明显低于亲本菌。(三)敏感亲本y0109S和耐药子代y0109R的ERG11基因均无点突变;耐药子代y0109R的ERG3基因存在D19E突变;耐药子代y0109R的TAC1基因存在L47K和N977K突变。(四)通过融合PCR扩增得到含有目的基因ORF19.1510上下游同源重组片段和标记HIS1、LEU2的敲除盒,通过基因组DNA套式PCR验证ORF19.1510基因一条等位基因敲除菌、两条等位基因敲除菌构建正确。按照同样方法和原理,成功构建ORF19.3216、ORF19.5518基因敲除菌。与亲本菌相比,ORF19.1510基因缺失菌在完全培养基和极限培养基中对数生长期的倍增时间无明显变化;ORF19.1510基因缺失菌单个细胞增大,可从细胞表面多个方位不规则出芽生长;ORF19.1510基因缺失菌在各种诱导条件下菌丝形成能力明显降低;ORF19.1510基因缺失菌对唑类药物、氯化钠的敏感性明显升高;ORF19.1510基因缺失菌对过氧化氢、山梨醇的敏感性无明显变化;ORF19.1510基因缺失菌对DNA单链抑制剂甲磺酸甲酯、羟基脲的敏感性明显降低,对DNA双链抑制剂腐草霉素的敏感性升高;ORF19.1510基因缺失菌的细胞周期在G2/S期转变发生阻滞。与亲本菌相比,ORF19.3216、ORF19.5518基因缺失菌形态、菌丝形成能力、细胞周期、对唑类药物、盐刺激、过氧化氢刺激、渗透刺激的敏感性等表型均无明显变化。
结论(一)白念珠菌经典氧化呼吸通路(细胞色素途径)受到抑制或阻断,转而使用交替氧化为主要电子传递通路时,可明显降低菌株对唑类药物的敏感性;使用氧肟酸化合物抑制白念珠菌交替氧化呼吸通路,可明显升高菌株对唑类药物的敏感性。白念珠菌在唑类药物作用下,其交替氧化酶可被药物作用后升高的内源性活性氧所诱导高表达,从而发挥电子分流作用,在一定程度上减少真菌细胞内活性氧水平,削弱药物的抑菌作用,进而降低菌株对唑类药物的敏感性。这是白念珠菌耐药性产生的新机制之一。白念珠菌根据生存环境变化及时调节电子在细胞色素途径和交替呼吸途径的流量,并减少活性氧产生,是其高适应性的表现之一。
(二)白念珠菌醛脱氢酶Ald5p主要参与细胞内乙醛代谢,不改变细胞周期进展,有助于维持真菌细胞在非发酵碳源环境中的生存。CaAld5p有助于维持白念珠菌细胞呈圆球形形态,略降低菌株的生物被膜形成能力;但是不改变细胞以一端或两端为主的极化出芽生长方式,不改变酵母态细胞向菌丝态细胞的转变,不改变菌株对哺乳动物巨噬细胞的敏感性,也不改变白念珠菌对外源性氧化刺激、盐刺激、渗透刺激的敏感性。白念珠菌Ald5p不影响其线粒体膜电位水平,但是,可以增加将线粒体内乙醛转变为乙酸时产生的还原态NADPH,从而增加细胞内活性氧水平,增加菌株对唑类药物的敏感性。
(三)白念珠菌TAC1基因可因N977K突变而使转录因子Tac1p活化,并调控其靶基因CDR1、CDR2、PDR17等高表达,进而表现出对唑类药物敏感性降低。白念珠菌ERG3基因可因D19E突变而使Erg3p功能受到抑制,并改变白念珠菌甾醇代谢通路,麦角甾醇含量减少,中间代谢产物和麦角甾醇替代物含量增加,进而表现出对唑类药物敏感性降低。
(四)白念珠菌新基因ORF19.1510有助于维持菌株的细胞周期G2/S期转变,有助于白念珠菌由酵母态向菌丝态的转变;可明显降低菌株对唑类药物、盐刺激、DNA双链抑制剂的敏感性,明显升高菌株对DNA单链抑制剂的敏感性。白念珠菌新基因ORF19.1510、ORF19.3216、ORF19.5518的功能有待进一步研究。
More recently, it has been widely accepted that Candida albicans can exploit several cellular responses to facilitate tolerance of antifungal agents and can further acquire resistance by multiple mechanisms. The response to oxidative stress is considered to contribute significantly to modulation of fluconazole (FLC) tolerance in C. albicans in response to antifungal stress. We previously presented evidence that a protective metabolic shift due to mitochondrial respiration deficiency may enable the FLC-resistant C. albicans strain to survive drug challenge partly due to a reduction in generation of intracellular reactive oxygen species (ROS). As a result, it is expected that mitochondrial functions that regulate metabolic behaviour are likely to contribute to fitness and flexibility of C. albicans strains in response to external challenges.
Goal To explore the antifungal susceptibility of C. albicans under conditions of mitochondrial respiration blockages and mitochondrial respiration enhancers. To investigate the function of Ald5 in C. albicans, a potential mitochondrial-function-related protein probably encoding an aldehyde dehydrogenase. To identify the potential point mutations in ERG3, ERG11, and TAC1 genes in the matched pair of FLC-resistant and FLC-sensitive C. albicans strains. To characterize and identify the unigenes of ORF19.1510, ORF19.3216, and ORF19.5518 in C. albicans.
Methods (Ⅰ) In order to investigate whether cyanide or SHAM would affect drug susceptibility in C. albicans, we used spot and filter disk assays to test C. albicans both clinical and laboratory strains in the presence of cyanide or SHAM. To evaluate possible differences in azole tolerance conferred by Aox1a and Aox1b activity, we compared azole sensitivities of four strains - the WT strain, the isogenetic aox1a/aox1a mutant strain, the aox1b/aox1b mutant strain, and the aox1a/aox1a aox1b/aox1b double mutant strain - by the filter disk method with the treatment of cyanide or SHAM. Alteration of ROS production as measured through DCF fluorescence over time was measured for these four strains. We explored the in vitro combination use of FLC and SHAM against clinical C. albicans isolates by using filter disk and microtiter plates methods. (Ⅱ) To investigate the function of ALD5, we constructed the ald5 null mutant using wild-type strain CAI4. Replacements of the ALD5 alleles with linear disruption fragments were monitored by PCR and southern-blotting with genomic DNA. Then, the susceptibility testing, endogenous ROS production, hyphae induction, biofilm induction, and the interaction with macrophage cell assays were investigated with the wild-type and ald5 null mutant. (Ⅲ) Point mutations in ERG3, ERG11, and TAC1 genes were further examined in the matched pair of C. albicans strains, y0109S and y0109R. (Ⅳ) We performed gene disruption of ORF19.1510, ORF19.3216 and ORF19.5518 in C. albicans using fusion PCR and heterologous markers. First, two pairs of primers were used to amplify genomic DNA on the 5’side and 3’side of the target gene separately. Another pair of primers was used to amplify the selectable markers C. dubliniensis HIS1 and C. maltose LEU2 separately. The first round of gene disruption was carried out with C. dubliniensis HIS1, and the second round was carried out with C. maltose LEU2. After selection of transformants on the appropriate single or double amino acid dropout medium, gene disruption candidates were screened by PCR for expected 5’and 3’junctions as well as the size of the disrupted gene. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. Then, morphosis, the susceptibility testing, and the cell cycle analysis were investigated with the wild-type and the target genes null mutants.
Results (Ⅰ) The addition of 1 mM cyanide to the culture medium resulted in decreased susceptibility to FLC, itroconazole (ITC), ketoconazole (KTC), and miconazole (MCZ) for both FLC-resistant and -sensitive C. albicans strains. The drug sensitivity was further confirmed for these four strains by the filter disk method, in which the diameters of the inhibition zones of FLC, ITC, KTC, and MCZ disks were significantly diminished in the presence of 1 mM cyanide. Meanwhile, all four strains were strikingly susceptible to the four azoles tested in the presence of 5 mM SHAM, with larger inhibition zones than those in the absence of SHAM. Furthermore, with the addition of 1 mM cyanide, the wild-type and the isogenetic aox1a/aox1a mutant strain strains with the induction of the Aox1b showed hyper-tolerance to all azoles tested, whereas the degree of reduced sensitivity to azole antifungals for aox1b/aox1b mutant strain with the Aox1a induction was less evident. With the treatment of the ROS-inducing agents, MCZ and benomyl, ROS generation was augmented in the aox1a/aox1a mutant, aox1b/aox1b mutant, and aox1a-aox1b/aox1a-aox1b mutant, to a greater degree than in the wild-type strain. The addition of SHAM remarkably reduced the MIC80s of FLC, as was evident from the checkboard analysis and filter disk assay. FLC MIC80s against resistant isolates decreased more than 128 fold, and the MIC80s against susceptible isolates decreased from 16 to 25 fold. Among the 20 clinical isolates, synergy between FLC and SHAM was observed in 17 (85%), and indifference was observed in 3 (15%) susceptible isolates. (Ⅱ) We successfully constructed the ald5 null mutant in C. albicans. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in cells. The results showed that the ald5 null mutant has no significant difference in forming hyphae in comparison to the wild-type strain. The susceptibility of the ald5 null mutant to macrophage cells, hydrogen peroxide, salt, and osmotic stresses was similar to it of the wild-type strain. In the non-fermentation medium, the ald5 null mutant grew slowly, while had no cell cycle arrest. The ald5 null mutant showed decreased susceptibility to azoles in a certain degree in the non-fermentation medium. (Ⅲ) For the strains y0109S and y0109R, the only one missense mutation in ERG3 gene was the single amino acid substitution D19E. Meanwhile, both strains harbored one nucleotide change at base position 304 (ACC) in ERG3 which had no effect in the amino acid sequence. There were no mutations in TAC1 in the strain y0109S. In contrast, TAC1 gene from the strain y0109R was showed a total of two missense mutations (L47K and N977K) in comparison with the published sequence dataset. In addition, there was no amino acid difference in Erg11 in these two strains. (Ⅳ) We performed the disruption of ORF19.1510, ORF19.3216, and ORF19.5518 using a two-step fusion PCR and heterologous markers. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. The orf19.1510 null mutant showed a changed budding pattern: in the middle-exponential phase, a small proportion of the orf19.1510 null mutant was unusually enlarged, and with random budding scars; in the stationary phase, most of the orf19.1510 null mutant cells were the same size as the wild-type strain, but there were still a proportion of cells that were enlarged, round, and with random budding scars. The orf19.1510 null mutant had a small effect on germ tube formation in YPD containing 10% FBS liquid culture at 37℃, and no hyphal growth was seen in spider or Lee’s medium, either on solid or in liquid culture at 37℃. The orf19.1510 null mutant was sensitive to azoles, sodium chloride, and double-strand DNA damage agent, phleomycin (PHL), but was resistant to single-strand DNA damage agents, methyl methane sulfonate (MMS), and hydroxyurea (HU). The orf19.1510 null mutant has the same sensitivity to peroxide oxygen and sorbitol. The DNA contents of many more single cells of the orf19.1510 null mutant were in 4N (G2) phase in the early-, middle-log growth phase. The DNA contents of a majority of single cells of the orf19.1510 null mutant in the stationary phase were in between the 2N (G1) and 4N (G2) phases. The ORF19.3216 and ORF19.5518 null mutants showed no difference in the phenotypes in our study.
Conclusion (Ⅰ) Our present study indicated that alternative, cyanide-insensitive, respiration in C. albicans leads to azole tolerance through the reduction of intracellular ROS production. This is a novel mechanism contributing to decreased FLC susceptibility and increased survival of C. albicans. We showed in this study that cyanide treatment, with the resulting induction of the alternative pathway in C. albicans, caused reduced susceptibility to antifungal azoles, and that the inhibition of alternative respiration by SHAM resulted in enhanced susceptibility to azoles. By testing the aox mutant C. albicans strains, we identified that Aox1b played a major role in conferring azole tolerance. Our analysis of intracellular ROS formation further proved that the Aox in C. albicans lowered mitochondrial ROS levels upon antifungal drug treatment. These findings provide a novel mechanistic insight into FLC tolerance in C. albicans and explain how the alternative oxidative pathway might regulate electron flux and mitochondrial respiration and thereby generate a survival advantage during antifungal stress.
(Ⅱ) We successfully constructed the ald5 null mutant in C. albicans and showed that the deletion of ald5 decreased susceptibility of C. albicans to azoles in a certain degree in the non-fermentation medium. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in C. albicans cells.
(Ⅲ) We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hyperactivity of Tac1 could up-regulate CDR1, CDR2, and PDR17 in C. albicans and result in resistance to azole. A single amino acid difference (D19E) in ERG3 that led to inactivation of Erg3 could accumulate sterol precursors along with changes in expression of ergosterol biosynthesis genes, which could contribute to azoles resistance in C. albicans.
(Ⅳ) We successfully constructed the orf19.1510 null mutant, orf19.3216 null mutant, and orf19.5518 null mutant of C. albicans. The results showed that the deletion of ORF19.1510 significantly increased susceptibility of C. albicans to azoles, sodium chloride, and double-strand DNA damage agent (PHL), but decreased sensitivity to single-strand DNA damage agents (MMS and HU). The deletion of ORF19.1510 was deficient in the yeast to hyphal switch. We proposed that the primary effect of ORF19.1510 depletion is accumulation of unrepaired DNA damage and the subsequent activation of the DNA-damage checkpoint. One of the activated molecules triggers the phenotypes. The deletion of ORF19.3216 and ORF19.5518 had no effect in the phenotypes in current study. Further experimental investigations are needed.
引文
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[2] Snydman DR. Shifting patterns in the epidemiology of nosocomial Candida infections [J]. Chest. 2003, 123(5 Suppl):500S.
[3]张秀珍. 1986-2003年深部真菌感染及耐药趋势.抗真菌药物与真菌感染诊治研究学术会议论文集. 2003年12月厦门:67-71.
[4]吴绍熙等.中国致病真菌10年动态流行病学研究.临床皮肤科杂志. 1999, 28(1):1-44.
[5] Odds FC, Brown AJ, Gow NA. Antifungal agents: mechanisms of action [J]. Trends Microbiol. 2003, 11(6):272-279.
[6] Chen D, Shan B, Yan PH. Analyses of 1225 cases of nosocomial eumycophyta infection [J]. Clin J Lab Med. 2005, 28(4):387-388.
[7] Cannon RD, Lamping E, Holmes AR, Niimi K, Tanabe K, Niimi M, Monk BC. Candida albicans drug resistance another way to cope with stress [J]. Microbiology. 2007, 153(Pt 10), 3211-3217.
[8] Bader T, Schroppel K, Bentink S, Agabian N, Kohler G, Morschhauser J. Role of calcineurin in stress resistance, morphogenesis, and virulence of a Candida albicans wild-type strain [J]. Infect Immun. 2006, 74(7):4366-4369.
[9] Ikner A, Shiozaki K. Yeast signaling pathways in the oxidative stress response. Mutat Res. 2005, 569(1-2):13-27.
[10] Du, W., M. Coaker, J. D. Sobel, and R. A. Akins. Shuttle vectors for Candida albicans: control of plasmid copy number and elevated expression of cloned genes [J]. Curr. Genet. 2004, 45(6): 390-398.
[11] White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans [J]. Antimicrob. Agents Chemother. 1997, 41(7): 1488-1494.
[12] Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents [J]. Antimicrob. Agents Chemother. 1998, 42(2):241-253.
[13] Kelly SL, Lamb DC, Kelly DE, Manning NJ, Loeffler J, Hebart H, Schumacher U, Einsele H. Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation [J]. FEBS. Lett. 1997, 400(1):80-82.
[14] Prasad R, De Wergifosse P, Goffeau A, Balzi E. Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals [J].Current Genetics. 1995, 27(4):320-329.
[15] Sanglard D, Ischer F, Monod M, Bille J. Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene [J]. Microbiology. 1997, 143(Pt2):405-416.
[16] Wirsching S, Michel S, Morschhauser J. Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates [J]. Mol Microbiol. 2000, 36(4):856-865.
[17] Mukherjee PK, Chandra J, Kuhn DM, Ghannoum MA. Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols [J]. Infect. Immun. 2003, 71(8):4333-4340.
[18] Dunkel N, Liu TT, Barker KS, Homayouni R, Morschh?user J, Rogers PD. A gain-of-function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate [J]. Eukaryot Cell. 2008, 7(7):1180-1190.
[19] Morschhauser J, Barker KS, Liu TT, Bla?-Warmuth J, Homayouni R, Rogers PD. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans [J]. PLoS Pathog. 2007, 3(11): e164
[20] Coste AT, Karababa M, Ischer F, Bille J, Sanglard D. TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2 [J]. Eukaryot. Cell. 2004, 3(6):1639-1652.
[21] Coste A, Turner V, Ischer F, Morschhauser J, Forche A, Selmecki A, Berman J, Bille J, Sanglard D. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans [J]. Genetics. 2006, 172(4):2139-2156.
[22] Karababa M, Valentino E, Pardini G, Coste AT, Bille J, Sanglard D. CRZ1, a target of the calcineurin pathway in Candida albicans [J]. Mol Microbiol. 2006, 59(5):1429-1451.
[23] Jain P, Akula I, Edlind T. Cyclic AMP signaling pathway modulates susceptibility of Candida species and Saccharomyces cerevisiae to antifungal azoles and other sterol biosynthesis inhibitors [J]. Antimicrob Agents Chemother. 2003, 47(10):3195-3201.
[24] Rogers PD, Barker KS. Evaluation of differential gene expression in fluconazole susceptible and resistant isolates of Candida albicans using cDNA microarray analysis [J]. Antimicrob Agents Chemother. 2002, 46(11):3412-3417.
[25] Rogers PD, Barker KS. Genome-wide expression profile analysis reveals coordinately regulated genes associated with stepwise acquisition of azole resistance in Candida albicans clinical isolates [J]. Antimicrob Agents Chemother. 2003, 47(4):1220-1227.
[26] Karababa M, Coste AT, Rognon B, Bille J, Sanglard D. Comparison of gene expression profiles ofCandida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters [J]. Antimicrob Agents Chemother. 2004, 48(8):3064-3079.
[27] Hooshdaran MZ, Barker KS, Hilliard GM, Kusch H, Morschh?user J, Rogers PD. Proteomic analysis of azole resistance in Candida albicans clinical isolates [J]. Antimicrob. Agents Chemother. 2004, 48(7):2733- 2735.
[28] Kobayashi D, Kondo K, Uehara N, Otokozawa S, Tsuji N, Yagihashi A, Watanabe N. Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect [J]. Antimicrob. Agents Chemother. 2002, 46(10):3113- 3117.
[29] Kontoyiannis DP. Modulation of fluconazole sensitivity by the interaction of mitochondria and Erg3p in Saccharomyces cerevisiae [J]. J Antimicrob Chemother. 2000, 46(2):191-197.
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