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Volume 40 Issue 4
Jul.  2022
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WANG Xinrong, LU Renyi, WANG Yan. The role of SDH2 gene in the environmental adaptability of Candida albicans[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(4): 309-313. doi: 10.12206/j.issn.1006-0111.202201096
Citation: WANG Xinrong, LU Renyi, WANG Yan. The role of SDH2 gene in the environmental adaptability of Candida albicans[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(4): 309-313. doi: 10.12206/j.issn.1006-0111.202201096

The role of SDH2 gene in the environmental adaptability of Candida albicans

doi: 10.12206/j.issn.1006-0111.202201096
  • Received Date: 2022-01-26
  • Rev Recd Date: 2022-06-23
  • Available Online: 2022-07-27
  • Publish Date: 2022-07-25
  •   Objective  To investigate the role of SDH2 gene in the environmental adaptability of Candida albicans.   Methods  Wild-type C. albicans strain SC5314, SDH2 gene knockout mutant sdh2 Δ/Δ and reintegrated strain sdh2 Δ /SDH2 were used as experimental objects. Spot assay was conducted to assess the sensitivity of the WT C. albicans strain SC5314, SDH2 gene knockout mutant sdh2 Δ/Δ and reintegrated strain sdh2 Δ /SDH2 to external stress stimulants and antifungal drugs. The effect of SDH2 gene deletion on drug efflux ability of C. albicans was determined by rhodamine 6G efflux assay.   Results  After SDH2 gene deletion, C. albicans showed slight tolerance to cell wall stress stimulants caffeine, oxidative stress stimulators diamide and menadione. Notably, the sensitivity of SDH2 gene knockout mutant sdh2 Δ/Δ to azole antifungal drugs was significantly increased. The drug efflux capacity of C. albicans was decreased due to the deletion of SDH2 gene.   Conclusion  SDH2 gene deletion lead to changes in environmental adaptability of C. albicans, including changes in response to external environmental stress and increased sensitivity to azole antifungal drugs. The development of fungal-specific inhibitor targeting SDH2 gene may lead to the discovery of new antifungal drugs which have synergistic effect with azole drugs.
  • [1] PFALLER M A, DIEKEMA D J. Epidemiology of invasive mycoses in North America[J]. Crit Rev Microbiol,2010,36(1):1-53. doi:  10.3109/10408410903241444
    [2] ENOCH D A, YANG H N, ALIYU S H, et al. The changing epidemiology of invasive fungal infections[J]. Methods Mol Biol,2017,1508:17-65.
    [3] BROWN G D, DENNING D W, GOW N A, et al. Hidden killers: human fungal infections[J]. Sci Transl Med,2012,4(165):165rv13.
    [4] ENE I V, ADYA A K, WEHMEIER S, et al. Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen[J]. Cell Microbiol,2012,14(9):1319-1335. doi:  10.1111/j.1462-5822.2012.01813.x
    [5] SUN N, FONZI W, CHEN H, et al. Azole susceptibility and transcriptome profiling in Candida albicans mitochondrial electron transport chain complex I mutants[J]. Antimicrob Agents Chemother,2013,57(1):532-542. doi:  10.1128/AAC.01520-12
    [6] THOMAS E, ROMAN E, CLAYPOOL S, et al. Mitochondria influence CDR1 efflux pump activity, Hog1-mediated oxidative stress pathway, iron homeostasis, and ergosterol levels in Candida albicans[J]. Antimicrob Agents Chemother,2013,57(11):5580-5599. doi:  10.1128/AAC.00889-13
    [7] BI S, LV Q Z, WANG T T, et al. SDH2 is involved in proper hypha formation and virulence in Candida albicans[J]. Future Microbiol,2018,13:1141-1156. doi:  10.2217/fmb-2018-0033
    [8] OYEDOTUN K S, LEMIRE B D. The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies[J]. J Biol Chem,2004,279(10):9424-9431. doi:  10.1074/jbc.M311876200
    [9] HANS S, FATIMA Z, HAMEED S. Insights into the modulatory effect of magnesium on efflux mechanisms of Candida albicans reveal inhibition of ATP binding cassette multidrug transporters and dysfunctional mitochondria[J]. Biometals,2021,34(2):329-339. doi:  10.1007/s10534-020-00282-w
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The role of SDH2 gene in the environmental adaptability of Candida albicans

doi: 10.12206/j.issn.1006-0111.202201096

Abstract:   Objective  To investigate the role of SDH2 gene in the environmental adaptability of Candida albicans.   Methods  Wild-type C. albicans strain SC5314, SDH2 gene knockout mutant sdh2 Δ/Δ and reintegrated strain sdh2 Δ /SDH2 were used as experimental objects. Spot assay was conducted to assess the sensitivity of the WT C. albicans strain SC5314, SDH2 gene knockout mutant sdh2 Δ/Δ and reintegrated strain sdh2 Δ /SDH2 to external stress stimulants and antifungal drugs. The effect of SDH2 gene deletion on drug efflux ability of C. albicans was determined by rhodamine 6G efflux assay.   Results  After SDH2 gene deletion, C. albicans showed slight tolerance to cell wall stress stimulants caffeine, oxidative stress stimulators diamide and menadione. Notably, the sensitivity of SDH2 gene knockout mutant sdh2 Δ/Δ to azole antifungal drugs was significantly increased. The drug efflux capacity of C. albicans was decreased due to the deletion of SDH2 gene.   Conclusion  SDH2 gene deletion lead to changes in environmental adaptability of C. albicans, including changes in response to external environmental stress and increased sensitivity to azole antifungal drugs. The development of fungal-specific inhibitor targeting SDH2 gene may lead to the discovery of new antifungal drugs which have synergistic effect with azole drugs.

WANG Xinrong, LU Renyi, WANG Yan. The role of SDH2 gene in the environmental adaptability of Candida albicans[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(4): 309-313. doi: 10.12206/j.issn.1006-0111.202201096
Citation: WANG Xinrong, LU Renyi, WANG Yan. The role of SDH2 gene in the environmental adaptability of Candida albicans[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(4): 309-313. doi: 10.12206/j.issn.1006-0111.202201096
  • 白念珠菌是一种条件致病真菌,在健康人体口腔或胃肠道中以无害共生菌的形式存在。在免疫功能低下的患者中,能够导致危及生命的全身性感染。近年来,随着癌症患者、器官移植接受者等免疫功能低下人群不断增加,白念珠菌感染引发的严重疾病的发病率不断攀升[1-2],死亡率粗略估计已高于40%[3]

    高适应性是白念珠菌传播和致病的关键因素之一,包括对宿主和外界环境的适应性、形态的转换以及对抗真菌药物的适应性。代谢的改变能够影响白念珠菌的药物敏感性,Ene等[4]发现培养基中碳源由葡萄糖转变为乳酸后白念珠菌对抗真菌药物(咪康唑、两性霉素B、卡泊芬净)的敏感性发生改变。线粒体作为重要的代谢细胞器, 其中的某些基因缺失或者功能缺陷,会导致白念珠菌药物敏感性的变化,Sun等[5]发现线粒体复合体I相关基因GOA1NDH51的缺失会导致白念珠菌对唑类药物敏感性上升,Edwina等[6]的研究发现线粒体关键基因FZO1的缺失能够增强白念珠菌对唑类药物的敏感性。

    本课题组前期发现SDH2基因缺失导致白念珠菌致病力显著下降,并发现白念珠菌无法利用非发酵碳源[7]。生物信息学分析显示SDH2基因编码琥珀酸脱氢酶(SDH)的铁硫亚基,琥珀酸脱氢酶在三羧酸循环和线粒体电子传递链中均发挥作用,是能量代谢中重要的一环。SDH2基因可能在白念珠菌代谢过程中发挥重要的作用,那么它是否能够通过影响代谢从而改变白念珠菌的环境适应性呢?本研究聚焦SDH2基因对白念珠菌环境适应性的影响,包括对外界压力应答和药物敏感性的影响,并探索其可能的机制。

    • 挑取少量冻存于−80℃、30% 甘油中的菌株,在YPD 液体培养基中活化,30℃,200 r/min振荡培养24 h后,吸取10 μl置于新的1 ml YPD液体培养基中,继续在 30℃,200 r/min振荡条件下培养 16 h,用 SDA 固体培养基划板,30℃培养 48 h,置于 4℃ 保存备用。实验时挑取 SDA 平板上的单克隆菌落置于1 ml YPD 液体培养基中,30℃,200 r/min振荡培养 16 h 过夜,使其处于对数生长期用于后续实验。

    • 白念珠菌活化16 h ,用YPD液体培养基调整菌浓度至3×106细胞/ml。制备每种菌株的系列稀释液,以10倍倍比稀释成5个浓度梯度,每种白念珠菌的系列浓度稀释物各取5 μl,点在事先加入了待考察的不同浓度的化学试剂和药物的YPD琼脂平板上,倒置于孵箱内,25、30、37 ℃培养24~48 h,期间观察各菌株之间生长的差异并拍照记录。

    • 收集处于对数生长期的白念珠菌细胞悬浮液,用无菌PBS洗涤3次。随后将细胞重悬于无菌PBS中调整其浓度至5×107细胞/ml,孵育2 h以耗尽能量,并加入终浓度为10 μmol/L的罗丹明6G。30℃、200 r/min振荡培养细胞悬浮液60 min,使罗丹明6G积累。洗涤白念珠菌细胞悬浮液3次,注意要保证白念珠菌细胞最终浓度为5×107细胞/ml。然后加入终浓度为20 mmol/L的葡萄糖。分别在25 、30、37℃ 条件下,200 r/min振荡培养白念珠菌细胞悬浮液2 h,取白念珠菌细胞样品(约1 ml)离心,收集上清液,在527 nm处测量吸光度。

    • 使用GraphPad Prism软件对全部数据进行分析,时序检验 (Mantel Cox) 方法比较各组差异 ,当P<0.05时,表示差异具有统计学意义。

    • 我们利用多种刺激剂考察了SDH2在白念珠菌应激反应中的作用。实验应用了sdh2Δ/Δ敲除菌、野生型菌和回复菌,考察在存在应激刺激剂的培养基中sdh2Δ/Δ敲除菌的生长情况与野生型菌和回复菌的区别,从而判断SDH2在白念珠菌应激反应中的作用。应激刺激剂包括细胞壁应激刺激剂(十二烷基硫酸钠和咖啡因)、渗透压应激刺激剂(氯化钠)、氧化应激刺激剂(过氧化氢、二酰胺和甲萘醌)。敏感性实验在25、30、37 ℃三种不同的温度下进行,将不同浓度相同体积的白念珠菌接种于琼脂培养板上,同样的培养板制备3份,白念珠菌生长形成明显的菌落后拍照。结果显示,与野生型菌或回复菌相比,sdh2Δ/Δ敲除菌于25 ℃在0.03%十二烷基硫酸钠培养板上生长得多(图1A)。此外,在所有培养温度下,sdh2Δ/Δ敲除菌在含咖啡因、二酰胺或甲萘醌的培养板上也较野生型菌/回复菌生长得多 (图1A、1B1C)。

    • 我们考察了sdh2Δ/Δ敲除菌、野生型菌和回复菌对抗真菌药物的敏感性,考察的药物包括唑类抗真菌药物特比萘芬、两性霉素B、氟胞嘧啶和阿尼芬净。将不同浓度相同体积的白念珠菌接种于琼脂培养板上,同样的培养板制备3份,白念珠菌生长形成明显的菌落后拍照。药物敏感性实验结果显示,与野生型菌/回复菌相比,sdh2Δ/Δ敲除菌在含有氟康唑、酮康唑和伊曲康唑的培养板上生长得明显减少(图2A2B2C),而在含有其他抗真菌药物,如特比萘芬、两性霉素B、氟胞嘧啶和阿尼芬净的培养板上没有明显差别。

    • 罗丹明6G是ABC(ATP-binding cassette)转运蛋白的特异性底物,在能量不足时以被动扩散的方式进入白念珠菌细胞,加入葡萄糖供能后,可通过药物外排泵以主动转运的方式泵出细胞外。我们通过罗丹明6G的外排实验考察了SDH2敲除对白念珠菌药物外排能力的影响。结果显示在30 ℃和37 ℃ 培养温度下,通过添加葡萄糖以启动能量代谢2 h后,与野生型菌相比,从sdh2Δ/Δ敲除菌的细胞中泵出的罗丹明6G显著减少(P<0.01,见图3)。有趣的是,在25 ℃ 的培养温度下,3种菌株泵出的罗丹明6G都较30 ℃和37 ℃条件下有所减少,但各菌株之间没有显著差异(图3)。

    • SDH2编码琥珀酸脱氢酶中的一个亚基,琥珀酸脱氢酶参与三羧酸循环,并且它位于线粒体内膜上,发挥电子传递的作用,对于能量代谢十分重要[8]。我们对SDH2基因在白念珠菌环境适应性中的作用进行了初步探索,实验选择在30 ℃(白念珠菌最适宜生长温度)、37 ℃(宿主温度)和25 ℃(室温)三种温度下进行。实验之所以选用三种温度,一方面是由于在不同环境温度下白念珠菌的代谢可能不同,选用不同的温度可以考察温度造成的差别;另一方面在三种温度下考察SDH2基因的功能,结果相互之间可以比较或者印证,增加信息量,有利于分析SDH2基因的功能。有意思的是SDH2基因缺失后菌株对唑类抗真菌药物(氟康唑、酮康唑、伊曲康唑)的敏感性显著上升,而对于其他类型的抗真菌药物包括丙烯胺类特比萘芬、多烯类两性霉素B、核苷类氟胞嘧啶和棘白菌素类阿尼芬净,其敏感性和野生型菌没有差异。唑类药物的敏感性与药物外排泵的功能密切相关,药物外排功能障碍可导致对唑类抗真菌药物敏感性增加[9]。药物外排泵包括ABC转运蛋白超家族和主要易化因子超家族,其中ABC转运蛋白中的Cdr1p、Cdr2p发挥作用都需要消耗能量。罗丹明6G是能量依赖型ABC转运蛋白的特异性底物,通过外排泵以能量依赖的主动转运方式排出细胞外。罗丹明6G外排实验的结果能显示能量依赖型ABC转运蛋白的功能。研究发现SDH2缺失后白念珠菌在30 ℃和37 ℃时外排能力都显著降低。SDH2对能量代谢非常重要,SDH2基因缺失后,菌株能量代谢异常,能量依赖型ABC转运蛋白的功能下降,缺失菌对唑类抗真菌药物的敏感性增加。

      SDH2基因缺失后白念珠菌对咖啡因、二酰胺和甲萘醌都表现出轻微的耐受,这提示SDH2基因在白念珠菌环境应激中有一定的作用,这些环境因素包括细胞壁压力的刺激以及氧化刺激,具体的作用机制还有待进一步探讨。

      综上所述,本研究发现SDH2基因缺失会导致白念珠菌对环境压力应激反应的改变;此外,SDH2基因缺失后菌株的药物外排能力降低,菌株对唑类抗真菌药物的敏感性增加。代谢对白念珠菌的环境适应能力和致病力十分重要,SDH2基因缺失导致白念珠菌对唑类抗真菌药物专特性地敏感,以SDH2为靶基因,开发真菌特异性SDH2抑制剂,有望发现与唑类药物协同的新型抗真菌药物。

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