Retigeric acid B的抗真菌机制及白色念珠菌中分子工具的构建
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摘要
近三十年,随着随着免疫缺陷患者的增多、器官移植的广泛开展、免疫抑制剂的使用以及肿瘤化疗和抗生素的广泛应用,临床上真菌感染的发生率逐年上升。与此同时,由于抗真菌药物在临床上的大量使用,导致真菌的耐药性迅速蔓延。另外,随着病人用药时间的延长,造成真菌耐受的出现,这又进一步加剧了临床真菌感染的治疗难度。而临床上有效的抗真菌药物品种(尤其是对系统性真菌感染有效的品种)非常有限。因此筛选新的抗真菌药物,寻找新的抗真菌感染治疗策略,探索真菌耐受的形成机制,并针对性的设计克服真菌耐受的药物就变得非常重要。
植物药一直是天然药物的重要来源,也是人类治疗疾病的重要来源之一。其中地衣(lichen)是植物界中一个特殊的类群,是藻类(algae)和真菌(fungi)高度结合的共生复合体。文献报道近些年科学家们已经从地衣植物中分离获得了许多具有多种生物活性的新化合物。本实验室研究发现对从云南光肺衣中分离得到的retigeric acid B(RAB)具有抗白色念珠菌的活性,此化合物具有中等强度的抗真菌活性,最低抑制浓度为8～16μg/ml。本实验室已有研究报道RAB与临床唑类药物(氟康唑、酮康唑、伊曲康唑)联合用药,对从临床分离得到的白色念珠菌菌株具有协同抗真菌活性。研究还发现RAB能够抑制外派泵的活性,通过下调ERG11的表达抑制麦角甾醇的合成,从而与唑类药物产生协同抗真菌的效果。为了深入研究RAB的抗真菌机制,本课题从RAB对白色念珠菌的毒力、被膜的形成以及细胞增殖、凋亡和坏死等各方面进行了系统的研究,并揭示了其抗真菌机制。此外还对临床上造成真菌感染复发的重要因素耐受进行了初步研究探索,并检测了RAB对念珠菌耐受的影响。
白色念珠菌作为临床上的一种机会致病性真菌,其生长形态具有二相性,它能够依外界环境的变化从酵母态变为菌丝态。而菌丝态因其有着粘附和侵袭的优势,容易造成宿主的感染,因而白色念珠菌酵母态到菌丝态的转变将显著增强它的毒力。实验研究发现RAB在白色念珠菌菌丝诱导的环境条件下能够抑制菌丝的形成,采用秀丽隐杆线虫作为白色念珠菌的动物感染模型,发现RAB能够通过抑制白色念珠菌菌丝的形成,降低其毒力,从而提高线虫的存活率。为了阐明RAB抑制白色念珠菌菌丝形成的分子机制,采用荧光定量PCR技术发现RAB能够下调菌丝调控的重要信号通路Ras1-cAMP-Efg1, cAMP在此信号通路中发挥着重要作用,采用cAMP ELISA试剂盒测定RAB作用后细胞内的cAMP含量,发现胞内cAMP含量随RAB剂量的增高而降低,当补加外源cAMP时,能够逆转RAB对菌丝的抑制作用,显示RAB是通过影响cAMP的合成来抑制菌丝的形成。白色念珠菌中合成cAMP的酶为腺苷酸环化酶Cdc35,将RAB作用后的白色念珠菌细胞进行裂解,将裂解产物作为粗酶,进行腺苷酸环化酶活性测定,发现RAB处理后的细胞内Cdc35的活力降低,并呈剂量依赖效应。将Cdc35进行外源表达然后测定RAB对Cdc35的酶活影响,发现RAB对体外表达的Cdc35无酶活抑制效果。因而推断RAB是通过间接的方式影响了Cdc35的活性。有研究报道法尼醇能够与Cdc35直接作用,抑制Cdc35的活性。采用气相色谱和质谱联用的技术检测RAB处理后细胞分泌的法尼醇含量,发现法尼醇的含量随RAB浓度的增高而增加,呈时间和剂量依赖效应。采用绿色荧光蛋白与编码法尼醇合成酶Dpp3进行融合表达,以绿色荧光蛋白的强度作为Dpp3表达量的指针,发现RAB的加入能够促进白色念珠菌Dpp3的表达量。此外通过荧光定量PCR和绿色荧光标记的方法发现RAB能够下调白色念珠菌粘附和侵袭相关因子如Als3的表达量,从而降低白色念珠菌对宿主细胞的侵袭性。基于以上结果得出这样的结论：RAB通过上调Dpp3的表达量,增加法尼醇的分泌,从而抑制Cdc35的活性,降低cAMP的合成,通过Ras1-cAMP-Efg1信号调节通路抑制白色念珠菌菌丝的形成,抑制粘附和侵袭相关因子,降低了白色念珠菌的毒力,提高了被真菌感染的宿主的存活率
鉴于RAB与临床唑类药物具有协同抗真菌的效果,本课题探索了RAB与唑类药物联合应用抑制白色念珠菌菌丝和被膜形成的效果。白色念珠菌菌丝的形成不仅增强了它的毒力,同时也是成熟被膜形成的必要过程。被膜形成后,它对所包裹的细胞形成一种保护,使其对临床上所用的抗真菌药物高度耐受,并且产生一些耐受菌,在药物撤去后,耐受菌继续分裂繁殖,造成患者的复发感染,因而被膜的形成极大的增加了临床上白色念珠菌感染的治疗难度。体外实验发现低剂量的RAB与唑类药物联用能够显著抑制白色念珠菌菌丝的形成。采用Balb/C小鼠作为白色念珠菌体内动物感染模型,结果显示RAB与氟康唑联用能够显著延长小鼠的存活时间；对小鼠肾组织中的菌量进行计数,发现联合用药可以显著降低白色念珠菌对肾组织的侵袭性；肾组织病理切片染色观察发现联合用药组中,白色念珠菌的形态为酵母态,而在对照组和药物单用组中,念珠菌的形态为菌丝态。据此推论RAB与唑类药物联合能够通过抑制菌丝的形成,降低白色念珠菌的侵袭性,从而提高被感染小鼠的存活率和存活时间。为了进一步验证RAB与氟康唑能够抑制白色念珠菌对宿主细胞的侵袭性,采用白色念珠菌与宿主细胞共培养,观察在药物作用下,白色念珠菌对宿主细胞的侵袭力和粘附性。实验结果显示RAB与氟康唑联用能够显著抑制白色念珠菌对宿主细胞的侵袭以及对宿主细胞的粘附。
基于RAB与唑类药物联用抑制白色念珠菌菌丝形成的效果,采用平板微量稀释法检测了RAB与唑类药物联用对白色念珠菌中的实验和临床菌株的被膜形成作用,并使用FICI模型评估了联用效果,结果显示RAB与唑类药物联用具有协同抑制真菌被膜形成的作用；采用激光共聚焦显微镜观察用二乙酸荧光素(FDA,活细胞染色)和碘化丙啶(PI,死细胞染色)双染的白色念珠菌被膜结构,发现单用RAB或者氟康唑处理的白色念珠菌所形成的被膜结构与对照组相比,较为稀疏,菌的形态主要为菌丝态,而药物联用组处理的白色念珠菌无法形成被膜,仅有一些散落的酵母态细胞粘附在介质表面,同时药物联用还表现出-定的杀菌效果。3维薄层扫描结果显示,对照组中白色念珠菌形成了致密且厚度较高的被膜,而单用RAB或FLC处理后所形成的被膜较对照组稀薄。以上结果显示RAB可以与唑类药物联用,通过抑制菌丝的形成,协同抑制成熟被膜的形成,并表现出一定的杀菌效果。实时荧光定量PCR结果显示RAB能够降低白色念珠菌多药耐药外排泵MDR1的表达,增强其与唑类药物联用的抗真菌效果。
白色念珠菌的酵母态虽然毒力较弱,但是酵母态的结构形态易于其随血液进行扩散,基于这种扩散特性,抑制白色念珠菌酵母态的增殖也是一种有效的抗真菌策略。本课题研究发现RAB能够通过调控白色念珠菌的细胞周期抑制其分裂,低剂量的RAB能够使细胞阻滞在G2/M期,而高剂量的RAB使细胞阻滞在G1期。RAB能够造成细胞内ROS的积累,从而造成DNA损伤,将分裂的细胞阻滞在G2/M期。有研究报道称一些抗真菌剂能够降低胞内cAMP的含量,抑制G1期相关蛋白的表达,使细胞阻滞在G1期,从而抑制细胞的分裂增殖。实验发现RAB也能够降低胞内cAMP的含量,并呈剂量依赖效应,通过外源cAMP回补的方法证明RAB确实是通过降低cAMP的含量使得细胞阻滞的G1期。以上结果表明RAB能够促使细胞内ROS含量的积聚,并降低胞内cAMP的含量,延长细胞周期,从而抑制白色念珠菌的增殖。
此外,研究发现当白色念珠菌细胞受到高剂量RAB (32μg/ml)作用时,细胞表现出坏死的特性。透射电镜结果显示经高剂量药物作用后细胞内染色质固缩或分散、细胞膜破裂、细胞器出现皱缩。对细胞进行凋亡染色,并用荧光显微镜进行观察,发现药物作用后有大量坏死细胞和少量的凋亡细胞。进一步的研究发现RAB所诱导产生的细胞坏死与胞内积聚的ROS相关,这与文献中报道的抗真菌药物通过促进ROS增多来发挥杀菌效果相一致。采用罗丹明123进行染色,并用流式细胞仪进行检测发现RAB能够使得细胞线粒体膜电势超极化,用ATP检测试剂盒发现RAB作用后的细胞内ATP含量降低,并呈剂量依赖递减效应。这表明RAB促进了胞内ROS的积聚,使得线粒体膜电势超极化,抑制了ATP的产生,这三个因素共同导致了念珠菌细胞的坏死。
为了研究白色念珠菌的耐受发生机制,首先要建立快速检测耐受菌(持留菌)的模型。将糖酵解代谢基因TDH3进行GFP标记,念珠菌细胞在受到高剂量杀菌剂两性霉素B处理后,用GFP阳性、PI阴性细胞作为持留菌的特征进行检测。同时还用该模型评价了RAB对念珠菌耐受的影响,发现RAB对降低耐受无明显作用效果,该结果与用传统的评价结果相一致。为了分选获得持留菌以便进行基因表达谱分析,进行了不同培养条件的优化,得到了持留菌比率较高的培养条件。
为了构建一个能够在白色念珠菌中稳定遗传的分子工具,以便于进行白色念珠菌的遗传学研究和抗真菌药物的筛选。在一个普通的质粒骨架上添加真菌抗性筛选标记NAT1(抗Nourseothricin),复制活性位点ORC984或着丝粒序列CEN4,并将端粒序列放在质粒的两端,用限制性内切酶线性化该质粒,就成为一个类染色体的人造染色体,将线性化的质粒转染白色念珠菌,采用轮廓钳位均匀电泳(CHEF)并结合southern杂交的方法确证该质粒转化所得的克隆子中，线性质粒依然能够保持游离态,未被整合到基因组中。进一步研究发现这些克隆子能够稳定遗传该质粒,将功能基因装载到该质粒上,在转化克隆子中能够表达该目的基因,并稳定遗传。以上结果显示上述质粒可以作为一种有效的分子工具在白色念珠菌中使用。
本论文详细论述了从地衣中分离得到的三萜类化合物RAB的抗真菌机制,为三萜类化合物的抗真菌机制提供了理论依据,并为临床上治疗真菌感染提供了一种可能性。论文中所述的白色念珠菌耐受模型的建立为真菌耐受发生机制的研究以及抗真菌耐受药物的筛选奠定了基础。在白色念珠菌中稳定遗传的质粒为白色念珠菌的基础研究以及抗真菌药物的筛选提供了一种有效的分子工具。
Over the last30years, the incidence of clinical fungal infection has dramatically increased due to the increased immune-compromised patients caused by organ transplantation, the application of immunosuppressive drugs, cancer chemotherapy, and widely used antibiotics. Additionally, the drug resistance appears as the antifungal agents are broadly used in clinic. With the long time treatment received by the patients, the persistence emerges, which makes the clinical therapy much more difficult. However, the antifungal drugs used are limited, especially for the fungicidal agents. The development of new antifungal drugs and therapitical strategies, investigating the mechanism of persistence and designing drugs targeting the persistence are therefore important.
Natural plants, as an important source of natural medicines are used to treat human diseases. Lichens are quite a special large group in the the plant kingdom as high degree of integration of the symbiotic complex between algae and fungi. Recently, abundant secondary metabolites from lichens with multiple bioactivities have been obtained as reported in the literature. Retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, has been isolated in our lab. It displays moderate antifungal action, with minimum inhibitory concentrations (MICs) ranging from8to16μg/ml. It also has synergistic antifungal activity when applied in combination with azoles including fluconazole, ketoconazole and itraconazole, especially for azoles-resistant strains. Our previous research showed the synergistic action was astributed to the inhibited activity of efflux pumps by RAB and reduced ergosterol synthesis through downregulation of ERG11. To better elucidate the antifungal mechanisms of RAB, we focused on the virulence, biofilm formation, proliferation, apoptosis and necrosis of Candida albicans affected by RAB. Besides that, the persistence of C. albicans, an important factor for the relapse of fungal infection, was also investigated.
C. albicans, an opportunistic human pathogen, undergoes yeast-to-hyphal switch according to the growing conditions. Hyphae filaments, having the advantage of adhering and penetrating tissue, are apt to invade the host. The yeast to hyphae conversion significantly increases its virulence. The study showed RAB could inhibit the yeast-to-hyphal transition in hyphae stimulating growing condition. The in-vivo study showed RAB could reduce the virulence of C. albicans through blocking the hyphae formation when Caenorhabditis elegans was utilized as an infection model, which conferred the improved longevity of the hosts. To elucidate the underlying mechanism of RAB against hyphae formation, real time quantitative PCR (qPCR) was applied. The results suggested RAB downregulated Rasl-cAMP-Efgl pathway, among which cAMP plays a critical role in regulating the hyphae formation. Furthern research showed RAB reduced the intracellular level of cAMP in a dose-dependent manner revealed by cAMP detection assay. The exogenerous cAMP could restore the hyphae formation inhibited by RAB, suggesting the reduced cAMP by RAB was responsible for the inhibited hyphae formation. In C. albicans, cAMP was synthized by Cdc35. To determine whether RAB has inhibitory effect on the activity of Cdc35, two independent methods were carried out. C. albicans cells treated with RAB were lysed to detect the Cdc35activity in the cell lysates. The results showed the Cdc35activity was inhibited by RAB in a dose-dependent way. To determine whether RAB directly inhibit Cdc35, the purified catalytic domain of Cdc35was used in the adenylyl cyclase activity inhibition assays. Experimental results suggested RAB did not influence the enzymatic activity, which suggested another factor induced by RAB may inhibit the activity of Cdc35. It has reported farnesol could directly interact with Cdc35, inhibiting cAMP synthesis. GC-MS (gas chromatography-mass spectrometry) was performed to detect the amounts of farnesol secreted by C. albicans when treated by RAB. The results demonstrated RAB could stimulate farnesol synthesis. It was also found RAB could induce the expression of Dpp3, a protein synthizing farnesol. The resulsts of qPCR and protein detection assay based on GFP fluorescence showed adesins and invasins such as Als3were also inhibited by RAB. Based on the above results, it is concluded that RAB upregulates the expression of Dpp3, which inhibites the activity of Cdc35, reducing the synthesis of cAMP, a critical factor in Rasl-cAMP-Efgl signaling pathway regulating hyphae formation, and then inhibites the adhesins and invasins, the virulence factors, to promote the survival of infected hosts.
Based on the synergesitic action of RAB and azoles against clinical C. albicans strains, we determine the effect of RAB and azoles on inhibiting the hyphae formation and preventing biofilm formation. Filamentation is not only increasing the virulence, but also a requirement for a mature biofilm formation. A biofilm is a protected niche for microorganisms in which they are less susceptible to antibiotic treatment and can create a source of persistent infection. When the antifungal drugs are removed, the persisters start to replicate, causing the recurrent infection, which makes biofilm-associated C. albicans infections much more difficult to treat. The in-vitro study showed low doses of RAB combined with azoles displayed additive or synergistic action against hyphae formation. The Balb/C infective mice model also showed RAB could prolong the longevity of the hosts when applied with fluconazole. The kidney fungal burden examination showed the combination treatment significantly reduced the pathogenesis of C. albicans. Histopathological analysis revealed that C. albicans under the combination treatment displayed only in yeast form, while the organisms in control group or drug-alone treated group grew mainly in hyphae form. It is concluded that the combination treatment could promote the longevity of the infected host through inhibiting the hyphae formation and reducing the pathogenesis. The in vitro co-culture of C. albicans cells and hosts'cells reveals the combination of RAB and fluconazole significantly inhibit the invasiveness and pathogenesis.
To dermine the effect of the combination treatment of RAB and fluconazole on biofilm formation, checkerboard microdilution assay was performed. FICI (fractional inhibitory concentration index) method used in the assay showed RAB displayed syngergistic manner against the biofilm formation together with azoles. Furtherly, the biofilm structure stained by fluorescein diacetate and propidium iodide was observed using confocal scanning laser microscope. The biofilm under single drug treatment is thinner compared with that in control. They all are mainly composed of filaments. However, no biofilm formes and only clusters of yeast cells adhere to the substratum when cells are exposed to combination treatment. Of the interest is the fungicidal effect under the combination treatment. Three dimentional scanning results showed non-treated C. albicans forms a thick biofilm with a tight structure, while single treated C. albicans forms a thinner one. Above results showed the combination of RAB and azoles could synergistically inhibit the biofilm formation through preventing the hyphae formation. qPCR results showed the downregulation of MDR1, one of drug pump, by RAB facilitates the antifungal effect of azoles.
For C. albicans, the yeast form, as a disseminating form, plays a critical role in the early stage of Candida infectious process for the suitable dimensions and physical properties to get access to the host's bloodstream. So inhibiting the replication is also a strategy for overcoming the Candida infection. RAB suppresses the proliferative rate of cells in C. albicans by regulating the cell cycle. Low doses of RAB treatment leads to accumulation of cells at the G2/M phase, whereas high concentrations of RAB intriguingly caused cells accumulated in G1phase of the cell cycle. The finding that RAB causes ROS accumulation in C. albicans suggestes the extra ROS could damage DNA and prevent the G2/M transition at the cell cycle checkpoint, and subsequently leading to G2/M cell cycle arrest. RAB treatment could also decrease the levels of cAMP in a dose-dependent manner, resulting in G1cell cycle arrest, which is in line with a previous report that the antifungal agents with an ability of reducing the cAMP level could prolong the cell cycle through delaying the expression of G1cyclins. This notion is further supported by the recovery of RAB-induced G1cell cycle arrest with addition of exogenous cAMP.
C. albicans cells exhibit features of death revealed by apoptosis and necrosis detection when they are exposed to32μg/ml of RAB. Transmission electron micrographs showed chromatin condensation, dispersion and nuclear fragmentation and plasma membrane breakage and organelle swelling in RAB-treated cells. RAB induced cells death in C. albicans is due to the enhanced ROS formation, which consistent with published studies that some antifungal agents exert the antifungal activity via ROS. RAB increases the mt△ψ, but decreases ATP level in a dose-dependent manner. The drop of ATP level is likely attributed to the mt△ψ hyperpolarization that is induced by enhanced ROS accumulation, which together contributes to the cell death.
To better understand the persistence of C. albicans, an effective model needs to be established to screen the persisters. The strain with TDH3tagged by GFP is treated by high dose of amphotericin B, then cells with GFP positive and PI negative are considered as persisters. This model revealed RAB had no action to eliminate the persister, in consistent with the conventional method. To isolate the persister for transcriptional profiling, different cultural conditions were screened to achieve high persister rate.
To construct a molecular tool used in C. albicans genetic research or screening antifungal agents, a plasmid backbone was inserted with NATl sequence (resistant to nourseothricin), an origin sequence ORC984or CEN4, and inverted telomeres at both ends. The constructed plasmids were linearized and transformed into C. albicans cells. The contour-clamped homogeneous electric field gel electrophoresis plus southern blot revealed some transformants have free plasmids in Candida cells, which are not integrative to the genomes. And the plasmids in the transformants could stably replicate in different generations. The plasmid carring a functional gene could also makes the tranformants display the phenotype, suggesting it is a good genetic tool for Candida research.
In this thesis, we described the antifungal mechanism of RAB, which is the basis for the mechanism of triterpene analogs. The combination treatment provides a possible application in overcoming clinical fungal infection. The investigation of persistence paves the way for understanding the mechanism of persistence and designing new drugs targeting the persister formation. The plasmid constructed for C. albicans works as an effective molecular tool in Candida genetic research and screening antifungal agents.
植物药一直是天然药物的重要来源,也是人类治疗疾病的重要来源之一。其中地衣(lichen)是植物界中一个特殊的类群,是藻类(algae)和真菌(fungi)高度结合的共生复合体。文献报道近些年科学家们已经从地衣植物中分离获得了许多具有多种生物活性的新化合物。本实验室研究发现对从云南光肺衣中分离得到的retigeric acid B(RAB)具有抗白色念珠菌的活性,此化合物具有中等强度的抗真菌活性,最低抑制浓度为8～16μg/ml。本实验室已有研究报道RAB与临床唑类药物(氟康唑、酮康唑、伊曲康唑)联合用药,对从临床分离得到的白色念珠菌菌株具有协同抗真菌活性。研究还发现RAB能够抑制外派泵的活性,通过下调ERG11的表达抑制麦角甾醇的合成,从而与唑类药物产生协同抗真菌的效果。为了深入研究RAB的抗真菌机制,本课题从RAB对白色念珠菌的毒力、被膜的形成以及细胞增殖、凋亡和坏死等各方面进行了系统的研究,并揭示了其抗真菌机制。此外还对临床上造成真菌感染复发的重要因素耐受进行了初步研究探索,并检测了RAB对念珠菌耐受的影响。
白色念珠菌作为临床上的一种机会致病性真菌,其生长形态具有二相性,它能够依外界环境的变化从酵母态变为菌丝态。而菌丝态因其有着粘附和侵袭的优势,容易造成宿主的感染,因而白色念珠菌酵母态到菌丝态的转变将显著增强它的毒力。实验研究发现RAB在白色念珠菌菌丝诱导的环境条件下能够抑制菌丝的形成,采用秀丽隐杆线虫作为白色念珠菌的动物感染模型,发现RAB能够通过抑制白色念珠菌菌丝的形成,降低其毒力,从而提高线虫的存活率。为了阐明RAB抑制白色念珠菌菌丝形成的分子机制,采用荧光定量PCR技术发现RAB能够下调菌丝调控的重要信号通路Ras1-cAMP-Efg1, cAMP在此信号通路中发挥着重要作用,采用cAMP ELISA试剂盒测定RAB作用后细胞内的cAMP含量,发现胞内cAMP含量随RAB剂量的增高而降低,当补加外源cAMP时,能够逆转RAB对菌丝的抑制作用,显示RAB是通过影响cAMP的合成来抑制菌丝的形成。白色念珠菌中合成cAMP的酶为腺苷酸环化酶Cdc35,将RAB作用后的白色念珠菌细胞进行裂解,将裂解产物作为粗酶,进行腺苷酸环化酶活性测定,发现RAB处理后的细胞内Cdc35的活力降低,并呈剂量依赖效应。将Cdc35进行外源表达然后测定RAB对Cdc35的酶活影响,发现RAB对体外表达的Cdc35无酶活抑制效果。因而推断RAB是通过间接的方式影响了Cdc35的活性。有研究报道法尼醇能够与Cdc35直接作用,抑制Cdc35的活性。采用气相色谱和质谱联用的技术检测RAB处理后细胞分泌的法尼醇含量,发现法尼醇的含量随RAB浓度的增高而增加,呈时间和剂量依赖效应。采用绿色荧光蛋白与编码法尼醇合成酶Dpp3进行融合表达,以绿色荧光蛋白的强度作为Dpp3表达量的指针,发现RAB的加入能够促进白色念珠菌Dpp3的表达量。此外通过荧光定量PCR和绿色荧光标记的方法发现RAB能够下调白色念珠菌粘附和侵袭相关因子如Als3的表达量,从而降低白色念珠菌对宿主细胞的侵袭性。基于以上结果得出这样的结论：RAB通过上调Dpp3的表达量,增加法尼醇的分泌,从而抑制Cdc35的活性,降低cAMP的合成,通过Ras1-cAMP-Efg1信号调节通路抑制白色念珠菌菌丝的形成,抑制粘附和侵袭相关因子,降低了白色念珠菌的毒力,提高了被真菌感染的宿主的存活率
鉴于RAB与临床唑类药物具有协同抗真菌的效果,本课题探索了RAB与唑类药物联合应用抑制白色念珠菌菌丝和被膜形成的效果。白色念珠菌菌丝的形成不仅增强了它的毒力,同时也是成熟被膜形成的必要过程。被膜形成后,它对所包裹的细胞形成一种保护,使其对临床上所用的抗真菌药物高度耐受,并且产生一些耐受菌,在药物撤去后,耐受菌继续分裂繁殖,造成患者的复发感染,因而被膜的形成极大的增加了临床上白色念珠菌感染的治疗难度。体外实验发现低剂量的RAB与唑类药物联用能够显著抑制白色念珠菌菌丝的形成。采用Balb/C小鼠作为白色念珠菌体内动物感染模型,结果显示RAB与氟康唑联用能够显著延长小鼠的存活时间；对小鼠肾组织中的菌量进行计数,发现联合用药可以显著降低白色念珠菌对肾组织的侵袭性；肾组织病理切片染色观察发现联合用药组中,白色念珠菌的形态为酵母态,而在对照组和药物单用组中,念珠菌的形态为菌丝态。据此推论RAB与唑类药物联合能够通过抑制菌丝的形成,降低白色念珠菌的侵袭性,从而提高被感染小鼠的存活率和存活时间。为了进一步验证RAB与氟康唑能够抑制白色念珠菌对宿主细胞的侵袭性,采用白色念珠菌与宿主细胞共培养,观察在药物作用下,白色念珠菌对宿主细胞的侵袭力和粘附性。实验结果显示RAB与氟康唑联用能够显著抑制白色念珠菌对宿主细胞的侵袭以及对宿主细胞的粘附。
基于RAB与唑类药物联用抑制白色念珠菌菌丝形成的效果,采用平板微量稀释法检测了RAB与唑类药物联用对白色念珠菌中的实验和临床菌株的被膜形成作用,并使用FICI模型评估了联用效果,结果显示RAB与唑类药物联用具有协同抑制真菌被膜形成的作用；采用激光共聚焦显微镜观察用二乙酸荧光素(FDA,活细胞染色)和碘化丙啶(PI,死细胞染色)双染的白色念珠菌被膜结构,发现单用RAB或者氟康唑处理的白色念珠菌所形成的被膜结构与对照组相比,较为稀疏,菌的形态主要为菌丝态,而药物联用组处理的白色念珠菌无法形成被膜,仅有一些散落的酵母态细胞粘附在介质表面,同时药物联用还表现出-定的杀菌效果。3维薄层扫描结果显示,对照组中白色念珠菌形成了致密且厚度较高的被膜,而单用RAB或FLC处理后所形成的被膜较对照组稀薄。以上结果显示RAB可以与唑类药物联用,通过抑制菌丝的形成,协同抑制成熟被膜的形成,并表现出一定的杀菌效果。实时荧光定量PCR结果显示RAB能够降低白色念珠菌多药耐药外排泵MDR1的表达,增强其与唑类药物联用的抗真菌效果。
白色念珠菌的酵母态虽然毒力较弱,但是酵母态的结构形态易于其随血液进行扩散,基于这种扩散特性,抑制白色念珠菌酵母态的增殖也是一种有效的抗真菌策略。本课题研究发现RAB能够通过调控白色念珠菌的细胞周期抑制其分裂,低剂量的RAB能够使细胞阻滞在G2/M期,而高剂量的RAB使细胞阻滞在G1期。RAB能够造成细胞内ROS的积累,从而造成DNA损伤,将分裂的细胞阻滞在G2/M期。有研究报道称一些抗真菌剂能够降低胞内cAMP的含量,抑制G1期相关蛋白的表达,使细胞阻滞在G1期,从而抑制细胞的分裂增殖。实验发现RAB也能够降低胞内cAMP的含量,并呈剂量依赖效应,通过外源cAMP回补的方法证明RAB确实是通过降低cAMP的含量使得细胞阻滞的G1期。以上结果表明RAB能够促使细胞内ROS含量的积聚,并降低胞内cAMP的含量,延长细胞周期,从而抑制白色念珠菌的增殖。
此外,研究发现当白色念珠菌细胞受到高剂量RAB (32μg/ml)作用时,细胞表现出坏死的特性。透射电镜结果显示经高剂量药物作用后细胞内染色质固缩或分散、细胞膜破裂、细胞器出现皱缩。对细胞进行凋亡染色,并用荧光显微镜进行观察,发现药物作用后有大量坏死细胞和少量的凋亡细胞。进一步的研究发现RAB所诱导产生的细胞坏死与胞内积聚的ROS相关,这与文献中报道的抗真菌药物通过促进ROS增多来发挥杀菌效果相一致。采用罗丹明123进行染色,并用流式细胞仪进行检测发现RAB能够使得细胞线粒体膜电势超极化,用ATP检测试剂盒发现RAB作用后的细胞内ATP含量降低,并呈剂量依赖递减效应。这表明RAB促进了胞内ROS的积聚,使得线粒体膜电势超极化,抑制了ATP的产生,这三个因素共同导致了念珠菌细胞的坏死。
为了研究白色念珠菌的耐受发生机制,首先要建立快速检测耐受菌(持留菌)的模型。将糖酵解代谢基因TDH3进行GFP标记,念珠菌细胞在受到高剂量杀菌剂两性霉素B处理后,用GFP阳性、PI阴性细胞作为持留菌的特征进行检测。同时还用该模型评价了RAB对念珠菌耐受的影响,发现RAB对降低耐受无明显作用效果,该结果与用传统的评价结果相一致。为了分选获得持留菌以便进行基因表达谱分析,进行了不同培养条件的优化,得到了持留菌比率较高的培养条件。
为了构建一个能够在白色念珠菌中稳定遗传的分子工具,以便于进行白色念珠菌的遗传学研究和抗真菌药物的筛选。在一个普通的质粒骨架上添加真菌抗性筛选标记NAT1(抗Nourseothricin),复制活性位点ORC984或着丝粒序列CEN4,并将端粒序列放在质粒的两端,用限制性内切酶线性化该质粒,就成为一个类染色体的人造染色体,将线性化的质粒转染白色念珠菌,采用轮廓钳位均匀电泳(CHEF)并结合southern杂交的方法确证该质粒转化所得的克隆子中，线性质粒依然能够保持游离态,未被整合到基因组中。进一步研究发现这些克隆子能够稳定遗传该质粒,将功能基因装载到该质粒上,在转化克隆子中能够表达该目的基因,并稳定遗传。以上结果显示上述质粒可以作为一种有效的分子工具在白色念珠菌中使用。
本论文详细论述了从地衣中分离得到的三萜类化合物RAB的抗真菌机制,为三萜类化合物的抗真菌机制提供了理论依据,并为临床上治疗真菌感染提供了一种可能性。论文中所述的白色念珠菌耐受模型的建立为真菌耐受发生机制的研究以及抗真菌耐受药物的筛选奠定了基础。在白色念珠菌中稳定遗传的质粒为白色念珠菌的基础研究以及抗真菌药物的筛选提供了一种有效的分子工具。
Over the last30years, the incidence of clinical fungal infection has dramatically increased due to the increased immune-compromised patients caused by organ transplantation, the application of immunosuppressive drugs, cancer chemotherapy, and widely used antibiotics. Additionally, the drug resistance appears as the antifungal agents are broadly used in clinic. With the long time treatment received by the patients, the persistence emerges, which makes the clinical therapy much more difficult. However, the antifungal drugs used are limited, especially for the fungicidal agents. The development of new antifungal drugs and therapitical strategies, investigating the mechanism of persistence and designing drugs targeting the persistence are therefore important.
Natural plants, as an important source of natural medicines are used to treat human diseases. Lichens are quite a special large group in the the plant kingdom as high degree of integration of the symbiotic complex between algae and fungi. Recently, abundant secondary metabolites from lichens with multiple bioactivities have been obtained as reported in the literature. Retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, has been isolated in our lab. It displays moderate antifungal action, with minimum inhibitory concentrations (MICs) ranging from8to16μg/ml. It also has synergistic antifungal activity when applied in combination with azoles including fluconazole, ketoconazole and itraconazole, especially for azoles-resistant strains. Our previous research showed the synergistic action was astributed to the inhibited activity of efflux pumps by RAB and reduced ergosterol synthesis through downregulation of ERG11. To better elucidate the antifungal mechanisms of RAB, we focused on the virulence, biofilm formation, proliferation, apoptosis and necrosis of Candida albicans affected by RAB. Besides that, the persistence of C. albicans, an important factor for the relapse of fungal infection, was also investigated.
C. albicans, an opportunistic human pathogen, undergoes yeast-to-hyphal switch according to the growing conditions. Hyphae filaments, having the advantage of adhering and penetrating tissue, are apt to invade the host. The yeast to hyphae conversion significantly increases its virulence. The study showed RAB could inhibit the yeast-to-hyphal transition in hyphae stimulating growing condition. The in-vivo study showed RAB could reduce the virulence of C. albicans through blocking the hyphae formation when Caenorhabditis elegans was utilized as an infection model, which conferred the improved longevity of the hosts. To elucidate the underlying mechanism of RAB against hyphae formation, real time quantitative PCR (qPCR) was applied. The results suggested RAB downregulated Rasl-cAMP-Efgl pathway, among which cAMP plays a critical role in regulating the hyphae formation. Furthern research showed RAB reduced the intracellular level of cAMP in a dose-dependent manner revealed by cAMP detection assay. The exogenerous cAMP could restore the hyphae formation inhibited by RAB, suggesting the reduced cAMP by RAB was responsible for the inhibited hyphae formation. In C. albicans, cAMP was synthized by Cdc35. To determine whether RAB has inhibitory effect on the activity of Cdc35, two independent methods were carried out. C. albicans cells treated with RAB were lysed to detect the Cdc35activity in the cell lysates. The results showed the Cdc35activity was inhibited by RAB in a dose-dependent way. To determine whether RAB directly inhibit Cdc35, the purified catalytic domain of Cdc35was used in the adenylyl cyclase activity inhibition assays. Experimental results suggested RAB did not influence the enzymatic activity, which suggested another factor induced by RAB may inhibit the activity of Cdc35. It has reported farnesol could directly interact with Cdc35, inhibiting cAMP synthesis. GC-MS (gas chromatography-mass spectrometry) was performed to detect the amounts of farnesol secreted by C. albicans when treated by RAB. The results demonstrated RAB could stimulate farnesol synthesis. It was also found RAB could induce the expression of Dpp3, a protein synthizing farnesol. The resulsts of qPCR and protein detection assay based on GFP fluorescence showed adesins and invasins such as Als3were also inhibited by RAB. Based on the above results, it is concluded that RAB upregulates the expression of Dpp3, which inhibites the activity of Cdc35, reducing the synthesis of cAMP, a critical factor in Rasl-cAMP-Efgl signaling pathway regulating hyphae formation, and then inhibites the adhesins and invasins, the virulence factors, to promote the survival of infected hosts.
Based on the synergesitic action of RAB and azoles against clinical C. albicans strains, we determine the effect of RAB and azoles on inhibiting the hyphae formation and preventing biofilm formation. Filamentation is not only increasing the virulence, but also a requirement for a mature biofilm formation. A biofilm is a protected niche for microorganisms in which they are less susceptible to antibiotic treatment and can create a source of persistent infection. When the antifungal drugs are removed, the persisters start to replicate, causing the recurrent infection, which makes biofilm-associated C. albicans infections much more difficult to treat. The in-vitro study showed low doses of RAB combined with azoles displayed additive or synergistic action against hyphae formation. The Balb/C infective mice model also showed RAB could prolong the longevity of the hosts when applied with fluconazole. The kidney fungal burden examination showed the combination treatment significantly reduced the pathogenesis of C. albicans. Histopathological analysis revealed that C. albicans under the combination treatment displayed only in yeast form, while the organisms in control group or drug-alone treated group grew mainly in hyphae form. It is concluded that the combination treatment could promote the longevity of the infected host through inhibiting the hyphae formation and reducing the pathogenesis. The in vitro co-culture of C. albicans cells and hosts'cells reveals the combination of RAB and fluconazole significantly inhibit the invasiveness and pathogenesis.
To dermine the effect of the combination treatment of RAB and fluconazole on biofilm formation, checkerboard microdilution assay was performed. FICI (fractional inhibitory concentration index) method used in the assay showed RAB displayed syngergistic manner against the biofilm formation together with azoles. Furtherly, the biofilm structure stained by fluorescein diacetate and propidium iodide was observed using confocal scanning laser microscope. The biofilm under single drug treatment is thinner compared with that in control. They all are mainly composed of filaments. However, no biofilm formes and only clusters of yeast cells adhere to the substratum when cells are exposed to combination treatment. Of the interest is the fungicidal effect under the combination treatment. Three dimentional scanning results showed non-treated C. albicans forms a thick biofilm with a tight structure, while single treated C. albicans forms a thinner one. Above results showed the combination of RAB and azoles could synergistically inhibit the biofilm formation through preventing the hyphae formation. qPCR results showed the downregulation of MDR1, one of drug pump, by RAB facilitates the antifungal effect of azoles.
For C. albicans, the yeast form, as a disseminating form, plays a critical role in the early stage of Candida infectious process for the suitable dimensions and physical properties to get access to the host's bloodstream. So inhibiting the replication is also a strategy for overcoming the Candida infection. RAB suppresses the proliferative rate of cells in C. albicans by regulating the cell cycle. Low doses of RAB treatment leads to accumulation of cells at the G2/M phase, whereas high concentrations of RAB intriguingly caused cells accumulated in G1phase of the cell cycle. The finding that RAB causes ROS accumulation in C. albicans suggestes the extra ROS could damage DNA and prevent the G2/M transition at the cell cycle checkpoint, and subsequently leading to G2/M cell cycle arrest. RAB treatment could also decrease the levels of cAMP in a dose-dependent manner, resulting in G1cell cycle arrest, which is in line with a previous report that the antifungal agents with an ability of reducing the cAMP level could prolong the cell cycle through delaying the expression of G1cyclins. This notion is further supported by the recovery of RAB-induced G1cell cycle arrest with addition of exogenous cAMP.
C. albicans cells exhibit features of death revealed by apoptosis and necrosis detection when they are exposed to32μg/ml of RAB. Transmission electron micrographs showed chromatin condensation, dispersion and nuclear fragmentation and plasma membrane breakage and organelle swelling in RAB-treated cells. RAB induced cells death in C. albicans is due to the enhanced ROS formation, which consistent with published studies that some antifungal agents exert the antifungal activity via ROS. RAB increases the mt△ψ, but decreases ATP level in a dose-dependent manner. The drop of ATP level is likely attributed to the mt△ψ hyperpolarization that is induced by enhanced ROS accumulation, which together contributes to the cell death.
To better understand the persistence of C. albicans, an effective model needs to be established to screen the persisters. The strain with TDH3tagged by GFP is treated by high dose of amphotericin B, then cells with GFP positive and PI negative are considered as persisters. This model revealed RAB had no action to eliminate the persister, in consistent with the conventional method. To isolate the persister for transcriptional profiling, different cultural conditions were screened to achieve high persister rate.
To construct a molecular tool used in C. albicans genetic research or screening antifungal agents, a plasmid backbone was inserted with NATl sequence (resistant to nourseothricin), an origin sequence ORC984or CEN4, and inverted telomeres at both ends. The constructed plasmids were linearized and transformed into C. albicans cells. The contour-clamped homogeneous electric field gel electrophoresis plus southern blot revealed some transformants have free plasmids in Candida cells, which are not integrative to the genomes. And the plasmids in the transformants could stably replicate in different generations. The plasmid carring a functional gene could also makes the tranformants display the phenotype, suggesting it is a good genetic tool for Candida research.
In this thesis, we described the antifungal mechanism of RAB, which is the basis for the mechanism of triterpene analogs. The combination treatment provides a possible application in overcoming clinical fungal infection. The investigation of persistence paves the way for understanding the mechanism of persistence and designing new drugs targeting the persister formation. The plasmid constructed for C. albicans works as an effective molecular tool in Candida genetic research and screening antifungal agents.
引文
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