【作者】 苏英；

【导师】 李春阳；

【作者基本信息】 山东大学 ， 皮肤病学， 2009， 博士

【摘要】 白念珠菌是人类最常见的条件致病性真菌,亦是院内感染的主要病原菌。目前,念珠菌感染性疾病呈日趋增高趋势,且念珠菌血症的病死率居高不下,深部白念珠菌感染病死率可高达67.9%,白念珠菌感染已成为院内感染特别是免疫缺陷病患者最主要死因之一。随着抗真菌药物的大量应用,真菌耐药的情况变得越来越严重,特别是对常用唑类药物的耐药,有研究显示15.5%乃至更高的白念珠菌对氟康唑耐药,这给有效控制日益增高的念珠菌感染带来严峻挑战。白念珠菌耐药与其毒力因子之间存在重要关系,耐药株通常较敏感株毒力强。研究发现耐药基因CDR1断裂突变的白念珠菌毒力明显下降,毒力增强的耐药菌株可有耐药基因CDR和MDR的过度表达。且念珠菌菌丝相形成能力和耐药之间存在一种正相关关系。随着白念珠菌对氟康唑耐药性的出现,其分泌蛋白酶和磷脂酶等毒力因子的能力亦同时增加。因此研究白念珠菌毒力因子与耐药发生间的关系,降低念珠菌毒力与致病性,防止念珠菌耐药的发生,最终有效控制念珠菌感染是21世纪面临的重大科研任务。念珠菌致病性与其毒力因子密切相关,念珠菌通过黏附来识别宿主,通过其利于侵入的酶增强毒力,通过形态发生及酵母相与菌丝相之间的转换使其利于侵入宿主,并对该微生物的适应性有利。因此,念珠菌毒力因子包括黏附素、利于侵入的酶、形态发生及表型转换等。其中细胞外磷脂酶是念珠菌入侵组织的重要毒力因子,它作用于分子中不同酯健,使甘油磷脂形成溶血磷脂从而引起组织细胞损伤。磷脂酶包括A、B、C、D四种类型,磷脂酶A有促进白念珠菌引发炎症的作用,磷脂酶C和D与白念珠菌表型转换和信息传递有关,磷脂酶B主要通过分解宿主细胞膜磷脂而引起膜的通透性增高和完整性受损,继而促进白念珠菌的早期入侵。磷脂酶B又分为磷脂酶B1和磷脂酶B2,磷脂酶B1是一种分泌型糖蛋白,分子量为84KD,由605个氨基酸组成,它同时具有水解酶和溶血磷脂转酰酶的活性,研究发现一个PLB1基因敲除的白念珠菌菌株在体外产生的磷脂酶减少且致病性降低,PLB1是念珠菌致病所必需的毒力因子。唑类抗真菌药物主要通过抑制14α-去甲基化酶活性作用影响14α去甲基化,从而使麦角固醇的合成受阻,导致真菌细胞膜的完整性受损而发挥抗真菌作用。患者的细胞免疫功能缺陷,药物的反复应用,机体的感染状况及可能存在的耐药遗传性等相关因素均可诱导耐药的发生。唑类的耐药机制主要包括药物流出泵的过度表达,药物靶酶的突变和过度表达,生物被膜的形成及念珠菌细胞内滤泡对药物分子的扣押机制等。但亦有研究发现有些MIC值较高的菌株,毒力因子的活性却降低。毒力高的菌株所致感染易于控制,而毒力低的菌株对FCZ的治疗出现耐药。念珠菌耐药与其毒力因子间存在复杂的相互关系,目前,有关念珠菌磷脂酶与耐药发生关系的研究报道国内外并不多见,国内报道通过诱导使白念珠菌耐药,进而研究比较耐药菌株、诱导耐药菌株、敏感菌株磷脂酶、蛋白酶活性及致病性强弱的报道。而从分子水平研究磷脂酶B与耐药的关系目前还是空白。本研究拟通过M27-A方案筛选白念珠菌耐药菌株和敏感菌株,研究其磷脂酶活性和致病性的不同,并进一步研究两组磷脂酶B1的mRNA和蛋白表达的不同,为磷脂酶在耐药发生中的作用提供研究基础,以期最终能够有效控制念珠菌耐药和感染的发生。目的:1.通过蛋黄平板法研究白念珠菌耐药菌株和敏感菌株磷脂酶活性的差异。2.通过动物毒力试验比较白念珠菌耐药菌株和敏感菌株致病性的强弱。3.通过RT-PCR比较研究两组间磷脂酶B1转录水平mRNA表达的差异。4.通过Western blot法比较研究两组间磷脂酶B1蛋白表达水平的差异。方法:1.白念珠菌氟康唑耐药菌株与敏感菌株的筛选实验用白念珠菌均分离自山东大学齐鲁医院门诊患者,经血清芽管试验、厚壁孢子试验和YBC酵母鉴定卡鉴定试验证实为白念珠菌。根据NCCLS制订的M27-A方案中的操作步骤进行药敏试验,氟康唑药物浓度范围0.125μg/mL-64μg/mL,接种菌量为0.5-2.5×10³CFU/mL,35℃培养48h判定结果。2.磷脂酶活性的测定制备5×10⁷CFU/mL菌悬液,微量移液器移取配好的菌悬液10μL滴于蛋黄平皿表面,每株菌测定两份,将平皿密封后置35℃培养48h观察结果。测量菌落直径及沉淀圈直径,利用菌落直径与沉淀圈直径的比值（Pz值）来定量表示磷脂酶的活性,Pz=菌落直径/总直径（菌落直径+沉淀圈直径）,Pz值愈低,菌株磷脂酶活性就愈强,Pz值=1.0时表示磷脂酶活性阴性。3.耐药菌株与敏感菌株对小白鼠致病性的对比研究清洁级小白鼠30只,随机分成耐药组和敏感组,每组15只。制备敏感菌株和耐药菌株茵悬液,浓度为1-1.5×10⁷CFU/mL。分别于尾静脉注射感染小鼠,注射菌量为0.2mL,观察30d内小鼠存活天数,计算比较两组小鼠的死亡率和平均存活期。4.RT-PCR检测耐药菌株与敏感菌株磷脂酶B1 mRNA的表达应用E.Z.N.A Yeast RNA Kit试剂盒提取白念珠菌总RNA,具体步骤样严格按照说明书进行。分光光度计测定A₂₆₀/A₂₈₀,检测其浓度和纯度。以5‘—TCACCAACGCCATAAGTCC—3’,5’-CATCTTCTTCAACAGCAGCTTG-3’为磷脂酶B1引物,EFB1为内参照,进行逆转录反应和PCR扩增反应,PCR反应条件:95℃预变性5min,95℃变性45sec,58℃退火45sec,72℃延伸45sec。35个循环扩增目的基因,72℃终延伸7min。取10μLPCR产物,1.5%琼脂糖凝胶电泳20min,EB显色,Gel Dos 2000凝胶图像系统成像,Molecular Analyst图像分析软件分析扩增条带的亮度,磷脂酶B1 mRNA表达的相对系数=目的基因强度/EFB1基因强度。5.Western blot检测耐药菌株和敏感菌株磷脂酶B1蛋白表达的不同实验用菌株接种于50mL改良YPD培养基中,接种菌量为6×10⁶CFU/mL,35℃、200rpm摇床培养12h。离心吸取上清液过滤,盐析法提取胞外蛋白,P0013B强R1PA裂解液裂解提取细胞内蛋白质,BCA法测定蛋白质浓度。SDS-PAGE凝胶电泳后电转移至硝酸纤维素薄膜上,封闭液室温下振荡封闭2h。分别加入一抗（1:500内参照β-actin抗体、1:3000磷脂酶B1抗体）与胞外磷脂酶B1蛋白、胞内磷脂酶B1蛋白及β-actin结合,在培养皿中4℃反应过夜,洗膜液洗膜。分别加入HRP标记的抗鼠二抗（1:1250）反应液,室温下振荡反应2h,洗膜液洗膜。应用ECL试剂盒显影、定影。凝胶成像系统成像。用TotalLab1.0软件进行半定量分析各条带的相对吸光值,以各目的条带的IOD与β-actin的IOD比值代表其蛋白的相对表达量。结果:1.药敏试验结果与生长对照孔相比将浊度被抑制80%以上孔的药物浓度判定为MIC值,筛选出氟康唑耐药菌株（MIC＞64μg/mL）及敏感菌株（MIC＜8μg/mL）各15株。2.耐药组和敏感组磷脂酶活性比较15株耐药菌中12株产生特异沉淀圈,磷脂酶阳性率为80.0%,Pz值为0.786±0.055;敏感菌株中7株产生特异沉淀圈,阳性率为46.7%,Pz值为0.913±0.078。统计学检验两者磷脂酶活性有显著性差异,P＜0.01,耐药菌株磷脂酶活性明显强于敏感菌株。3.耐药组和敏感组小鼠平均存活期比较耐药组小鼠30d内死亡12只,敏感组小鼠30d内死亡6只。耐药组和敏感组小鼠的死亡率分别为80%和40%。两组小鼠死亡率和平均存活天数之间的差异有统计学意义,P＜0.05。耐药菌株对小鼠的致病性明显强于敏感菌株。4.耐药菌株与敏感菌株磷脂酶B1 mRNA表达水平的比较提取白念珠菌总RNA,检测OD₂₆₀/OD₂₈₀比值在1.8-2.0范围内,总RNA浓度和纯度符合试验要求。RT-PCR逆转录扩增耐药菌株和敏感菌株磷脂酶B1基因和内参照EFB1基因片段,扩增产物符合相应目的基因片段大小,预期目的基因片段大小约311bp。Molecular Analyst软件分析并计算耐药菌株和敏感菌株磷脂酶B1的mRNA相对表达强度,统计学检验显示耐药组和敏感组磷脂酶B1mRNA表达有显著性差异,P＜0.05,耐药菌株磷脂酶B1 mRNA表达强于敏感菌株。5.Western blot比较白念珠菌耐药菌株和敏感菌株磷脂酶B1蛋白表达水平以β-actin为内参照,白念珠菌耐药菌株胞内和胞外PLB1蛋白相对表达量为0.4145±0.2773和0.2720±0.2194,敏感菌株为0.1825±0.1831和0.0688±0.0631。耐药菌株胞内和胞外PLB1蛋白表达水平均高于敏感菌株,差异有统计学意义（P＜0.05）。结论:白念珠菌耐药菌株毒力因子磷脂酶的活性、菌株致病性、磷脂酶B1转录及翻译水平的表达均强于敏感菌株。白念珠菌磷脂酶与其耐药的发生可能存在一定相关性。更多还原

【Abstract】 Candida albicans is the most common human opportunistic pathogenic fungus and the most important organism of nosocomial infection.Recently the infection caused by Candida has dramatically increased,.The mortality rate of bloodstream infections was always at the high level which systemic infections caused by C.albicans had been estimated to be up to 67.9%.C.albicans had become a major lethal cause of hospital-acquired infectious patients especially in immunocompromised patients or immunologically debilitated individuals.With the wide use of antifungal agents,the condition of resistance in fungus became more and more severe,especially the resistance to azole antifungal agents.It was reported that 15.5%or more C.albicans was resistance to fluconazole.It was a big challenge to control the increased candidal infections effectively.There was an important correlation between antidrug of candida and virulent factors.Study indicated that gene disruption of a multidrug resistance MDR₁ leaded to dramatic attenuation of virulence in C.albicans.Resistant candidal strains with high-virulence exhibited over-expression of MDR and CDR,which encoded an ABC drug efflux pump.The resistant isolates of C.albicans were easier to translate from yeast cells to hyphae.The extracellular secreted proteinase and phospholipase activities were found to be higher with the emergence of fungal resistance.The virulence of resistant strains was usually more active than that of susceptible strains.It was an important and severe scientific task in the 21 century to study the relationship between resistance to antifungal drug and virulent factors of Candida,to reduce the virulence and pathogeny,and to prevent the emergency of resistance and cure the candidosis at lastA close correlation existed between pathogenicity of candida and virulent factors. Candida discriminated and invaded the host by adhesion,secreted enzymes which could enhance the virulence,phenotypic switching and the change between hyphal forms and spore forms which was good to adapt fungus to various environment and physiological conditions.Therefore,attributes that putatively contribute to candida virulence included adhesion,hyphal formation,phenotypic switching and extracellular hydrolytic enzyme production.Extracellular phospholipase played an important role in the pathogenesis of candida.Phospholipase targeted membrane phospholipids and digested these components,leading to cell lysis and direct host cell damage that lysis had been proposed as a major mechanism contributing to fungal virulence.Four types of phospholipase had been reported in C.albicans including phospholipase A,phospholipase B,phospholipase C and phospholipase D. Phospholipase A played a role in accelerating inflammation during candidal infection. Phospholipase C and phospholipase D were associated with the phenotypic switching and signal transduction.Phospholipase B could damage the integrality of host cell membrane and facilitate the penetration of the infecting agent by digesting membrane phospholipids components,which was beneficial to invade host in early stages. Phospholipase B was divided into phospholipase B1（PLB1） and phospholipase B2（PLB2）.PLB1 was a secreted protein,which displayed a molecular weight of 16KD and consisted of 605 amino acids.It had function of hydrolyst and lbysophospholipase-transacylase（LPTA）.A few studies had shown PLB1-deficient strains attenuated candidal virulence and pathogenicity,therefore,PLB1 was necessary during candidal infection as one of virulent factors.The 14a-lanosterol demethylase of fungus was involved in an important step in the biosynthesis of ergosterol which was essential for fungus to build their plasma membrane and was the target enzyme of azole drugs.Azole drugs acted efficiently by inhibiting the enzyme to damage the integrity of cell membrane.There were many related reasons why the fungus was resistance to antifungal drugs including immune dysfunction,taking the same medicine repeatly,infectious severity of the patient, resistance heredity to a antifungal agent and so on.Some molecular mechanisms of resistance to the azole drugs had been elucidated:overexpression of efflux pumps had been implicated in azole,of which encoding gene were CDR₁,CDR₂ and MDR₁; Point mutations in the gene(ERG₁₁) encoding the target of the azoles increased expression of ERG₁₁ which had been identified to be associated with azole resistance; Other mechanism included formation of biofilm and azole sequestration caused by vesicular vacuoles of the resistant isolates.However,it was suggested that decreased virulence of C.albicans isolates was associated with increasing the value of MICs to fluconazole（FCZ） in some cases but not in others and showed that the more virulent isolates caused infections which could be successfully treated,whereas the less virulent isolates caused infections which were refractory to fluconazole therapy. Clearly,the relationship was complex between virulence and resistance to azole.There were a limited number of reports on the correlation between phospholipase and resistance to antifungal agents.Only one in our country which determined the relationship between virulence production of secreted aspartyl proteinase and phospholipase of C.albicans and the developments of the resistance to FCZ by experimentally inducing resistant strains in liquid medium where FCZ concentration was increased gradually.We sought to determine if difference in phospholipase activity,pathogenicity and expression of PLB1 mRNA and PLB1 protein existed between fluconazole-susceptible and fluconazole-resistant C.albieans strains.We hoped the study was good to elucidate the role of phospholipase on emergence of candidal resistance to azole,so as to effectively control candidal infection and antifungal drug resistanceObjectives:1.Study the difference of phospholipase activity between fluconazole-resistant and fluconazole-susceptible C.albicans isolates. 2.Comparative study pathogenicity difference of C.albicans between fluconazole -resistant and fluconazole-susceptible strains by mice models.3.Comparative study expression difference of PLB1 mRNA between fluconazole-resistant and fluconazole-susceptible C.albicans strains by semi-quantitative RT-PCR4.Comparative study expression difference of PLB1 protein secreted by C.albicans between fluconazole-resistant and fluconazole-susceptible strains by Western blot analysisMethods:1.Screening the fluconazole-resistant and fluconazole-susceptible C.albicans strainsAll testing isolates were obtained from outpatients in QiLu Hospital,ShanDong University.C.albicans was confirmed by germ tube test,chlamydospore formation test and YBC identification test.Susceptibility testing to FCZ was performed by using the broth microdilution assay as described in the NCCLS document M27-A （National Committee for Clinical Laboratory Standards）.The inoculum suspension was prepared with a final inoculum of 0.5-2.5×10³CFU/mL.The final concentration of the antifungal agent was 0.125-64μg/mL The microplates were incubated at 35℃and MIC endpoints were read after 48h of incubation.Drug free and yeast control were included.C.albicans ATCC22029 was tested as a quantity control.2.Determination of phospholipase activityThe yeast suspension was adjusted to 5×10⁷CFU/mL.About 10μL of droplets of suspension of each C.albicans isolate was plated on the surface of the egg yolk medium.The plates sealed were incubated at 37℃for 48h.The phospholipase activity of the isolates was interpreted positive when a precipitation zone was visible around the growth colony formed on the plate.The value of phospholipase production（Pz） was determined by the ratio of the diameter of the colony to the total diameter of the colony plus the precipitation zone.Each candidal isolate was tested in duplicate.A Pz value of one indicated negative for phospholipase,and less than one（Pz<1） indicated the degree of phospholipase positivity.3.Determination of pathogenicity by mice models30 mice used in the study were divided into fluconazole-resistant group and fluconazole-susceptible group by randomization,and each group included 15 mice. each isolate was prepared inoculum suspension（1-1.5×10⁷CFU/mL）.Those mice were infected with fluconazole-resistant and fluconazole-susceptible strains respectively at the same time.Each mouse was injected with 0.2mL of cell suspension into the tail vein.The survival time of the mice were monitored twice a day for 30 days.4.RT-PCR analyses:Total RNA was extracted from C.albicans using E.Z.N.A Yeast RNA kit reagent kit according to the manufacturers instmctions.Spectrophoto-metry detected the value of OD₂₆₀/OD₂₈₀ to determine the purity and concentration of total RNA.In the semiquantitative analysis of PLB1 mRNA expression,the oligonucleotide PLB1 primers used were:5’-TCACCAACGAA GTCCCATCT-3’（forward）and 5’-CAACGAAGCGGTGTTGTCTA-3’（reverse）.EFB1 gene was amplified in the same PCR reaction condition as the internal control.The EFB1 primers were: 5’-ATTGAACGAATTCTTGGCTGAC-3’（forward）and 5’-CATCTTCTTCAACAGCAGCTTG-3’（reverse）,and expected PCR product,526 bp.The cycling conditions were as follows:preparation denaturation at 95℃for 5 min.denaturation at 95℃for 45sec,annealing at 58℃for 45see,extension at 72℃for 45see and 35 cycling was amplified for the target regions of PLB1,311 bp. final extension at 72℃for 7 min.The PCR products were examined on a 1.5% agarose gel with ethidium bromide staining.The gel image was captured with a GeI Dos 2000 Camera system.IOD value of PLB1 and EFB1 were determined by Molecular Analyst Quantification Software.5.Western blot analyses:C.albicans isolates（6×10⁶CFU/mL） were grown in YPD broth medium containing 4%glucose at 35℃for 12h on a rotary shaker（200rpm）.The cell-free supematant was obtained by centrifuging the medium at 3000gfor 10 min and was then filtered.Solid ammonium sulfate was then added to 65%saturation.The salted-out proteins were redissolved and dialyzed and then applied to a DEAE-cellulose column.The cells were lyses using P0013B RIPA Lysis Buffer to obtain the intracellular protein.Protein concentration was determined using BCA Protein Assay Kit.Equal amount of protein were separated on the polyacrylamide gel by SDS-PAGE and transferred to a nitrocellulose membrane.The transferred blots were incubated with 5%non-fat dry milk and probed with an primary antibody at 4℃ovemight.Anti-PLB1 antibody and anti-β-actin antibody were used at 1:3000 and 1:500 dilution respectively.After several washes,the membranes were incubated at room temperature for 2h with the secondary antibody conjugated to HRP, and then washed again.The blots were then visualized with enhanced chemiluminescence（ECL）.The IOD values of PLB1 andβ-actin protein expression were determined by the TotalLab1.0 Analyst Quantification software.Results:1.Determination of the MIC values:The MIC of FCZ was read as the lowest concentration at which 80%decrease in turbidity relative to the growth control was observed,and 15 fluconazole-resistant strains（MIC>64μg/mL） and 15 fluconazole-susceptible strains（MIC<8μg/mL） were obtained respectively.2.Comparison of phospholipase activity:12 of 15 fluconazole-resistant strains and 7 of 15 fluconazole-susceptible strains produced distinct precipitation zone implicated the phospholipase activity.The positive rate of phospholipase was 80.0% in fluconazole-resistant strains and 46.7%in fluconazole-susceptible strains.The mean values of Pz were（0.786±0.055） and（0.913±0.078） respectively.Significant difference was observed in the phospholipase activity between fluconazole-resistant strains and fluconazole-susceptible strains statistically（P<0.01）.The phospholipase activity of fluconazole-resistant strains was proved to be more superior than that of fluconazole-susceptible strains.3.Comparison of pathogenicity:12 of 15 infected mice in resistant group died within 30 days,40%of mice died in susceptible group.Significant differences were proved in the mortality and average survive time between two groups（P<0.05）.The virulence and pathogenicity of fluconazole-resistant group were stronger than that of fluconazole-susceptible group.4.Comparison of PLB1 mRNA expression:The value of OD₂₆₀/OD₂₈₀ detected by spectrophotometry comply with the standards.The target regions of PLB1 contained 311bp examined on agarose gel.There was significant difference in PLB1 mRNA expression between resistant and susceptible strains（P<0.05）.The level of PLB1 mRNA expression was higher in fluconazole-resistant C.albicans strains.5.Comparison of PLB1 protein expression:β-actin was selected as an internal control.IOD value of the band intensity of extracellular and intracellular PLB1 protein showed a significant increase in fluconazole-resistant C.albicans strains.The level of extracellular and intracellular PLB1 protein in fluconazole-resistant strains were higher than those in fluconazole-susceptible strains（p<0.05）.Conclusions:Phospholipase activity,pathogenicity to mice,expression of PLB1 mRNA and expression of PLB1 protein in fluconazole-resistant isolates of C.albieans were higher than those in fluconazole-susceptible isolates.There was relationship between the phospholipase and resistance to antifungal drugs in C.albicans.Phospholipase maybe play a role in the emergence of resistance to antifungal drug.更多还原