以CpG-ODN为佐剂的新型隐球菌疫苗研究

新型隐球菌感染尚无有效的预防手段。本文探讨以Cp G为佐剂开发抗新型隐球菌疫苗的可行性。将经新型隐球菌甘露糖蛋白(mannoprotein,MP)或/和Cp G-ODN刺激过的DCs经气管接种于实验鼠,检测支气管肺泡灌洗液(BALF)中IFN-γ的产生。将新型隐球菌接种于实验鼠气管内,取气管旁淋巴结细胞与经新型隐球菌MP(mannoprotein)或/和Cp G-ODN预处理过的DCs共培养,检测培养上清中IFN-γ的产生。结果显示,经MP刺激过的DCs共培养可诱导淋巴细胞产生IFN-γ。以MP为抗原,Cp G-ODN为佐剂的疫苗可以保护小鼠抵御新型隐球菌感染。本研究证实了隐球菌甘露糖蛋白MP通过DCs诱导了Th1型反应,并提示了以Cp G-ODN为佐剂基于MP制备新型隐球菌疫苗的可能性。     

小檗碱抗新生隐球菌活性和作用机制

【目的】研究小檗碱对新生隐球菌的抗菌活性及其作用机制。【方法】采用微量肉汤稀释法测定小檗碱对新生隐球菌标准菌株和临床分离菌株的最小抑菌浓度,通过棋盘法测定小檗碱与氟康唑、两性霉素B的协同作用,测定小檗碱对隐球菌重要毒力因子的表达,以及对巨噬细胞和隐球菌互作的影响,采用隐球菌感染大蜡螟模型测定小檗碱的体内杀菌活性。【结果】小檗碱是一种杀真菌化合物,在测试的菌株中,最小抑菌浓度(minimum inhibitory concentration, MIC)范围为8-16μg/mL。亚致死剂量小檗碱能够抑制隐球菌荚膜大小、产黑色素能力和有性生殖能力,并能增强巨噬细胞的杀菌能力。锌指转录因子Nrg1介导了上述重要的过程。在隐球菌感染动物模型中,小檗碱能够延长感染大蜡螟的存活时间。【结论】小檗碱在体内外具有优异的抗隐球菌活性,有望作为抗隐球菌药物开发的起始化合物。     

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

Background  

Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and C. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. C. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of C. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes.     

一种制备新生隐球菌单细胞的方法

【目的】建立流式细胞仪分选新生隐球菌单细胞的方法,确定新生隐球菌单细胞恢复生长的条件和能力。【方法】利用Moflo XDP流式细胞分析分选仪,体外测定不同条件下新生隐球菌恢复生长的比率。【结果】建立了流式细胞仪新生隐球菌单细胞分选流程。确定流式细胞仪分选得到的新生隐球菌单细胞具有恢复生长的能力,恢复生长的能力受营养条件和菌株差异的影响。在营养丰富的条件下,新生隐球菌JEC21和H99单细胞恢复生长比率分别为74%和89%。在寡营养条件下,JEC21和H99单细胞恢复生长比率分别为37%和80%。JEC21生长比率随细胞数的增加而升高,细胞数为100个时,生长比率为55%;细胞数为1000个时,生长比率为97%。【结论】流式细胞仪分选得到新生隐球菌单细胞具有恢复生长的能力,生长能力受营养条件、菌株差异的影响。     

新生隐球菌一个新的产孢相关蛋白Srp1的鉴定与功能分析

【目的】研究产孢相关蛋白Srp1在新生隐球菌有性产孢和致病性中的作用及机理。【方法】采用基因枪转化技术构建新生隐球菌SRP1基因缺失突变体及其互补菌株,并通过小鼠致病性实验和菌株交配实验检测Srp1在新生隐球菌有性产孢和致病性中的作用。【结果】与野生型菌株相比,srp1Δ突变体小鼠致病性无差异;srp1Δ突变体能够交配并形成双核菌丝,但丧失产生担孢子的能力;初步机理分析表明srp1Δ突变体交配后其减数分裂过程被阻断,从而导致srp1Δ突变体不能产生担孢子。【结论】产孢相关蛋白Srp1不影响新生隐球菌的致病性,但可通过调控减数分裂过程影响新生隐球菌的有性生殖。     

新生隐球菌半胱氨酸转运蛋白Mup1鉴定及其表达调控研究

【目的】鉴定新生隐球菌(Cryptococcus neoformans)的半胱氨酸转运蛋白及其对致病性的影响。【方法】构建候选基因敲除株,检测突变株以半胱氨酸为唯一硫源的生长情况;检测半胱氨酸转运蛋白Mup1对新生隐球菌毒力因子表达和不同胁迫条件下生长的影响;通过新生隐球菌大蜡螟(Galleria mellonella)和小鼠感染模型分析Mup1对致病性的影响;通过转录组分析和酵母单杂交研究硫代谢核心转录因子Cys3与Mup1的调控关系。【结果】Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的能力。基因MUP1缺失不影响毒力因子表达和细胞对应激的反应。大蜡螟和小鼠隐球菌感染模型表明Mup1对新生隐球菌的致病性无显著影响。转录组分析和酵母单杂交实验显示Cys3可能间接调控MUP1的转录。【结论】新生隐球菌Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的功能,但不影响致病性,基因转录可能受Cys3的间接调控。     

新型隐球酵母U6启动子的鉴定、克隆及功能验证

【目的】鉴定及克隆新型隐球酵母(Cryptococcus neoformans)中PolⅢ型的U6启动子(Cn U6启动子),并验证CnU6启动子能否有效转录shRNA及CRISPR/Cas9系统中gRNA。【方法】结合Gen Bank数据库已公布的新型隐球酵母基因组信息和本实验室RNA-seq文库数据,利用生物信息学技术分析得到新型隐球酵母中具有高转录水平的U6RNA序列。使用重叠PCR和Easy Geno方法将预测的CnU6启动子分别克隆到sh RNA及gRNA的上游区域,通过观察shRNA对靶基因的RNAi效果及gRNA引导Cas9核酸酶对靶位点的切割结果,确定CnU6启动子能否转录短RNA。【结果】CnU6启动子能够转录形成shRNA对靶基因进行沉默,并且能转录形成g RNA引导Cas9核酸酶对靶点进行切割。【结论】新型隐球酵母的CnU6启动子被成功鉴定及克隆,它能有效驱动shRNA和gRNA的转录。