【作者】 孔庆涛；

【导师】 桑红；

【作者基本信息】 南京大学 ， 内科学， 2012， 硕士

【摘要】 背景和目的新生隐球菌(C.neoformans)在环境中广泛存在,是单倍雌雄异株的担子菌类机会性致病性真菌,它是一种重要的能导致系统感染的病原真菌,主要侵犯AIDS、器官移植、细胞毒药物化疗和皮质类固醇长期应用等造成的免疫缺陷患者,也可感染免疫功能正常的人群,主要导致致命性的脑膜脑炎,感染死亡率很高,而且这种感染会造成慢性化或潜伏感染状态。C.neoformans主要的致病因素为荚膜、产黑素、尿素酶和磷脂酶的毒性作用及其在宿主体温环境生长的能力等。C.neoformans是专性需氧致病酵母菌,在实验室条件下,新生隐球菌在大气氧含量(21%)条件下生长最佳。吸入的C.neoformans可以通过血液播散到达中枢神经系统主要导致致命性的脑膜脑炎。众所周知,人脑组织的氧浓度明显低于大气氧含量(21%),不同的解剖部位也不同。此外,炎症、血栓形成以及感染相关的坏死也导致组织氧含量下降。因此,C.neoformans在致病过程中必须面对并适应宿主组织氧含量的变化。国内外最新研究认为C.neoformans这种专性需氧致病酵母菌可以适应缺氧环境并生存,并且在中枢神经系统的生长在于调整其复制速率以适应相应的氧含量的变化,并描述了C.neoformans两种可能感应缺氧的途径,即SREBP(甾醇调节因子连接蛋白家族)和Tco1途径,但缺氧适应对C.neoformans毒性因子的影响尚未被关注。我们前期研究中发现新生隐球菌在相对缺氧组织中荚膜生成缺陷,因此我们提出假设：缺氧可能导致新生隐球菌重要致病表型发生变化,使新生隐球菌在宿主中进入慢性或潜伏感染状态,导致感染迁延不愈。本研究通过建立不同氧含量的培养条件,探讨氧含量变化对C.neoformans毒力因子的影响,以期为揭示缺氧在新生隐球菌导致潜伏感染或慢性化感染的机制中的作用提供新思路。方法本研究以厌氧、微需氧和二氧化碳产气袋建立不同氧含量培养条件,以大气条件为正常氧含量培养条件,以新生隐球菌标准株5株(BLS71、BLS63、ATCC32609、ATCC3487、YD53,分别为血清型A、B、C、D、AD),临床株1株为研究对象,利用形态学观察培养基(YPD和YES)、产黑素培养基、尿素酶培养基和卵黄培养基等对不同菌株进行培养,通过观测菌落直径、荚膜厚度、产黑培养基和尿素酶培养基颜色变化情况以及卵黄培养基中的PZ值等指标,探讨不同氧含量培养条件对新生隐球菌毒力因子的影响。结果1.形态学结果各个菌株在不同氧含量条件下均有生长,正常氧含量条件下菌落生长最好,受试菌株菌落生长速度随氧含量的递减而减慢,所有菌株厌氧条件下生长明显减慢,未出现明显的乳白色酵母样菌落,菌落直径比其他三组明显减小。同种培养条件下各个菌株之间菌落直径无明显差异。YPD和YES两种培养基对隐球菌生长无明显影响,菌落大小基本一致。2.产黑素结果在多巴培养基中,3天时正常氧、二氧化碳及微含氧培养条件下的所有受试菌株均使整个培养基变为深褐色,厌氧条件下培养基颜色未出现明显变化,7天时培养基颜色变化程度比3天时加深。在咖啡酸培养基中,3天时除正常氧条件下所有受试菌株菌落及周围出现咖啡色外,其余条件下均未出现颜色变化,7天时结果与3天基本一致。3.荚膜测量结果除菌株YD53(血清型AD)外,其余受试菌株均可形成明显的荚膜。YD53在同样培养条件下,大多以假菌丝样形式生长。在非诱导培养条件下,菌株BLS71、BLS63、ATCC32609和临床株在四种氧含量培养条件下荚膜大小差异有统计学意义。在诱导培养条件下,菌株BLS71、BLS63和临床株在厌氧培养条件下的荚膜厚度比其余氧含量条件下的荚膜明显变小。在所有氧含量培养条件下,菌株BLS71和临床株诱导条件下荚膜比非诱导条件下荚膜大,差异有统计学意义。4.尿素酶和磷脂酶活性培养7天时,正常氧含量培养条件下除菌株ATCC32609未出现颜色变化,其他菌株均出现不同程度的颜色变化,微含氧和厌氧培养条件下所有受试菌株均未出现颜色变化；将观察时间延长至14天,可见正常氧含量及二氧化碳培养条件下所有受试菌株都出现不同程度的颜色变化,而厌氧组培养条件下均未出现颜色变化。培养7天时,除厌氧培养条件下受试菌株未出现沉淀圈外,其余培养条件下的所有受试菌株均出现了特异性的白色沉淀圈。菌株BLS71和ATCC34874在正常氧培养条件下PZ值小于其他培养条件,差异有统计学意义。同一菌株培养条件随氧含量的降低,PZ值有增大趋势。同一种培养条件下受试菌株PZ值差异无统计学意义。结论新生隐球菌可以在厌氧条件下生长,低氧可能对新生隐球菌荚膜、产黑素、尿素酶和磷脂酶活性具有抑制作用。不同血清型的新生隐球菌菌株对氧含量变化的反应可能不同。更多还原

【Abstract】 Background and PurposeC. neoformans is an environmental pathogen found primarily in pigeon droppings and soil contaminated with avian guanos throughout the world. It is a heterothallic, basidiomycetous, pathogenic fungus that infects both immunocompetent and immunocompromised individuals. The prevalence of disease caused by this organism has increased dramatically as a result of human immunodeficiency virus (HIV) infection, organ transplantation, cytotoxic chemotherapy and corticosteroid use. And the infection caused by C. neoformans can result in chronic dormant infection with the potential for acute outbreaks. C. neoformans has several well established virulence factors:capsule, melanin, expression of urease and phospholipase, and the ability to grow at human body temperature.C. neoformans is strictly an obligate aerobic pathogenic yeast. In laboratory conditions, atmospheric levels of oxygen (21%) are required for optimal growth of C. neoformans and lower oxygen concentrations lead to a significant reduction in cell growth. The inhaled C. neoformans cells reach the central nervous system by haematogenous dissemination and cause life-threatening meningoencephalitis. It is well known that oxygen concentrations in the human brain are reported to be drastically lower than in the atmosphere and vary significantly among anatomical sites. Moreover, inflammation, thrombosis, and necrosis associated with infection are thought to lead to increased degrees of hypoxia. Thus, in order to establish infection in the host, C. neoformans cells must sense and adapt to rapidly changing oxygen level. Recent studies have demonstrated the obligate aerobic pathogenic yeast C. neoformans can adapt to and survive in hypoxic conditions, and indicated that the central strategy of C. neoformans lies in adjustment of its proliferation rate to the available oxygen levels in CNS, Sterol-response element binding protein (SREBP) and Tco pathways seem to be two sensing systems involved in hypoxia sensing in C. neoformans, while there is still a lack of information on the effect of hypoxia on virulence of C. neoformans. We found that C. neoformans did not form apparent capsule under hypoxia conditions. Hence we propose a hypothesis that cryptococcal response to hypoxia might be the driving force for developing a state of dormant infection which is characterized by extensive changes in phenotype and virulence factors.This study will focus on the effect of different oxygen concentrations caused by AnaeroPack Series on virulence of C. neoformans, in order to provide new ideas for the role of hypoxia in chronic dormant infection caused by C. neoformans.MethodsC. neoformans type strains (BLS71, BLS63, ATCC32609, ATCC34874and YD53, serotype A, B, C, D and AD, respectively) and clinical isolates were grown in YPD at37℃. The cultures were then spotted on to YPD, YES, DOPA (dihydroxyphenylalanine) and caffeic acid agars, urea agar and egg yolk agar under various oxygen levels caused by AnaeroPack Series. The colony diameter, capsule size, pigmentation, urease and phospholipase activity of strains were observed and the effect of different oxygen concentrations on virulence factors of C. neoformans was investigated.Results1. Morphology observationThe test strains can grow under different oxygen concentrations and grow better under normoxic conditions than other conditions. C. neoformans slows its growth as the oxygen concentration declines. All test strains did not form visible yeast-like colony under anaerobic condition and colony diameters were significantly smaller than other three groups. No differences of colony diameters of all strains were found under the same oxygen level culture conditions. No differences of growth rates and colony sizes between the YPD and YES media were found.2. Melanin productionWith exception of anaerobic condition, all species were able to produce melanin on DOPA media after3and7days under other three culture conditions. Conversely, most strains showed no evidence of pigmentation on caffeic acid agar under different oxygen concentrations except normoxic conditions.3. CapsuleWith exception of YD53, all strains form polysaccharide capsules under any of oxygen levels. In capsule non-inducing conditions, there was significant difference between the four oxygen concentrations in BLS71、BLS63、ATCC32609and clinical isolates. BLS71、BLS63and clinical isolates have a smaller capsule under anaerobic condition. BLS71and clinical isolates have a greater capsule in capsule-inducing conditions than non-inducing conditions under any of the four oxygen concentrations.4. Urease and Phospholipase activityAll strains except ATCC32609display pink colour intensity after incubation in atmospheric conditions for7days. All tested strains showed no urease activity under anaerobic condition after incubation for14days. With exception of anaerobic condition, all C. neoformans in this study expressed phospholipase activity after7days. The PZs of BLS71, ATCC32609and ATCC34874in normoxic conditions were smaller than in other conditions. The PZs of the same strains increased as the oxygen concentration decline. There was no significant correlation of phospholipase activity with different strains in the same culture condition.ConclusionC. neoformans can grow under anaerobic condition. Hypoxia may have some inhibiting effect on capsule formation, melanin production and the activities of phospholipase and urease of C. neoformans. The strains tested may have different susceptibility to oxygen. This study suggested that serotypes may affect the susceptibility of C. neoformans to various oxygen concentrations.更多还原