氧含量对新生隐球菌形态学及毒力因子影响的初步研究

发布时间：2017-12-31 06:26

本文关键词：氧含量对新生隐球菌形态学及毒力因子影响的初步研究　出处：《南京大学》2012年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background and purpose
Cryptococcus neoformans (C.neoformans) exists widely in the environment, is a heterothallic basidiomycete opportunistic pathogenic fungi, it is a kind of important fungal pathogens can lead to infection, mainly involving AIDS, organ transplantation, caused by cytotoxic chemotherapy and long-term corticosteroid application in immunodeficient patients, can be infected people with normal immune function, which lead to fatal encephalitis infection, the mortality rate is very high, and the infection can cause chronic or latent infection of the main pathogenic factors for.C.neoformans capsule, melanin production, toxicity of urease and phospholipase and temperature in the host environment for the growth of ability.
C.neoformans is an obligate aerobic pathogenic yeast, under laboratory conditions, Cryptococcus neoformans in atmospheric oxygen content (21%) under the conditions of growth of the best. Inhalation of C.neoformans can reach the central nervous system through the blood spread mainly cause life-threatening meningoencephalitis. As everyone knows, the oxygen concentration of brain tissue was significantly lower than the atmospheric oxygen content (21%), anatomy the different parts are different. In addition, inflammation, thrombosis and necrosis associated with infection also leads to decreased oxygen content. Therefore, C.neoformans must face and adapt to changes in the oxygen content of the host tissue in the pathogenic process at home and abroad. According to the latest research from C.neoformans this obligate aerobic pathogenic yeast can adapt to hypoxic environment and survival, and is adjust the replication rate to adapt to changes in the oxygen content of the corresponding growth in the central nervous system, and describes two possible C.neoformans Way of induction of hypoxia, namely SREBP (sterol regulatory element junction protein family) and Tco1 pathway, but the effect of preconditioning on C.neoformans virulence factors have not been concerned. Cryptococcus neoformans in relative hypoxia tissue defects generated in our previous study found that the capsule, so we hypothesized that hypoxia may lead to important pathogenic phenotype of Cryptococcus neoformans the change of Cryptococcus neoformans in the host into chronic or latent infection, leading to delayed healing of infection.
This study through the establishment of different culture conditions on oxygen content, oxygen content effect on C.neoformans virulence factors, in order to reveal hypoxia in Cryptococcus neoformans cause latent infection or chronic infection mechanism to provide new ideas.
Methods in this study, the anaerobic, microaerophilic and carbon dioxide gas generating bag to establish culture conditions with different oxygen content, with atmospheric conditions for cultivating conditions of normal oxygen content, with 5 strains of Cryptococcus neoformans strains (BLS71, BLS63, ATCC32609, ATCC3487, YD53, respectively, serotype A, B, C, D, AD), clinical 1 strains as the research object, using the morphological observation of culture medium (YPD and YES), producing melanin medium, urease medium and egg yolk medium of different strains were cultured by observing colony diameter, the thickness of the capsule, black culture medium and culture medium and color change of urease yolk medium PZ value, to investigate the effect of culture conditions on Cryptococcus neoformans virulence factors with different oxygen content.
Result
1. morphological results
All the strains in the conditions of different oxygen contents were normal growth, oxygen content under the condition of colony growth, the tested strains colony growth rate with decreasing oxygen content decreased, the growth slowed down all the strains under anaerobic conditions, no obvious white yeast like colonies, the colony diameter was significantly reduced than in the other three groups of the same culture. Under the condition of each strain between colony diameter had no significant difference in.YPD and YES two kinds of medium on the growth of Cryptococcus neoformans had no obvious effect on colony size is basically the same.
2. production of melanin
In DOPA medium, normal oxygen for 3 days, carbon dioxide and oxygen micro culture all isolates were the medium to dark brown color of the medium conditions, there was no significant change in culture under anaerobic conditions, the 7 day training degree based color change 3 days than in the coffee acid burn. The medium for 3 days in addition to the normal oxygen condition of all the subjects appear Brown colony and around, under other conditions there were no color change, 7 days and 3 days were basically the same.
3. capsular measurement results
Except for the bacteria strain YD53 (serotype AD), the other tested strains could form a distinct capsule.YD53 under the same culture conditions, mostly in the form of false mycelium like growth.
Under non inducing conditions, strain BLS71, BLS63, ATCC32609 and clinical isolates in four kinds of oxygen content in culture has significant difference in size of capsular condition. Under the condition of strain BLS71 cultured BLS63 and clinical strains in anaerobic culture conditions than the rest of the thickness of the capsule of oxygen under the condition of the capsule was significantly smaller in all culture conditions. The oxygen content, strain BLS71 and strain induced clinical conditions than non capsular inducing conditions capsule, the difference was statistically significant.
4. activity of urease and phospholipase
When cultured for 7 days, normal oxygen culture conditions except strain ATCC32609 without color change, other strains have different degrees of color change, containing micro aerobic and anaerobic culture conditions of all subjects were not changed color strains; the observing time is extended to 14 days, all the tested strains have different degrees of the color change under normal oxygen and carbon dioxide visible and anaerobic culture conditions, group culture conditions showed no color change.
When cultured for 7 days, in addition to anaerobic culture conditions tested strains did not appear precipitation outside the ring, the culture conditions of all tested strains showed a white precipitate ring specific. Strains BLS71 and ATCC34874 culture conditions, PZ value is less than the other culture conditions in normal oxygen, statistically significant differences between the same strain. Culture conditions with oxygen content decreased, PZ value increased. The same culture conditions tested strains PZ were no significant difference.
conclusion
Cryptococcus neoformans can grow under anaerobic conditions. Hypoxia may inhibit the production of melanin, urease and phospholipase activity in Cryptococcus neoformans. The reactions of different serotypes of Cryptococcus neoformans to oxygen content may be different.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R379

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