Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis
Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis
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DOI：
10.1128/iai.00551-10
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发表时间：
2010-10-01
影响因子：
3.1
通讯作者：
Perfect, John R.
中科院分区：
文献类型：
--
作者：
Lee, Anthony;Toffaletti, Dena L.;Perfect, John R.
新型隐球菌是一种真菌病原体，在哺乳动物宿主的生长过程中会遇到各种微环境，包括细胞内液泡、血液和脑脊液（CSF）。由于脑脊液被血脑屏障隔离，我们假设脑脊液表现出独特的压力，新生C.必须克服这些压力才能建立感染。我们检测了1201个突变体在生长培养基、生理盐水和人脑脊液中的存活缺陷。我们评估了csf特异性突变体(i)突变体在人支气管肺泡灌洗液（BAL）和胎牛血清（FBS）中的存活，（ii）在巨噬细胞中的存活，以及（iii）在秀丽隐球菌和兔隐球菌模型中的毒力。13个突变体表现出CSF特有的显著生存缺陷。在临床血清型A菌株H99中重现了其中三个突变体的突变：阳离子atp酶转运蛋白（ena1 Delta）、推测的NEDD8泛素样蛋白（rub1 Delta）和磷脂酰肌醇4-激酶（pik1 Delta）的基因缺失。突变体在酵母培养基、生理盐水和BAL液中的存活率与野生型相似；然而，FBS的存活率降低，但没有降低到CSF的水平。这些突变菌株在巨噬细胞中也表现出细胞内存活率下降，在线虫中表现出不同程度的毒力，在兔脑膜炎模型中表现出严重的存活率下降。我们用质谱法分析脑脊液中导致生存缺陷的候选化合物。我们的研究结果表明，新生C.在脑脊液中存活所需的基因是在这种独特的宿主环境中生存和感染所必需的。
Cryptococcus neoformans is a fungal pathogen that encounters various microenvironments during growth in the mammalian host, including intracellular vacuoles, blood, and cerebrospinal fluid (CSF). Because the CSF is isolated by the blood-brain barrier, we hypothesize that CSF presents unique stresses that C. neoformans must overcome to establish an infection. We assayed 1,201 mutants for survival defects in growth media, saline, and human CSF. We assessed CSF-specific mutants for (i) mutant survival in both human bronchoalveolar lavage (BAL) fluid and fetal bovine serum (FBS), (ii) survival in macrophages, and (iii) virulence using both Caenorhabditis elegans and rabbit models of cryptococcosis. Thirteen mutants exhibited significant survival defects unique to CSF. The mutations of three of these mutants were recreated in the clinical serotype A strain H99: deletions of the genes for a cation ATPase transporter (ena1 Delta), a putative NEDD8 ubiquitin-like protein (rub1 Delta), and a phosphatidylinositol 4-kinase (pik1 Delta). Mutant survival rates in yeast media, saline, and BAL fluid were similar to those of the wild type; however, survival in FBS was reduced but not to the levels in CSF. These mutant strains also exhibited decreased intracellular survival in macrophages, various degrees of virulence in nematodes, and severe attenuation of survival in a rabbit meningitis model. We analyzed the CSF by mass spectrometry for candidate compounds responsible for the survival defect. Our findings indicate that the genes required for C. neoformans survival in CSF ex vivo are necessary for survival and infection in this unique host environment.