Alanio Alexandre, Vernel-Pauillac Frédérique, Sturny-Leclère Aude, Dromer Françoise
mBio. 2015 Mar 31;6(2):e02580-14. doi: 10.1128/mBio.02580-14.
Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studied C. neoformans infection at the macrophage level (in vitro H99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing several C. neoformans populations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over time in vivo and in vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could exist in vitro and in vivo. C. neoformans exhibits a huge plasticity and adaptation to hosts that deserves further study. In vitro generation of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.
Cryptococcus neoformans is a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment. C. neoformans causes cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying an in vitro model of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy in C. neoformans.
隐球菌病是由无处不在的新型隐球菌酵母引起的机会性感染。这种酵母与先天免疫细胞密切相互作用，导致多种结果，包括真菌在细胞内持续存在，从而使得酵母细胞有可能通过特洛伊木马策略随单核细胞传播。在人类中，感染的自然史始于儿童期的初次感染，随后进入休眠状态，在某些个体中，免疫抑制时会重新激活。为了解决休眠问题，我们在巨噬细胞水平（体外H99-巨噬细胞相互作用）和隐球菌病小鼠模型的器官水平上研究了新型隐球菌感染。我们通过基于流式细胞术（定量流式细胞术、多光谱成像流式细胞术、分选）、显微镜检查（动态成像）和基因表达分析的新方法对几个新型隐球菌群体进行特征描述，分析了酵母对宿主适应的多样性。根据增殖和应激反应参数，在体内和体外随时间观察到了不同的酵母细胞群体。细胞分选使得能够鉴定出一个亚群，该亚群在标准条件下比其他群体更不易生长，添加血清可增强其生长。基因表达分析表明，该群体具有可能反映休眠的特定代谢特征。我们的数据表明，休眠酵母细胞可能存在于体外和体内。新型隐球菌表现出巨大的可塑性和对宿主的适应性，值得进一步研究。体外产生休眠细胞是目前克服我们模型中回收的酵母细胞数量有限这一问题的主要挑战。
新型隐球菌是一种有荚膜的单细胞真菌，它与各种细胞和生物体密切相互作用，包括变形虫、线虫和哺乳动物的免疫细胞。这种酵母能够在细胞内环境中增殖和存活。新型隐球菌会引起隐球菌病，根据多年前获得的流行病学证据，有人提出人类体内存在酵母休眠现象。通过研究酵母-巨噬细胞相互作用的体外模型和隐球菌病小鼠模型，我们观察到酵母细胞在感染期间基于整体代谢活性在异质群体中进化。我们比较了这两种模型中不同群体酵母细胞的生长能力和基因表达。我们最终发现了一群代谢低的酵母细胞，它们符合一些休眠细胞的标准。这为进一步表征新型隐球菌的休眠铺平了道路。
