Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30–40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.代谢静止的病原体可维持存活但不复制的状态达数月乃至数年。对于肺结核、隐球菌病、组织胞浆菌病等部分传染病而言，潜伏感染正是这类休眠状态的必然结果，且存在复激活并引发临床疾病的风险。在小鼠隐球菌感染及巨噬细胞吞噬过程中，应激与宿主免疫会诱导新生隐球菌（Cryptococcus neoformans）产生异质性，形成一类酵母亚群，其表型符合休眠特征（低应激反应、生长潜伏期）。该亚群的线粒体转录活性受到调控，这一表型曾被认为是干细胞静止状态的标志性特征。基于上述发现，我们致力于在体外重现该表型，随后标准化实验条件，以稳定诱导新生隐球菌进入休眠状态。我们发现，将静止期酵母（STAT, stationary phase yeasts）置于营养限制条件与缺氧环境中培养8天（8D-HYPOx），可获得模拟体内所得表型的酵母细胞。与常氧培养8天（8D-NORMOx）组中30%~40%酵母发生凋亡或死亡相比，该处理组的酵母死亡率/凋亡率低于5%。8D-HYPOx组酵母的应激反应更低、生长延迟，且在琼脂平板上的可培养率低于1%，表明此类酵母属于活的但不可培养细胞（VBNC, viable but non culturable cells）。这些VBNC细胞在泛酸（pantothenic acid）存在时可被复激活——泛酸是一种已知参与群体感应的维生素，同时也是乙酰辅酶A（acetyl-CoA）的前体。与8D-NORMOx及STAT组相比，8D-HYPOx细胞的全局代谢存在特异性需求，且整体代谢通路处于抑制状态。线粒体分析显示，相较于8D-NORMOx组，8D-HYPOx组的线粒体质量增加，且线粒体大多处于去极化状态，同时线粒体基因的表达水平升高。对8D-HYPOx组进行蛋白质组学与转录组学分析后发现，与8D-NORMOx及STAT组相比，其检测到的分泌蛋白与转录本数量均有所减少，且其蛋白质组、分泌组与转录组在培养仅4天时就已形成特异性特征谱。尤为重要的是，乙酰辅酶A及涉及线粒体的脂肪酸通路，对于VBNC的产生与存活维持必不可少。综上，本研究首次在新生隐球菌中成功诱导出VBNC表型。该VBNC状态与特异性代谢模式相关，有待进一步研究以阐明该酵母的休眠/静止机制。