新生隐球菌是一种环境获得性真菌病原体,每年导致140,000多人死亡。当感染性颗粒沉积到肺部时,就会发生隐球菌感染,在那里他们遇到宿主吞噬细胞。新生梭菌可能被这些吞噬细胞吞噬,感染的重要步骤,导致从感染终止到隐球菌传播的结果。为了研究这个关键过程,我们筛选了大约4,700个隐球菌基因缺失突变体,以改变摄取,使用原代小鼠和人吞噬细胞。在这两个屏幕的热门歌曲中,我们在两个系统中都鉴定了93个摄取受干扰的突变体,以及其他仅由一种细胞类型吸收差异的细胞。我们进一步筛选了胶囊厚度的变化,细胞周围的保护性多糖层是重要的隐球菌毒力因子。我们三个筛选的组合产生了45个突变体,包括缺乏磷脂酰肌醇-4-磷酸磷酸酶Sac1的。在这项工作中,我们暗示Sac1参与宿主细胞的摄取和囊的产生。我们发现sac1突变体表现出脂质运输缺陷,分泌系统功能的减少,以及胶囊大小和成分的变化。这些变化中有许多特别发生在组织培养基中,强调Sac1磷酸酶活性在响应宿主样条件的胁迫中的作用。总的来说,这些发现显示了基因组规模筛查如何鉴定有助于我们理解隐球菌生物学的细胞因子,并证明了Sac1在确定真菌毒力中的作用.新形式的IMPORTANCECryptococus是一种对全球健康具有重大影响的真菌病原体。从环境中吸入的隐球菌细胞沉积到肺部,他们第一次接触人体免疫系统的地方.新生梭状芽孢杆菌和宿主细胞之间的相互作用是关键的,因为感染的该步骤可以确定真菌细胞是否在人宿主内死亡或增殖。尽管这个感染阶段很重要,我们对影响其结果的隐球菌因素的了解有限。在这项研究中,我们确定了影响人类和小鼠细胞摄取的隐球菌基因。我们还鉴定了胶囊改变的突变体,围绕细胞的保护涂层,以保护它们免受宿主免疫系统的影响。最后,我们描述了一个基因的作用,SAC1,在这些过程中。总的来说,这项研究有助于我们理解新型梭菌是如何与宿主细胞相互作用并保护自身免受宿主细胞侵害的。
Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these
phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1, in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.