Upadhya Rajendra, Lam Woei C, Hole Camaron R, Vasselli Joseph G, Lodge Jennifer K
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, United States.
Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, United States.
Front Fungal Biol. 2023;4. doi: 10.3389/ffunb.2023.1183291. Epub 2023 May 12.
is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence.
The purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response.
Yeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection.
We observed that the growth of wild-type KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells.
These findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the cell wall, as well as their impact on fungal virulence and the host response.
新型隐球菌是一种担子菌纲真菌，可引起脑膜脑炎，尤其是在免疫功能低下的患者中。新型隐球菌能在多种不同培养基中生长，不过人们很少关注生长条件对新型隐球菌细胞壁或毒力的作用。
本研究的目的是确定不同培养基如何影响细胞壁中几丁质和壳聚糖的含量，进而影响细胞壁结构和宿主反应。
酵母提取物、蛋白胨和葡萄糖（YPD）培养基以及酵母氮源（YNB）培养基是在体外或体内实验中培养新型隐球菌常用的两种培养基。因此，新型隐球菌在YPD或YNB中培养，这两种培养基要么不进行缓冲，要么用吗啉代丙烷磺酸（MOPS）缓冲至pH 7。然后用细胞壁特异性荧光探针标记这些细胞，以确定各种细胞壁成分的含量。此外，使用小鼠吸入感染模型，将这些细胞用于动物毒力研究。
我们观察到，野生型新型隐球菌KN99在生长过程中会显著改变未缓冲培养基的pH值。在未缓冲的YPD中生长时，pH值会升至8.0，而在未缓冲的YNB（YNB-U）中生长时，pH值会降至2.0。重要的是，细胞壁的组成受到不同培养基中生长的显著影响。与在YPD中生长的细胞相比，在YNB-U中生长的细胞中壳聚糖（几丁质的脱乙酰形式）减少了90%。与在YPD或pH 7的YNB中生长的细胞相比，YNB-U中生长的细胞pH值和壳聚糖的降低与细胞表面一些病原体相关分子模式的显著增加有关。这种改变的细胞壁结构在使用小鼠感染模型进行测试时导致毒力显著降低。此外，当使用热灭活细胞作为接种物时，在YNB-U中生长的KN99细胞在肺部引起异常的过度炎症反应，导致动物迅速死亡。相比之下，在pH 7的YNB中生长的热灭活KN99细胞在宿主肺部几乎不引起炎症反应，但用作疫苗时，它们对随后的强毒KN99细胞攻击感染提供了强大的保护反应。
这些发现强调了生长过程中培养基和pH值在塑造新型隐球菌细胞壁的成分和组织方面的重要性，以及它们对真菌毒力和宿主反应的影响。
