Department of Genetics & Biochemistry, Eukaryotic Pathogens Innovation Center (EPIC), Clemson University, Clemson, SC 29634, USA.
Department of Biology, Furman University, Greenville, SC 29613, USA.
Genetics. 2021 Mar 3;217(1):1-15. doi: 10.1093/genetics/iyaa009.
The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.
人类真菌病原体新生隐球菌依赖于复杂的信号网络来适应和在宿主温度下存活。钙调神经磷酸酶蛋白磷酸酶是在 37°C 下增殖的核心，但它的确切贡献仍不清楚。为了更好地定义对新生隐球菌温度耐受性的遗传贡献，对 4031 个基因敲除体进行了筛选，以确定在 37°C 下和使钙调神经磷酸酶失活的条件下必需的基因。鉴定出 83 个候选菌株，这些菌株可能对 37°C 敏感，随后对其进行了技术简单但稳健的检测，即在该检测中，细胞暴露在温度梯度下。这导致鉴定出 46 个基因有助于新生隐球菌增殖的最高温度 (Tmax)。在无药物培养基上具有不同 Tmax 的 46 个突变体进一步在抑制钙调神经磷酸酶的条件下评估 Tmax，这导致鉴定出几个以前未表征的敲除体与钙调神经磷酸酶抑制的合成相互作用。缺乏隔膜蛋白 Cdc11 的突变体是 Tmax 最低的突变体之一，在没有钙调神经磷酸酶活性的情况下无法增殖。为了进一步定义钙调神经磷酸酶和隔膜蛋白在高温生长中的关系，对 46 个突变体在 37°C 下的细胞形态和在破坏细胞壁和细胞膜的药物存在下的生长进行了测试。对钙调神经磷酸酶抑制敏感的突变体进行了测试，以确定与隔膜蛋白编码基因 CDC12 的缺失和隔膜蛋白 Cdc3-mCherry 的定位的合成致死相互作用。这里描述的分析表明，以前未表征的基因在标准生长测定中被遗漏，这表明温度梯度测定是阐明微生物增殖温度范围的遗传基础的有价值的补充工具。
