FKS1 Is Required for Cryptococcus neoformans Fitness In Vivo: Application of Copper-Regulated Gene Expression to Mouse Models of Cryptococcosis.
FKS1 Is Required for Cryptococcus neoformans Fitness In Vivo: Application of Copper-Regulated Gene Expression to Mouse Models of Cryptococcosis.
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目前迫切需要新的抗真菌药物来治疗隐球菌性脑膜脑炎，这是艾滋病毒/艾滋病感染者死亡的主要原因。抗真菌药物开发的一个重要方面是验证靶点，以确定它们是否需要在动物疾病模型中存活。在新型隐球菌中，铜调控启动子（pCTR4-2）先前已被用于调节体内基因表达。这些实验的前提是铜的浓度取决于宿主的生态位。在这里，我们直接验证了这一前提，并证实了用于调节基因表达的启动子CTR4的表达在小鼠肺部比在大脑中要低得多。为了进一步探索这种方法，我们将其应用于编码1,3-β-葡聚糖合成酶FKS1的基因。在体外，FKS1的表达减少对生长影响不大，但确实激活了细胞壁完整性应激反应，增加了对caspofungin （FKS1的直接抑制剂）的敏感性。这些数据表明，减少新生芽孢杆菌耐药性的代偿途径是通过转录后效应实现的。然而，在体内，FKS1表达的不太明显的减少导致肺真菌负荷的显著减少（~1 log10 CFU），表明FKS1表达减少的代偿反应在体内不如在体外有效。总之，在体外和体内使用铜调控的假定药物靶点表达可以深入了解感染期间靶点活性降低的生物学后果。条件表达系统被广泛用于在小鼠感染模型中遗传验证抗真菌药物靶点。使用CTR4基因启动子的铜调控表达在新生生物中已被零星地用于此目的。在这里，我们发现CTR4在肺中的表达低，在脑中的表达高，建立了这种方法背后的基本前提。我们将该方法应用于研究FKS1基因，该基因编码棘白菌素类1,3-β-葡聚糖合成酶抑制剂的靶基因。我们的体外和体内研究表明，新生C.能耐受极低水平的FKS1表达。这一观察结果为1,3-β-葡聚糖合成酶抑制剂对新生芽孢杆菌活性差提供了可能的解释。
There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used previously to modulate gene expression in vivo. The premise for these experiments is that copper concentrations differ depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied it to the gene encoding 1,3-β-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through posttranscriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. IMPORTANCE Conditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied the approach to the study of FKS1, the gene encoding the target of the echinocandin class of 1,3-β-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-β-glucan synthase inhibitors toward C. neoformans.
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DOI：
10.1038/s41579-021-00511-0
发表时间：
2021-07
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Iyer KR;Revie NM;Fu C;Robbins N;Cowen LE
通讯作者：
Cowen LE
影响因子：
3.3
作者：
Fu, Ci;Beattie, Sarah R.;Cowen, Leah E.
通讯作者：
Cowen, Leah E.
影响因子：
8
作者：
Goldman, DL;Khine, H;Casadevall, A
通讯作者：
Casadevall, A
影响因子：
4.4
作者：
Gushiken, Alexis C.;Saharia, Kapil K.;Baddley, John W.
通讯作者：
Baddley, John W.
影响因子：
4.9
作者：
Meletiadis, Joseph;Pournaras, Spyros;Walsh, Thomas J.
通讯作者：
Walsh, Thomas J.