Universidade de São Paulo, Biotechnology Graduate Program, São Paulo, SP, Brazil.
Universidade Federal de São Paulo, Campus Diadema, Department of Biological Sciences, Diadema, SP, Brazil.
PLoS One. 2019 Jan 25;14(1):e0211393. doi: 10.1371/journal.pone.0211393. eCollection 2019.
Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.
隐球菌病是一种由新生隐球菌引起的侵袭性真菌感染（IFI），主要发生在免疫功能低下的患者中。由于病原体耐药性、治疗不规律和抗真菌药物选择有限导致治疗失败是一个问题。研究氨基酸的生物合成和摄取为探索新型抗真菌药物的开发提供了机会。新生隐球菌有 10 种氨基酸转运蛋白，其中两种（Aap3 和 Aap7）在测试条件下不表达，有五种以前已经研究过（Aap2、Aap4、Aap5、Mup1 和 Mup3）。我们之前的研究结果表明，Aap4 和 Aap5 是具有重叠功能的主要转运蛋白。aap4Δ/aap5Δ 双突变体不能在氨基酸作为唯一氮源的条件下生长，在动物模型中也没有毒力。在这里，我们删除了剩余的氨基酸转运蛋白（AAP1、AAP6、AAP8），这些转运蛋白的基因表达受到营养条件的调节，并创建了一个双突变体（aap1Δ/aap2Δ）。我们研究了这些突变体的毒力特性，并探索了新生隐球菌中氨基酸摄取的调节机制。aap1Δ/aap2Δ 菌株在 37°C 的 L-氨基酸中生长缓慢，荚膜生成减少，在 Galleria mellonella 动物模型中减毒。我们的数据，连同以前的研究，（i）补充了对所有 10 种氨基酸转运蛋白突变体的分析，（ii）证实了这些转运蛋白作为全局转运蛋白的作用，（iii）在热和营养应激时需要，（iv）对毒力很重要。我们的研究还表明 Ras1 信号和氨基酸摄取之间存在新的可能联系。
