Rizzo Juliana, Oliveira Débora L, Joffe Luna S, Hu Guanggan, Gazos-Lopes Felipe, Fonseca Fernanda L, Almeida Igor C, Frases Susana, Kronstad James W, Rodrigues Marcio L
Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Michael Smith Laboratories, Department of Microbiology and Immunology, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, Canada.
Eukaryot Cell. 2014 Jun;13(6):715-26. doi: 10.1128/EC.00273-13. Epub 2013 Dec 13.
Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.
翻转酶是膜不对称性和分泌机制的关键调节因子。囊泡多糖分泌对于新型隐球菌的致病机制至关重要。基于植物中多糖分泌需要翻转酶以及假定的Apt1翻转酶是隐球菌毒力所必需的这些观察结果，我们使用先前表征的apt1Δ突变体分析了该酶在新型隐球菌多糖释放中的作用。突变体和野生型（WT）细胞具有重要的表型特征，包括荚膜形态和尺寸、葡糖醛酸木甘露聚糖（GXM）组成、分子大小和血清学特性。然而，与WT细胞相比，apt1Δ突变体产生的细胞外囊泡（EVs）的GXM含量较低且大小分布不同。我们的数据还表明，除了高尔基体结构的变化外，突变体细胞内的GXM合成存在缺陷。这些发现与体外生长、巨噬细胞感染和肺部定植期间GXM产量的降低相关。这种表型与突变体在感染小鼠肺部的存活率降低、白细胞介素-6（IL-6）细胞因子水平的诱导减少以及脑部定植无效有关。综上所述，我们的结果表明，APT1的缺失导致新型隐球菌在GXM合成和向细胞外环境的囊泡输出方面出现缺陷，这些过程显然与该真菌在动物感染期间使用的致病机制有关。
