National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Parasit Vectors. 2020 Feb 18;13(1):85. doi: 10.1186/s13071-020-3967-9.
Lipid metabolism is pivotal for the growth of apicomplexan parasites. Lipid synthesis requires bulk carbon skeleton acyl-CoAs, the transport of which depends on the acyl-CoA binding protein (ACBP). In Neospora caninum, the causative agent of neosporosis, the FASII pathway is required for growth and pathogenicity. However, little is known about the fatty acid transport mechanism in N. caninum.
We have identified a cytosolic acyl-CoA binding protein, with highly conserved amino acid residues and a typical acyl-CoA binding domain in N. caninum. The recombinant NcACBP protein was expressed to verify the binding activities of NcACBP in vitro, and the heterologous expression of NcACBP in Δacbp yeast in vivo. Lipid extraction from ΔNcACBP or the wild-type of N. caninum was analyzed by GC-MS or TLC. Furthermore, transcriptome analysis was performed to compare the gene expression in different strains.
The NcACBP recombinant protein was able to specifically bind acyl-CoA esters in vitro. A yeast complementation assay showed that heterologous expression of NcACBP rescued the phenotypic defects in Δacbp yeast, indicating of the binding activity of NcACBP in vivo. The disruption of NcACBP did not perturb the parasite's growth but enhanced its pathogenicity in mice. The lipidomic analysis showed that disruption of NcACBP caused no obvious changes in the overall abundance and turnover of fatty acids while knockout resulted in the accumulation of triacylglycerol. Transcriptional analysis of ACBP-deficient parasites revealed differentially expressed genes involved in a wide range of biological processes such as lipid metabolism, posttranslational modification, and membrane biogenesis.
Our study demonstrated that genetic ablation of NcACBP did not impair the survival and growth phenotype of N. caninum but enhanced its pathogenicity in mice. This deletion did not affect the overall fatty acid composition but modified the abundance of TAG. The loss of NcACBP resulted in global changes in the expression of multiple genes. This study provides a foundation for elucidating the molecular mechanism of lipid metabolism in N. caninum.
脂质代谢对顶复门寄生虫的生长至关重要。脂质合成需要大量的碳骨架酰基辅酶 A，其运输依赖于酰基辅酶 A 结合蛋白（ACBP）。在刚地弓形虫中，脂肪酸合成 II 途径对于生长和致病性是必需的。然而，关于刚地弓形虫的脂肪酸转运机制知之甚少。
我们已经鉴定了一种胞质酰基辅酶 A 结合蛋白，它在刚地弓形虫中具有高度保守的氨基酸残基和典型的酰基辅酶 A 结合结构域。表达重组 NcACBP 蛋白以验证 NcACBP 在体外的结合活性，并在体内表达Δacbp 酵母中的 NcACBP 异源表达。通过 GC-MS 或 TLC 分析ΔNcACBP 或野生型刚地弓形虫的脂质提取。此外，进行转录组分析以比较不同菌株的基因表达。
NcACBP 重组蛋白能够在体外特异性结合酰基辅酶 A 酯。酵母互补测定表明，NcACBP 的异源表达挽救了Δacbp 酵母的表型缺陷，表明 NcACBP 在体内具有结合活性。NcACBP 的敲除并未扰乱寄生虫的生长，但增强了其在小鼠中的致病性。脂质组学分析表明，NcACBP 的敲除并未导致脂肪酸的整体丰度和周转率发生明显变化，而敲除则导致三酰甘油的积累。ACBP 缺陷型寄生虫的转录分析显示，涉及广泛生物学过程的差异表达基因，如脂质代谢、翻译后修饰和膜生物发生。
我们的研究表明，NcACBP 的基因缺失并未损害刚地弓形虫的存活和生长表型，但增强了其在小鼠中的致病性。这种缺失不影响脂肪酸的整体组成，但改变了 TAG 的丰度。NcACBP 的缺失导致多个基因的表达发生全局变化。本研究为阐明刚地弓形虫脂质代谢的分子机制提供了基础。
