Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii.
Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii.
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DOI：
10.1371/journal.pgen.1005434
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发表时间：
2015-08
期刊：
影响因子：
4.5
通讯作者：
Sibley LD
中科院分区：
文献类型：
--
作者：
Behnke MS;Khan A;Lauron EJ;Jimah JR;Wang Q;Tolia NH;Sibley LD
弓形虫已经进化出许多策略来逃避其许多宿主的免疫反应。先前对流行于欧洲和北美的1、2和3株弓形虫克隆株之间杂交的遗传图谱发现，两种杆状蛋白ROP5和ROP18共同发挥作用，阻止小鼠宿主中干扰素伽马(IFNG)激活的先天免疫机制。然而，这些毒力因子和其他毒力因子在遗传差异更大的南美菌株中的作用尚不清楚。在这里，我们利用中等毒力的北美2型ME49毒株和高度毒力的南美10型Vand毒株之间的杂交，来定位小鼠毒力差异的遗传基础。对这个新组合的数量性状基因座(QTL)分析表明，在第12号染色体上有一个跨越ROP5基因座的峰。CRISPR-Cas9介导的ROP5在Vand株中的所有拷贝的缺失使其成为无毒株，互补证实了ROP5是导致2型和10型毒株差异的主要毒力因子。为了将这些观察扩展到代表不同遗传群体的其他南美强毒菌株，我们在8型TgCtBr5和4型TgCtBr18型菌株中剔除了ROP5，导致这两个背景下的毒力完全丧失。与此一致的是，在ROP5中表现出强烈的正选择特征的多态被证明对应于已知的与宿主免疫因子相互作用的区域。由于ROP5和ROP18共同发挥抵抗天然免疫机制的作用，并且在两个位点扫描中发现它们之间存在显著的相互作用，我们还评估了ROP18在南美毒株毒力中的作用。与之前描述的1型寄生虫中ROP18基因敲除的部分毒力丧失相反，南美4、8和10型毒株中ROP18的缺失导致了完全的减毒。这些数据表明，ROP5和ROP18在来自北美和南美的遗传多样性株中是保守的毒力因子，表明它们进化为抵抗弓形虫祖先株的天然免疫防御，随后在正选择下发生了多样性。寄生虫和它们感染的宿主之间不断地为生存而斗争。一方面，宿主需要控制寄生虫的生长，而寄生虫需要足够长的时间躲避宿主的反应，以便进行有效的传播。弓形虫寄生虫进化了毒力因子ROP5和ROP18，以逃避其天然中间宿主小啮齿动物的先天免疫机制。这些基因最初是在从欧洲和北美分离的克隆寄生虫类型中鉴定出来的，但导致遗传差异的南美菌株毒力的因素尚未得到测试。在这里，我们使用正向和反向遗传分析表明，ROP5和ROP18也是遗传上不同的南美强毒株的主要毒力因子。鉴于ROP5和ROP18在北美、欧洲和南美的毒株中作为毒力因子发挥作用，它们很可能在弓形虫辐射到目前的全球种群结构之前获得它们的功能。
Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection. Parasites and the hosts they infect are in constant struggle with each other for survival. On the one hand, the host needs to control parasite growth, while the parasite needs to evade the host response long enough to allow for efficient transmission. The parasite Toxoplasma gondii has evolved virulence factors ROP5 and ROP18 to evade innate immune mechanisms of its natural intermediate host, small rodents. These genes were initially identified in clonal parasite types isolated from Europe and North America, but the factors that contribute to virulence in genetically divergent South American strains have not been tested. Here we used forward and reverse genetic analyses to show that ROP5 and ROP18 are also major virulence factors in genetically distinct virulent South American strains. Given that ROP5 and ROP18 function as virulence factors in strains from North America, Europe, and South America they likely acquired their functions before Toxoplasma gondii radiated into its present global population structure.