De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes

Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion.

近期针对间日疟原虫（Plasmodium vivax）野外分离株与猴适应株的测序工作，实现了全基因组范围内单核苷酸多态性（Single Nucleotide Polymorphism, SNP）的系统解析。本研究前期的相关分析均基于将短读长序列比对至以猴适应株萨尔瓦多I（Salvador I）的DNA构建的间日疟原虫参考基因组展开，但该策略存在固有局限：若萨尔瓦多I株存在基因组位点缺失，则对应序列会在参考基因组中遗漏，进而无法被既往分析所检测到。本研究报道了一株间日疟原虫野外分离株的基因组从头组装结果。在2857个组装得到的重叠群（contig）中，我们鉴定出362个重叠群，每个均包含超过5kb的连续DNA序列，且这些序列未出现在已发表的参考基因组中。这些全新的间日疟原虫DNA序列总长380万个核苷酸，共包含792个预测基因。大多数此类重叠群携带多基因家族成员，且大概率源自端粒区域。值得关注的是，我们鉴定到两个重叠群，其中的预测蛋白编码基因与已知的疟原虫红细胞入侵蛋白高度相似。其中一个基因编码的网织红细胞结合蛋白基因，与食蟹猴疟原虫（Plasmodium cynomolgi）RBP2e及诺氏疟原虫（Plasmodium knowlesi）NBPXb为直系同源基因。另一个基因则具备疟原虫红细胞结合蛋白的全部典型特征，包括保守的类达菲结合结构域与C端半胱氨酸富集结构域。系统发育分析显示，该全新基因与所有已知疟原虫达菲结合蛋白基因自成独立进化分支。额外实验分析表明，该基因存在于大多数间日疟原虫基因组中，且在红内期疟原虫中发生转录，这提示间日疟原虫的红细胞入侵机制可能比当前学界的认知更为复杂。本研究采用的分析策略弥补了既往基因组研究的不足，充分利用了下一代测序数据，实现了这一重要人体疟原虫物种遗传变异的全面解析。通过从头组装发现的全新蛋白编码基因的后续功能研究，有望鉴定出影响间日疟原虫生物学关键特征的基因，包括人类红细胞入侵的替代机制。