Little Timothy S, Cunningham Deirdre A, Christophides George K, Reid Adam James, Langhorne Jean
The Francis Crick Institute, Midland Road, London, UK.
Department of Life Sciences, Imperial College London, South Kensington, London, UK.
BMC Genomics. 2025 May 29;26(1):544. doi: 10.1186/s12864-025-11752-1.
The plasmodium interspersed repeats (pir) multigene family is found across malaria parasite genomes, first discovered in the human-infecting species Plasmodium vivax, where they were initially named the virs. Their function remains unknown, although studies have suggested a role in virulence of the asexual blood stages. Sub-families of the P. vivax pir/virs have been identified, and are found in isolates from across the world, however their transcription at different localities and in different stages of the life cycle have not been quantified. Multiple transcriptomic studies of the parasite have been conducted, but many map the pir reads to existing reference genomes (as part of standard bioinformatic practice), which may miss members of the multigene family due to its inherent variability. This obscures our understanding of how the pir sub-families in P. vivax may be contributing to human/vector infection.
To overcome the issue of hidden pir diversity from utilising a reference genome, we employed de novo transcriptome assembly to construct the pir 'reference' of different parasite isolates from published and novel RNAseq datasets. For this purpose, a pipeline was written in Nextflow, and first tested on data from the rodent-infecting P. c. chabaudi parasite to ascertain its efficacy on a sample with a full, genome-based set of pir gene sequences. The pipeline assembled hundreds of pirs from the studies included. By performing BLAST sequence identity comparisons with reference genome pirs (including P. vivax and related species) we found a clustered network of transcripts which corresponded well with prior sub-family annotations, albeit requiring some updated nomenclature. Mapping the RNAseq datasets to the de novo transcriptome references revealed that the transcription of these updated pir gene sub-families is generally consistent across the different geographical regions. From this transcriptional quantification, a time course of mosquito bloodmeals (after feeding on an infected patient) highlighted the first evidence of ookinete stage pir transcription in a human-infective malaria parasite.
De novo transcriptome assembly is a valuable tool for understanding highly variable multigene families from Plasmodium spp., and with pipeline software these can be applied more easily and at scale. Despite a global distribution, P. vivax has a conserved pir sub-family structure-both in terms of genome copy number and transcription. We suggest that this indicates important roles of the distinct sub-families, or a genetic mechanism maintaining their preservation. Furthermore, a burst of pir transcription in the mosquito stages of development is the first glint of ookinete pir expression for a human-infective malaria parasite, suggesting a role for the gene family at a new stage of the lifecycle.
疟原虫散布重复序列（pir）多基因家族存在于疟原虫基因组中，最初在感染人类的间日疟原虫中被发现，最初被命名为virs。尽管研究表明它们在无性血液阶段的毒力中起作用，但其功能仍然未知。间日疟原虫pir/virs的亚家族已被鉴定，并且在来自世界各地的分离株中都有发现，然而它们在不同地点和生命周期不同阶段的转录尚未被定量。已经对该寄生虫进行了多项转录组学研究，但许多研究将pir读数映射到现有的参考基因组（作为标准生物信息学实践的一部分），由于其固有的变异性，这可能会遗漏多基因家族的成员。这掩盖了我们对间日疟原虫中的pir亚家族如何可能导致人类/媒介感染的理解。
为了克服使用参考基因组隐藏pir多样性的问题，我们采用从头转录组组装来构建来自已发表和新的RNAseq数据集的不同寄生虫分离株的pir“参考”。为此，在Nextflow中编写了一个管道，并首先在感染啮齿动物的约氏疟原虫查巴迪亚种的数据上进行测试，以确定其对具有完整的、基于基因组的pir基因序列集的样本的有效性。该管道从纳入的研究中组装了数百个pir。通过与参考基因组pir（包括间日疟原虫和相关物种）进行BLAST序列同一性比较，我们发现了一个转录本聚类网络，这与先前的亚家族注释非常吻合，尽管需要一些更新的命名法。将RNAseq数据集映射到从头转录组参考表明，这些更新的pir基因亚家族的转录在不同地理区域通常是一致的。从这种转录定量中，蚊子吸食血液后的时间进程（在吸食感染患者后）突出了人类感染性疟原虫中动合子阶段pir转录的首个证据。
从头转录组组装是理解疟原虫属高度可变多基因家族的宝贵工具，并且借助管道软件可以更轻松、大规模地应用。尽管间日疟原虫分布全球，但其在基因组拷贝数和转录方面都具有保守的pir亚家族结构。我们认为这表明不同亚家族具有重要作用，或者存在一种维持其保存的遗传机制。此外，在蚊子发育阶段pir转录的爆发是人类感染性疟原虫动合子pir表达的首个迹象，表明该基因家族在生命周期的新阶段发挥作用。
