Van Tyne Daria, Tan Yan, Daily Johanna P, Kamiza Steve, Seydel Karl, Taylor Terrie, Mesirov Jill P, Wirth Dyann F, Milner Danny A
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA USA.
Broad Institute, Cambridge, MA USA ; Graduate Program in Bioinformatics, Boston University, Boston, MA USA.
Genome Med. 2014 Nov 29;6(11):110. doi: 10.1186/s13073-014-0110-6. eCollection 2014.
During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.
We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy.
We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.
Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.
在人类恶性疟原虫感染的自然48小时红细胞内生命周期的后半段，寄生虫会隐匿于组织内皮深处，远离脾脏且外周血无法触及。这些晚期寄生虫可能会导致组织损伤，并可能引发临床疾病，因此需要更全面地了解它们的生物学特性。由于这些生命周期阶段因组织隐匿而不易采样，测量滋养体和裂殖体阶段寄生虫的体内基因表达一直是一项挑战。
我们开发了一个定制的nCounter®基因表达平台，并使用该平台测量体外和体内疟原虫的基因表达谱。我们还使用插补法生成全局转录谱，并评估体外生长的寄生虫与从尸检时收集的疟疾感染患者组织中回收的寄生虫之间的差异基因表达。
我们首次展示了隐匿于人体组织中的体内疟原虫的全局转录表达谱。我们发现寄生虫生理学可以与现有生命周期数据集的体外数据相关联，并且隐匿组织中的寄生虫呈现出预期的裂殖体样转录谱，在同一患者的不同组织中是保守的。基于60个标志性基因的插补生成了与通过微阵列测量的相同样本的全基因组表达模式高度相关的全局转录谱。最后，差异表达揭示了一组有限的体内上调转录本，这可能表明参与人类临床感染的独特寄生虫基因。
我们的研究突出了定制的nCounter®恶性疟原虫探针集的实用性、疟原虫物种内插补的验证以及人体组织中体内裂殖体阶段表达模式的记录。
