Lapp Stacey A, Mok Sachel, Zhu Lei, Wu Hao, Preiser Peter R, Bozdech Zybnek, Galinski Mary R
Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Malar J. 2015 Mar 13;14:110. doi: 10.1186/s12936-015-0612-8.
Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While a zoonosis in humans, this simian malaria parasite species infects macaque monkeys and serves as an experimental model for in vivo, ex vivo and in vitro studies. It has underpinned malaria discoveries relating to host-pathogen interactions, the immune response and immune evasion strategies. This study investigated differences in P. knowlesi gene expression in samples from ex vivo and in vitro cultures.
Gene expression profiles were generated using microarrays to compare the stage-specific transcripts detected for a clone of P. knowlesi propagated in the blood of a rhesus macaque host and then grown in an ex-vivo culture, and the same clone adapted to long-term in vitro culture. Parasite samples covering one blood-stage cycle were analysed at four-hour intervals. cDNA was generated and hybridized to an oligoarray representing the P. knowlesi genome. Two replicate experiments were developed from in vitro cultures. Expression values were filtered, normalized, and analysed using R and Perl language and applied to a sine wave model to determine changes in equilibrium and amplitude. Differentially expressed genes from ex vivo and in vitro time points were detected using limma R/Bioconductor and gene set enrichment analysis (GSEA).
Major differences were noted between the ex vivo and in vitro time courses in overall gene expression and the length of the cycle (25.5 hours ex vivo; 33.5 hours in vitro). GSEA of genes up-regulated ex vivo showed an enrichment of various genes including SICAvar, ribosomal- associated and histone acetylation pathway genes. In contrast, certain genes involved in metabolism and cell growth, such as porphobilinogen deaminase and tyrosine phosphatase, and one SICAvar gene, were significantly up-regulated in vitro.
This study demonstrates how gene expression in P. knowlesi blood-stage parasites can differ dramatically depending on whether the parasites are grown in vivo, with only one cycle of development ex vivo, or as an adapted isolate in long-term in vitro culture. These data bring emphasis to the importance of studying the parasite, its biology and disease manifestations in the context of the host.
诺氏疟原虫是已知可导致人类疟疾的五种疟原虫之一，可引发严重疾病甚至死亡。虽然它是一种人畜共患的疟疾寄生虫，但会感染猕猴，并作为体内、体外和离体研究的实验模型。它为有关宿主 - 病原体相互作用、免疫反应和免疫逃避策略的疟疾研究奠定了基础。本研究调查了诺氏疟原虫在离体和体外培养样本中的基因表达差异。
使用微阵列生成基因表达谱，以比较在恒河猴宿主血液中繁殖后再进行离体培养的诺氏疟原虫克隆，以及适应长期体外培养的同一克隆所检测到的阶段特异性转录本。每隔四小时分析一次涵盖一个血液阶段周期的寄生虫样本。生成 cDNA 并与代表诺氏疟原虫基因组的寡核苷酸阵列杂交。从体外培养中开展了两个重复实验。使用 R 和 Perl 语言对表达值进行过滤、归一化和分析，并应用于正弦波模型以确定平衡和幅度的变化。使用 limma R/Bioconductor 和基因集富集分析（GSEA）检测离体和体外时间点的差异表达基因。
在整体基因表达和周期长度方面（离体为 25.5 小时；体外为 33.5 小时），离体和体外时间进程之间存在显著差异。对离体上调基因进行的 GSEA 显示多种基因富集，包括 SICAvar、核糖体相关基因和组蛋白乙酰化途径基因。相比之下，某些参与代谢和细胞生长的基因，如胆色素原脱氨酶和酪氨酸磷酸酶，以及一个 SICAvar 基因，在体外显著上调。
本研究表明，诺氏疟原虫血液阶段寄生虫的基因表达会因寄生虫是在体内生长（仅在体外进行一个发育周期），还是作为适应的分离株进行长期体外培养而有显著差异。这些数据凸显了在宿主背景下研究该寄生虫及其生物学和疾病表现的重要性。
