Department of Biochemistry and Sports Sciences, College of Natural Sciences, Makerere University, Kampala, Uganda.
Kamuzu University of Health Sciences, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi.
PLoS Negl Trop Dis. 2024 May 3;18(5):e0011516. doi: 10.1371/journal.pntd.0011516. eCollection 2024 May.
Sleeping sickness caused by Trypanosoma brucei rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi.
Blood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNP calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure.
24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a less symptomatic disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi.
Our results suggest that the differences in disease presentation in the two foci is mainly driven by genetic differences in the parasites in the two major endemic foci of Rumphi and Nkhotakota rather than differences in the environment or host response.
由布氏冈比亚锥虫引起的昏睡病是一种致命疾病，流行于南部和东部非洲。迫切需要开发新的诊断和控制工具来消除罗得西亚昏睡病，这可能通过更好地了解使用地方分离株的锥虫基因表达和遗传学来实现。在这里，我们描述了马拉维人体血液中地方流行的 T. b. rhodesiense 分离株的转录组图谱和种群结构。
从 Nkhotakota 和 Rumphi 焦点的 r-HAT 病例中采集血液样本，在开始 r-HAT 治疗前用 PaxGene 管提取 RNA。每个样本获得 1 亿个读数，首先使用 HiSat2 将读数初始映射到人类基因组参考 GRCh38，然后使用 HiSat2 将未映射的读数映射到锥虫参考转录组（TriTrypDB54_TbruceiTREU927）。使用 R 中的 DeSeq2 包进行差异基因表达分析。使用 GATK 从映射到 T. brucei 基因组的读数中进行 SNP 调用，以识别 T.b. rhodesiense 种群结构。
从 r-HAT 病例中收集了 24 个样本，其中 8 个来自 Rumphi，16 个来自 Nkhotakota 焦点。Nkhotakota 的分离株富含细胞周期停滞和短体标记的转录物，而 Rumphi 焦点的分离株富含叶酸生物合成和抗原变异途径的转录物。这些寄生虫焦点特异性转录组图谱与在 Rumphi 观察到的更具毒性的疾病以及与非分裂短体相关的在 Nkhotakota 中症状较轻的疾病一致。有趣的是，与乌干达 T.b. rhodesiense 分离株相比，马拉维 T.b. rhodesiense 分离株表达的基因富集了细胞增殖减少。使用从 RNAseq 数据中调用的 SNPs 进行 PCA 分析表明，来自 Nkhotakota 的 T. b. rhodesiense 寄生虫在遗传上与在 Rumphi 收集的寄生虫不同。
我们的结果表明，两个焦点中疾病表现的差异主要是由 Rumphi 和 Nkhotakota 两个主要流行焦点中寄生虫的遗传差异驱动的，而不是环境或宿主反应的差异。
