Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.
Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.
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DOI：
10.1371/journal.ppat.1010346
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发表时间：
2022-03
期刊：
影响因子：
6.7
通讯作者：
Lawniczak MKN
中科院分区：
文献类型：
--
作者：
Howick VM;Peacock L;Kay C;Collett C;Gibson W;Lawniczak MKN
早期分化谱系，如锥虫，可以为真核生物有性生殖的进化提供线索。在引起非洲人类锥虫病的布鲁氏锥虫中，有性繁殖发生在昆虫宿主的唾液腺中，但对定义这些性形式的分子特征的分析是复杂的，因为它们与更多的有丝分裂发育阶段混合在一起。我们使用单细胞rna测序（scRNAseq）分析了388个来自受感染采采蝇的中肠、前室和唾液腺的个体锥虫，从而确定了组织特异性细胞类型。对唾液腺寄生虫转录组的进一步研究揭示了基因表达在发育过程中的细微变化，从假定的性形式到表达变异表面糖蛋白基因的元环。该细胞簇可能包含有性形式，其特征是配子融合蛋白HAP2的高水平转录，以及一系列表面蛋白和几个功能未知的基因。我们利用免疫荧光分析和报告基因表达将这些表达模式与不同的形态形式联系起来，以证明着丝质体保守基因Tb927.10.12080只在减数分裂中间体和配子中高水平表达。需要进一步的实验来确定这种目前功能未知的蛋白质是否在配子形成和/或融合中起作用。非洲锥虫是单细胞原生动物寄生虫，可在人类和牲畜中引起疾病。它们有一个复杂的生命周期，跨越哺乳动物和采采蝇宿主。在采采蝇体内，当采采蝇吸食有传染性的血液时，寄生虫首先进入中肠。随着它的发育，它进入前心室，然后是唾液腺，在这些组织中呈现出不同的形态。在唾液腺中，寄生虫可以通过减数分裂和产生配子进行非强制性的有性繁殖。然而，这种性发育的生物学过程和定义这些形态形式的分子特征仍然难以捉摸，因为它们存在于异质群体中，也包含有丝分裂形式。在这里，我们使用单细胞RNAseq来分析采采过程中寄生虫的转录组，重点是识别定义这些性阶段的表达模式。我们发现有性形式具有独特的转录谱，并使用荧光报告将这些表达模式与性发育的特定形态阶段联系起来。这使我们能够确定一个可能与生殖有关的新基因。阐明有性生殖和遗传交换的机制是理解毒力和耐药性等关键性状进化的基础。
Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion. African Trypanosomes are single-celled protozoan parasites that cause disease in humans and livestock. They have a complex life cycle that spans a mammalian and tsetse fly host. Within the tsetse fly, the parasite first travels into the midgut when the fly takes up an infectious blood meal. As it develops it moves into the proventriculus followed by the salivary glands taking on distinct morphological forms in each of these tissues. In the salivary glands, the parasite can undergo non-obligatory sexual reproduction via meiosis and the production of gametes. However, the biological processes that underly this sexual developmental and the molecular signatures that define these morphological forms remain elusive because they are found within heterogeneous populations that also contain mitotically dividing forms. Here we have used single-cell RNAseq to profile the transcriptomes of parasites across development in the tsetse with a focus on identifying the patterns of expression that define these sexual stages. We showed that the sexual forms have a unique transcriptional profile and we connect these expression patterns to specific morphological stages of sexual development using a fluorescent reporter. This allowed us to identify a new gene that may be involved in reproduction. Elucidating the mechanisms underlying sexual reproduction and genetic exchange is fundamental to understanding the evolution of key traits such as virulence and drug resistance.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
4.6
作者：
Attar M;Sharma E;Li S;Bryer C;Cubitt L;Broxholme J;Lockstone H;Kinchen J;Simmons A;Piazza P;Buck D;Livak KJ;Bowden R
通讯作者：
Bowden R
影响因子：
1.5
作者：
Bütikofer, P;Vassella, E;Roditi, I
通讯作者：
Roditi, I
影响因子：
3.2
作者：
Gibson, Wendy;Peacock, Lori;Bailey, Mick
通讯作者：
Bailey, Mick
影响因子：
1.5
作者：
Biebinger, S;Wirtz, LE;Clayton, C
通讯作者：
Clayton, C