Application of single-cell transcriptomics to kinetoplastid research

Briggs, Emma; Warren, Felix S. L.; Matthews, Keith R.; McCulloch, Richard; Otto, Thomas D.

doi:10.1017/s003118202100041x

Cited by 11 publications

(16 citation statements)

References 158 publications

(291 reference statements)

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“…We found a mean of 2.6x10 6 mapped reads per cell and a mean detection of 1756 genes per cell (S1A and S1B Fig) , which is a greater number of genes per cell than recently published data from Hutchinson et al 2021 [20] that used a droplet-based method on the same parasite stage (1258 genes per cell). This further supports the use of Smart-seq2 to get in depth transcriptomes (high-coverage and full-length) in a low-throughput, targeted fashion compared to droplet-based methods that have fewer genes detected but are higher throughput [12,30]. Additionally, we observed high expression of genes that encode known procyclic surface antigens including GPEET and EP1-3 [2,31] (S1C Fig) . These data support the utility of our protocol to profile single-cell expression profiles in kinetoplastids.…”

Section: Generation Of High-quality Transcriptomes From In Vitro Proc...supporting

confidence: 76%

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages

et al. 2022

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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.

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Section: Generation Of High-quality Transcriptomes From In Vitro Proc...supporting

confidence: 76%

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages

et al. 2022

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“…In order to identify hybridization-competent cells present in Leishmania cultures, we performed single-cell RNA sequencing of L747 and MA37 cultured cells exposed or not to γ-radiation. To our knowledge, this is the first application of single-cell transcriptomics in Leishmania , although it has been applied to other kinetoplastid pathogens to reveal the heterogeneity of the parasite populations arising in their mammalian hosts or insect vectors (reviewed in Briggs et al, 2021 ). Our analyses revealed transcriptomic heterogeneity within and between the parental L. tropica lines in the promastigote cultures.…”

Section: Discussionmentioning

confidence: 99%

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq

Louradour

Ferreira

Duge

et al. 2022

eLife

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Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

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“…This behavior may explain the different and peculiar responses to the DSBs previously mentioned. In this scenario, single-cell analyses (e.g., single-cell transcriptomics) can be a valuable tool to reveal possible cryptic populations capable of dealing with DSBs differently (Briggs et al, 2021). Profiling gene expression of individual cells with singlecell RNA sequencing may detect rare cell types in heterogeneous populations previously challenged with DSBs source agents, such as IR.…”

Section: Discussionmentioning

confidence: 99%

DNA Double-Strand Breaks: A Double-Edged Sword for Trypanosomatids

Silva

2021

Front. Cell Dev. Biol.

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For nearly all eukaryotic cells, stochastic DNA double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions. DSB processing and repair can cause sequence deletions, loss of heterozygosity, and chromosome rearrangements resulting in cell death or carcinogenesis. However, trypanosomatids (single-celled eukaryotes parasites) do not seem to follow this premise strictly. Several studies have shown that trypanosomatids depend on DSBs to perform several events of paramount importance during their life cycle. For Trypanosoma brucei, DSBs formation is associated with host immune evasion via antigenic variation. In Trypanosoma cruzi, DSBs play a crucial role in the genetic exchange, a mechanism that is still little explored but appear to be of fundamental importance for generating variability. In Leishmania spp., DSBs are necessary to generate genomic changes by gene copy number variation (CNVs), events that are essential for these organisms to overcome inhospitable conditions. As DSB repair in trypanosomatids is primarily conducted via homologous recombination (HR), most of the events associated with DSBs are HR-dependent. This review will discuss the latest findings on how trypanosomatids balance the benefits and inexorable challenges caused by DSBs.

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Application of single-cell transcriptomics to kinetoplastid research

Abstract: Abstract

Cited by 11 publications

References 158 publications

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq

DNA Double-Strand Breaks: A Double-Edged Sword for Trypanosomatids

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