Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics

Abstract

African trypanosomes proliferate as bloodstream forms and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated transcriptomes of both procyclic and slender bloodstream form Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single cell transcriptomic analysis of cryopreserved T. brucei. Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form-specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the cell cycle regulated transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance

“…After 24 to 72 h, parasites were moved to liquid nitrogen storage. For Chromium scRNA-seq (single cell RNA sequencing) (10× Genomics), samples were transported on dry ice before slow defrosting to limit cell death using the previously validated protocol ( 47 ). For each sample, parasites were combined from 2 to 3 mice in equal proportion depending on sample viability, only samples with >90% live, motile, parasites were used for sequencing.…”

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

“…Nonetheless, programming of DNA replication to predominantly initiate from a single origin or locus per chromosome would explain the chromosome size-dependent timing of L. major DNA replication we describe here and previously 82 . As we have argued 70, 81, 141, 142 , it is unlikely that whole genome duplication in Leishmania can be accomplished during S-phase using a single origin per chromosome, and so DNA replication may be supported by further, less efficient initiation activities. Such organisation could then explain Leishmania DNA replication timing, feeding into the other genome features we describe.…”

Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites

“…To further investigate whether SUMO chain mutants are primed for differentiation to stumpy forms, we challenged the parasites with high concentrations of cis -aconitate (CA) at low temperature to stimulate differentiation to PF, which is thought to occur via a stumpy-like intermediate stage [ 23 , 24 ]. During this transformation, the VSG coat is released and replaced by a different coat consisting of invariant GPEET and EP procyclins.…”