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

Abstract

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative “slender” forms differentiate into cell cycle arrested “stumpy” forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

m6A landscape is more pervasive whenTrypanosoma bruceiexits the cell cycle

“…In this study, we performed m 6 A immunoprecipitation without prior RNA fragmentation to obtain a complete set of methylated transcripts, regardless of the distribution of m 6 A modifications in poly(A) tails [16] or in internal locations [15]. Therefore, we did not assess which transcripts are internally or poly(A) methylated, and whether that impacts their expression and/or stability.…”

“…We also detected another RNA binding protein, a zinc-finger protein, a hypothetical protein, and a cyclinlike F-box 2 (CFB2) protein within the most abundant transcripts. Analysis of transcripts from procyclic forms showed again a silent VSG as the transcript most enriched in m 6 A, followed by a hypothetical protein, a zinc-finger protein, separase and one expression site-associated gene 2 (ESAG2) (Fig. 3D).…”

“…The function of m 6 A varies between organisms, including parasites, where it might be important for life cycle progression. In Toxoplasma gondii, m 6 A methylation is prevalent in asexual life cycle stages [12] and is involved in proper mRNA 3' end processing, likely affecting developmental gene regulation [13]. In Plasmodium falciparum, m 6 A is highly developmentally regulated, mediating translational repression of transcripts involved in regulation of gene expression in blood stages [14].…”

“…Their study showed how both life cycle stages differed in terms of methylated transcripts and identified in which region of the transcript (coding or untranslated) m 6 A peaks were located [15]. More recently, our lab identified m 6 A can also be found in the poly(A) tail, where it stabilizes VSG transcripts in slender forms and whose removal preceded VSG downregulation during parasite differentiation to procyclic forms [16]. However, whether m 6 A methylation is important for gene expression regulation in other stages of the T. brucei life cycle remains unknown.…”

“…When slender forms reach a critical cell density in the bloodstream, they differentiate into non-replicative stumpy forms through a quorum-sensing mechanism triggered by oligopeptides, generally known as the stumpy induction factor (SIF). Such peptides are generated by parasite-released peptidases in a cell density-dependent manner and activate a signaling pathway that leads to gene expression remodeling, including reduction of transcription and translation [5][6][7][8]. Uptake of bloodstream parasites by the tsetse fly triggers further differentiation into procyclic forms, which lead to broad transcriptomic and morphological changes, including the replacement of the VSG by a procyclin surface coat [9].…”