Single-cell transcriptomics reveals life cycle and cell cycle gene expression dynamics during in vitro growth of two Leishmania species

Abstract Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro . In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana , we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, in vitro L. major promastigotes development does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei . These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species. Importance Leishmania parasites cause a group of neglected tropical diseases that can be fatal or cause life-altering lesions and are transmitted by biting sand flies. Much of our understanding of the complex developmental changes these parasites undergo within sand flies comes from laboratory-grown parasites, yet it is unclear whether these cultures accurately reproduce development. By examining gene activity in thousands of individual parasites, we discovered that two Leishmania species follow different developmental pathways under laboratory conditions, with only L. mexicana accurately recapitulating development in the sand fly. We also identified hundreds of genes whose activity changes during development and cell division. Some developmental transitions are closely linked to cell division, whereas others occur independently. Our results provide a detailed map of parasite development and markers for identifying different parasite forms. This resource will support future studies of how Leishmania parasites proliferate, develop, and prepare for transmission.