title: Life and cell cycle progression analysis in Leishmania mexicana by single cell RNA-sequencing
Creators: name: Warren, Felix Sean Lee
Publisher: name: University of Glasgow
Year: 2023

description: Leishmaniasis is a neglected tropical disease, which is estimated to produce approximately 1.3 million new cases annually. As yet, no vaccines are available and current effective chemotherapeutic strategies are lacking, due to drug toxicity, resistance, and social and economic barriers affecting availability. Pivotal in the discovery of new interventions for infectious diseases is the understanding of the fundamental biology of the pathogen and its role in infection. Here, the application of single cell RNA sequencing (scRNA-seq) was employed to investigate the fundamental biology behind transcriptomic changes as the parasite progresses through life cycle stages, from promastigote to metacyclic forms before differentiating into amastigotes. The transcriptomic dynamics underpinning these multifarious developmental transitions through life cycle stages are yet to be fully described. To investigate these changes, we employed scRNA-seq over five time-points as Leishmania mexicana (L. mexicana) differentiated from promastigotes to axenicamastigotes in vitro. With clustering and marker analysis of over 16,500 parasites across three experiments, revealing thousands of stage specific markers. Of note during gene marker analysis was the discovery of a transitional cluster placed between promastigote and amastigote stages. This cluster, named here Trans A, displayed overlap between both promastigote and amastigote markers, and potentially representing a new intermediate life cycle stage, defined transcriptionally. Additionally in these data was the identification of over 1,500 differentially expressed markers for a metacyclic like cluster. To examine the timing and the patterns of gene expression over the life and cell cycle, pseudotime analysis was used for the first time in Leishmania, so that we may further explain the order of these transitional events and any potential stage specific patterns in gene expression, providing an unprecedented understanding of the life cycle transitions. Analysis indicated the development between promastigote and amastigote stages was possible by progression through this Trans A cluster, circumventing the infective metacyclic form. Thus, potentially revealing a new developmental strategy for life cycle progression. Furthermore, cell cycle labelling analysis was performed using phase marker orthologues, also revealing new cell cycle phase specific markers. When combined with promastigote stage marker orthologues, a striking overlap between promastigote life stages and cell cycle stages was found, potentially providing further evidence that promastigote morphologies are intrinsically linked with cell cycle stages. To validated how transient gene expression changes may be represented at the protein level, 96 fluorescently tagged cell lines were produced using a highthroughput CRISPR-Cas9 system. Of the 96 cell lines produced, 91 were hypothetical proteins identified as having transient expression patterns for developmental trajectories drawn over promastigote to metacyclic and axenic amastigote stages. Initial assessments by fluorescent microscopy revealed 80 out of the 96 tagged proteins matched overall pseudotime expression profiles, indicating that dynamics of RNA levels detected by scRNA-seq could reflect changes in protein levels. Furthermore, these 91 cell lines of tagged hypothetical proteins provide the opportunity for further research into new biology. So that this transitional gene progression may be explored in broader contexts, scRNA-seq of L. mexicana-infected human macrophages was undertaken. Allowing the comparison of the RNA populations found in differentiation to axenic amastigotes in vitro, to those of the amastigote forms infecting a host cell. This crucial life-cycle development stage of Leishmania within the macrophage revealed infection response associated genes, linked with parasite removal strategies. This project aimed to provide an example of investigating fundamental Leishmania biology by applying scRNA-seq for the study of life and cell cycle transitions, with the application of pseudotime analysis for the first time. Results of differential gene expression analysis revealed new biology not previously described in previous methods. Thereby demonstrating how the application of scRNA-seq may further disseminate parasite biology and infection dynamics.