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Could inositol pyrophosphates be central to metacyclogenesis in Leishmania?

Abstract

Inositol pyrophosphates (PP-IPs) are water-soluble metabolites that have at least one diphosphate moiety attached to an inositol ring and have been associated with a wide range of molecular pathways, meaning understanding of their specific roles is a riddle. In model eukaryotes, the best-characterized PP-IPs are the so-called IP7 and IP8, which are involved in processes such as homologous recombination and microbial pathogenicity. IP7 and IP8 are synthesized through complementary pathways involving IP6K and PP-IP5K kinases, respectively. Trypanosomatids, such as Leishmania braziliensis (the causative agent of mucocutaneous leishmaniasis), have orthologous genes for IP6K, but genes encoding PP-IP5K have not been identified in any trypanosomatid genome. It is unknown if trypanosomatids do not, therefore, synthesize IP8, and what consequences such an absence might have. Interestingly, trypanosomatids share several diverged cellular characteristics relative to other eukaryotes, including the virtual absolute absence of introns, almost universal use of transcription from long polycistronic clusters by RNA polymerase II, the absence of the classical non-homologous end joining DNA repair pathway, and tolerance of considerable genomic plasticity. These peculiar features may have arisen due to a genome streamlining process during the transition from a free-living to a parasitic lifestyle. However, it is still unclear, in molecular terms, what allowed this transition, though it is likely that the loss of genes essential for a free-living style drove or consolidated this adaptive event. A close-related family to trypanosomatids that deserves attention is the Bodonidae, which encompass only free-living organisms. They are commonly represented by Bodo saltans, the closest known free-living relative of trypanosomatids. Thus, comparative molecular analyzes between B. saltans and trypanosomatids can provide insight into important genomic gains and losses that occurred during the evolutionary transition to parasitism. To date, no work has asked about the correlation of PP-IPs with the emergence of parasitism in trypanosomatids. Also, few studies have investigated PP-IPs in trypanosomatids, and none has compared PP-IPs pathways in bodonids and trypanosomatids. Curiously, although trypanosomatids lack homologs for the kinase PP-IP5K, preliminary results from our group show that B. saltans has an ortholog for PP-IP5K with 42% identity to human PP-IP5K1. Moreover, our findings suggest while that P. confusum (a trypanosomatid) also has an orthologous gene for PP-IP5K. However, tertiary structure predictions indicate disruption of the catalytic site, suggesting the kinase is not functional in this organism. This finding makes us wonder if loss of PP-IP5K was a key feature of the emergence of parasitism by trypanosomatids, perhaps due to changes in DNA metabolism and/or host/vector interaction. To test this hypothesis, we first intend to add the PP-IP5K gene (from B. saltans) to L. braziliensis (episomal knock-in), confirm the expression of PP-IP5K, and characterize the generated lineage, including metabolomic analyses. Subsequently, we will stimulate metacyclogenesis and perform comparative single-cell transcriptomics (scRNA-seq) analyzes, aiming to reveal cryptic alterations in transitional forms (from procyclic and metacyclic promastigotes) due to the presence of PP-IP5K. Finally, in future research resulting from this project, we will investigate possible differences in pathogenicity and virulence of lineages generated through infection assays. This project will allow us to clarify whether PP-IPs have any relationship with parasitism in trypanosomatids, contributing to a better understanding of these metabolites that seem to be fundamental in several cellular pathways. (AU)

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