Biology subjects

Lima, A. P. C. A.

Publications and source records attributed to Lima, A. P. C. A..

4 recordsLinked to original sources

bioRxiv2025

First Genome-Wide Centromere Map of Trypanosoma cruzi Reveals Linear and 3D Compartment Boundaries and Spatial Clustering

Background: Trypanosoma cruzi, the etiological agent of Chagas disease, possesses a highly repetitive genome that has historically hindered high-quality assembly and structural characterization. Despite significant advances in assembling T. cruzi genomes, major gaps remain. Among these, the complete repertoire of centromeric sequences has remained elusive, representing a critical missing piece in our understanding of chromosome structure and inheritance. Results: Here, we generated high-coverage Hi-C (genome-wide chromosome conformation capture) data for the widely used T. cruzi Dm28c strain improving its genome assembly, reducing the number of scaffolds and producing a more contiguous and accurate genome. To investigate centromere organization, we performed ChIP-seq using the mNeonGreen-myc-tagged kinetochore proteins KKT2 and KKT3, resulting in the identification of 40 KKT-enriched peaks across 29 scaffolds. These peaks were located in regions enriched in retrotransposable elements, particularly L1Tc and VIPER, near strand switch regions, areas of high GC content, and at the boundaries between conserved genes and virulence-factor multigene families. Conclusion: Notably, Hi-C analysis revealed that centromeres may act as structural boundaries contributing to genome compartmentalization and frequently engage in 3D spatial clustering, suggesting a role in higher-order nuclear architecture. Overall, our study provides a high-quality reference genome for the Dm28c strain, presents the first genome-wide centromere map in T. cruzi, and offers novel insights into centromere-mediated 3D genome organization

genomics↗

bioRxiv2025

The mitotic spindle kinase MSK co-ordinates segregation of the nucleus and kinetoplast in Leishmania mexicana

Replication and segregation of the nucleus and kinetoplast, the mitochondrial DNA, are tightly coordinated in trypanosomatid parasites, but the signalling pathways that govern this process are unknown. Here, we characterise the mitotic spindle kinase (MSK), a key regulator of this coordination in Leishmania. Using chemical genetics, we engineered an analog-sensitive MSK to inhibit its activity. We show that inhibition of MSK impairs mitotic spindle elongation and blocks both nuclear and kinetoplast segregation, halting cell cycle progression and leading to cell death. We combined chemical genetics with proximity-based phosphoproteomics to identify four substrates: two GTPase-activating proteins, a nuclear segregation protein, and a hypothetical protein. We demonstrate that MSK co-localises with these four proteins in the nucleus, mitotic spindle, kinetoplast, and cytoplasm. Our findings establish MSK as a critical kinase that controls the co-ordinated segregation of the nucleus and kinetoplast, providing a new avenue for understanding cell cycle regulation in Leishmania.

cell biology↗

bioRxiv2025

Chemical genetics reveals Leishmania KKT2 and CRK9 kinase activity is required for cell cycle progression

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

molecular biology↗

bioRxiv2022

Tissue specific dual RNA-seq defines host-parasite interplay in murine visceral leishmaniasis caused by Leishmania donovani and Leishmania infantum

Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and haematological parameters in both pre-clinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFN{gamma} signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1{beta} and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFN{gamma} signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and [~]10% of host DEGs vary in infections with either parasite species. ImportanceVisceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.

microbiology↗