Biology subjects

Carnielli, J. B. T.

Publications and source records attributed to Carnielli, J. B. T..

5 recordsLinked to original sources

bioRxiv2025

Bi-directional highways and super-seeder tissues underpin parasite dissemination in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease caused by Leishmania donovani and L. infantum. Although VL is characterised by organ-specific immunopathology and parasitism of the spleen, liver and bone marrow, other tissues may harbour parasites without overt pathology. The mechanisms governing patterns of within-host dissemination and tissue tropism are largely unknown. We used a barcoded library of isogenic L. donovani and an ecological analysis framework to evaluate parasite population diversity across tissues and to map parasite dissemination in a murine infection model. We reveal an unexpected high degree of inter-connectivity between parasite populations demonstrating: i) continual bi-directional dissemination between viscera and skin; ii) the existence of "super-seeder" sites that act as hubs fuelling systemic spread; and iii) rerouting of dissemination "highways" following immune perturbation. These findings change our understanding of L. donovani pathogenesis, providing critical insights into infection dynamics with direct implications for relapse, treatment failure and transmission.

microbiology↗

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

Discovery and characterization of cell-permeable inhibitors of Leishmania mexicana CLK1 using an in-cell target engagement assay

Leishmaniasis is a neglected tropical disease with limited treatment options and significant unmet medical need. Here, we report the development of a bioluminescence resonance energy transfer (BRET)-based target engagement assay in live intact cells to identify and validate cell-permeable, ATP-competitive inhibitors of Leishmania mexicana (Lmx)CLK1. LmxCLK2, a closely related paralogue with an identical protein kinase domain, is also considered in our analysis. Genetic and pharmacological evidence indicates that simultaneous deletion or inhibition of both LmxCLK1/2 is lethal to the parasite. Using our newly developed assay, we screened a library of human kinase inhibitors and identified WZ8040, a third-generation EGFR inhibitor, as a potent LmxCLK1 ligand. WZ8040 demonstrated robust target engagement in both promastigotes and macrophage-internalized amastigotes, with low micromolar EC values for parasite killing and minimal toxicity to host macrophages. Biochemical assays confirmed that WZ8040 covalently binds and inhibits LmxCLK1, with mass spectrometry identifying Cys172 as the primary site of modification. Genetic validation using overexpression and knockout lines supports LmxCLK1 as the primary target of WZ8040. However, the retained activity of WZ8040 in mutant lines with the Cys172Ala substitution suggests that covalent binding is not essential for compound efficacy. Our findings highlight the utility of BRET-based assays for target validation in kinetoplastid parasites and underscore the potential of CLK1 as a druggable kinase in Leishmania. This integrated approach provides a framework for accelerating the discovery of novel anti-leishmanial agents through target engagement-guided strategies. Highlights- A live-cell NanoBRET assay enables in situ target engagement of CLK1 in Leishmania mexicana. - The covalent inhibitor WZ8040 effectively targets LmxCLK1 in both promastigote and intracellular amastigote stages. - CLK1 inhibition by WZ8040 does not depend on covalent modification of a single cysteine residue. - Combined target engagement and phenotypic data support LmxCLK1 as a druggable kinase in Leishmania.

cell biology↗

bioRxiv2021

Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome.

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

cell biology↗

bioRxiv2020

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation and survival

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here we applied kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 were dispensable, 44 protein kinase genes were refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering revealed functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

microbiology↗