Biology subjects

Catta-Preta, C. M. C.

Publications and source records attributed to Catta-Preta, C. M. C..

5 recordsLinked to original sources

bioRxiv2026

Reduced levels of inositol hexakisphosphate kinase (IP6K) impair life-cycle transitions and the intracellular development of Trypanosoma cruzi within human cardiomyocytes

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, T. cruzi undergoes several key differentiation processes that are essential for its survival. The precise mechanisms that regulate these processes remain elusive, and any interference in this cycle would represent a breakthrough in the development of effective therapy against Chagas disease. Here, after depleting a single IP6K allele of T. cruzi, we observed that key differentiation processes (metacyclogenesis, amastigogenesis and trypomastigogenesis) were profoundly impaired. Epimastigote forms of IP6K-deficient T. cruzi exhibited morphological alterations and reduced metacyclogenesis. IP6K-deficient metacyclic forms had reduced infective potential in human cardiomyocytes. IP6K-deficient amastigote forms showed impaired ability to transform into trypomastigotes, with most of the population egressing from human cardiomyocytes without completing trypomastigogenesis. Together, our results suggest that IP6K is critical to sustain the T. cruzi life cycle. Since disruption of both IP6K alleles was lethal and the primary structure of IP6K shares only [~]25% similarity with its human homolog, this kinase emerges as a promising target for drug development against Chagas disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/700787v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1cb37f0org.highwire.dtl.DTLVardef@c59fb8org.highwire.dtl.DTLVardef@791cb7org.highwire.dtl.DTLVardef@14c68fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

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↗

bioRxiv2024

IFT and BBSome proteins are required for Leishmania mexicana pathogenicity, but flagellar motility is dispensable

Protists of the order Kinetoplastida possess a single multifunctional flagellum, which powers cellular displacement and mediates attachment to tissues of the arthropod vector. The kinetoplastid flagellar cytoskeleton consists of a nine-microtubule doublet axoneme; further structural elaborations, which can vary between species and life cycle stages, include the assembly of axonemal dynein complexes, a pair of singlet microtubules and the extra-axonemal paraflagellar rod. The intracellular amastigote forms of Leishmania spp. build a short, non-motile cilium whose function has remained enigmatic. Here we used a panel of 25 barcoded promastigote cell lines, including mutants lacking genes encoding flagellar assembly proteins, cytoskeletal proteins required for normal motility, or flagellar membrane proteins to examine how these defects impact on their virulence in macrophages and mice. Mutants lacking intraflagellar transport (IFT) protein 88 were severely attenuated indicating that assembly of a flagellum is necessary to allow for Leishmania survival in a mammalian host. A similarly severe loss of virulence was observed upon deletion of BBS2, a core component of the BBSome complex, which may act as a cargo adapter for IFT. By contrast, promastigotes that were unable to beat their flagella due to loss of PF16 could establish an infection and only showed a small reduction of parasite burden in vivo compared to the parental cell lines. These results confirm that flagellar motility is not necessary for mammalian infection but flagellum assembly and the integrity of the BBSome are essential for pathogenicity.

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↗

bioRxiv2020

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division. Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

cell biology↗