Biology subjects

Leitao, A.

Publications and source records attributed to Leitao, A..

5 recordsLinked to original sources

bioRxiv2025

Bumped kinase inhibitor BKI-1708 interferes in cytokinesis and drives baryzoite stage conversion in the cyst-forming apicomplexan parasites Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti

Bumped kinase inhibitors (BKIs) have demonstrated safety and promising efficacy against various apicomplexan pathogens both in vitro and in vivo. However, in the closely related cyst-forming coccidians T. gondii, Neospora caninum and Besnoitia besnoiti, in vitro treatments with a range of BKIs induced the conversion of intracellular tachyzoites into atypical multinucleated complexes (MNCs), also named "baryzoites". In this study, baryzoites of T. gondii, N. caninum and B. besnoiti generated through exposure of tachyzoites to 2.5 {micro}M BKI-1708 were comparatively assessed. TEM showed that baryzoites contained multiple nuclei, clustered together and separated from the cytoplasmic organelles of newly formed zoites. These zoites do not have outer tachyzoite plasma membrane, were unable to complete cytokinesis, remained intracellular, and were enclosed by a parasitophorous vacuole membrane. TEM demonstrated the presence of an electron-dense cyst wall-like components only in T. gondii baryzoites. Species-specific differences in antigen expression were observed by immunofluorescence using specific antibodies. Comparative proteomic analysis revealed consistent downregulation of ribosomal proteins, proteins associated with secretory organelles, as well as of transcription and translation factors in all baryzoites. Bradyzoite-specific markers were upregulated only in T. gondii baryzoites. In addition, common orthologues of two alveolin-domain filament proteins (IMC7 and IMC12) and a hypothetical protein (TGME49_236950, NCLIV_050850, BESB_060040) were detected at higher abundance in all treated parasites. Overall, baryzoites exhibit distinct phenotypic and proteomic profiles, with ambiguous expression of tachyzoite and bradyzoite antigens, and lacking complete cellular division under drug pressure, suggesting a reversible response to stress rather than progression into a fully differentiated form. SignificanceApicomplexan parasites cause serious diseases worldwide, yet treatment options remain limited. A promising group of drugs are BKIs. We investigated how BKI-1708 affects threclosely related T. gondii, N. caninum, and B. besnoiti. Instead of killing the parasites, the drug induced the formation of multinucleated structures termed "baryzoites". These baryzoites exhibited ambiguous characteristics during the actively growing and dormant stages of the parasite life cycle and were unable to complete normal cell division. Moreover, we observed other key similarities and differences among species including downregulation of ribosomal proteins and transcription/translation factors, while only T. gondii displayed cyst wall formation. Microscopy and proteomics demonstrated that baryzoites represent a distinct stage that is formed upon drug pressure and promotes parasite survival during prolonged drug exposure. These findings highlight the unexpected ways parasites adapt to drug treatment and provide new insights into how BKIs exert their activities.

systems biology↗

bioRxiv2021

Selectivity of Protein Interactions along the Aggregation Pathway of a-Synuclein

The aggregation of -SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinsons Disease in neurons. Whilst -synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of -SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of -SYN. In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by -SYN. We measured binding to monomeric -SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of -SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant -SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with -SYN monomers at detectable levels, and only a subset recognizes the oligomeric -SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation. Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, -SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different -SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of -SYN.

biochemistry↗

bioRxiv2020

Constitutive activation of cellular immunity underlies the evolution of resistance to infection

Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When we exposed populations of Drosophila melanogaster to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, we found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring an induced immune response to become constitutive.

evolutionary biology↗

bioRxiv2019

Mapping the S1 and S1 subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds waswere designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1' subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. The three inhibitors had an EC50 value of ca. 4 M, thus being equipotent to benznidazole according to the anti-trypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.\n\nAuthor SummaryDespite many achievements in identifying novel agents for the treatment of tropical and neglected diseases, further research continues to be of fundamental importance. Our research groups have been using the cruzipain cysteine protease in its recombinant form, cruzain (Cz), to identify new trypanocidal agents. Considering the possible interchangeability with other cysteine proteases, the same series of dipeptidyl nitriles was tested in Leishmania mexicana LmCPB. Other potential targets for such inhibitors are human cysteine cathepsins, which are involved in different disease states. Thus, the inhibitors were also tested against cathepsins B, L, K, and S. Our results demonstrate that inhibition of these cysteine proteases can be achieved by appropriate structural modifications of dipeptidyl nitriles. It was also possible to identify trypanocidal agents, equipotent to benznidazole, the current drug of choice used for the treatment of Chagas disease.

biochemistry↗

bioRxiv2019

Ligand-induced Conformational Selection Predicts the Selectivity of Cysteine Protease Inhibitors

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.

bioinformatics↗