Role of Macrophage PIST Protein in Regulating Leishmania major Infection.

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently emerged as a key player in regulating viral pathogenesis. Nevertheless, the involvement of PIST in parasitic infections remains unexplored. Leishmania parasites infiltrate their host macrophage cells through phagocytosis, where they subsequently multiply within the parasitophorous vacuole (PV). Host cell autophagy has been found to be important in regulating this parasite infection. Since PIST plays a pivotal role in triggering autophagy through the Beclin 1-PI3KC3 pathway, it becomes interesting to identify the status of PIST during Leishmania infection. We found that while macrophage cells are infected with Leishmania major (L. major), the expression of PIST protein remains unaltered; however, it traffics from the Golgi compartment to PV. Further, we identified that in L. major-infected macrophage cells, PIST associates with the autophagy regulatory protein Beclin 1 within the PVs; however, PIST does not interact with LC3. Reduction in PIST protein through siRNA silencing significantly increased parasite burden, whereas overexpression of PIST in macrophages restricted L. major infectivity. Together, our study reports that the macrophage PIST protein is essential in regulating L. major infectivity.

The apo-acyl coenzyme A binding protein of Leishmania major forms a unique 'AXXA' motif mediated dimer.

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed 'AXXA' motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 - to C20-CoA, and the data could be best fit to a 'two sets of sites'/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics.

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Elucidating miR-146a-3p as a key player in autophagy and lipid metabolism in Leishmania major infection.

Autophagy is a key step involved in many unicellular eukaryotic diseases, including leishmaniasis, for cellular remodelling and differentiation during parasite's lifecycle. Lipids play a significant role in the infection process that begins with Leishmania major invading host cells. MicroRNAs (miRNAs), a family of small, 22-24 nucleotide noncoding regulatory RNAs, target mRNAs to modify gene expression and, subsequently, proteome output may have a regulatory role in altering the host cell processes. We observed miR-146a-3p expression increases in a time-dependent manner post Leishmania major infection. Transfecting miR-146a-3p mimic increases the expression of ATG7, an autophagy gene that encodes an E1-like enzyme in two ubiquitin-like conjugation systems required for autophagosome progression. HPGD (15-hydroxyprostaglandin dehydrogenase) operates as an enzyme, converting prostaglandin to its non-active form. Microarray data and western studies reveal that miR-146a-3p targets and inhibits HPGD, thereby increasing prostaglandin activity in lipid droplets. Herein, our research focuses on miR-146a-3p, which boosts ATG7 expression while reducing HPGD post Leishmania major infections helping us comprehend the intricate network of microRNA, autophagy, and lipid metabolism in leishmaniasis.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase.

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP.

Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl-acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.

The dynamic subcellular localisation of Rad1 is cell cycle dependent in Leishmania major.

The subcellular localisation of Rad1, a subunit of the Leishmania major 9-1-1 complex, remains unexplored. Herein, we reveal that Rad1 localises predominantly to the nucleus. Upon hydroxyurea treatment, the diffuse nuclear localisation of Rad1 becomes more punctate, suggesting that Rad1 is responsive to replication stress. Moreover, Rad1 localisation correlates with cell cycle progression. In the majority of G1 to early S-phase cells, Rad1 localises predominantly to the nucleus. As cells progress from late-S phase to mitosis, Rad1 relocalizes to both the nucleus and the cytoplasm in ∼90 % of cells. This pattern of distribution is different from Rad9 and Hus1, which remain nuclear throughout the cell cycle, suggesting Leishmania Rad1 may regulate 9-1-1 activities and/or perform relevant functions outside the 9-1-1 complex.

Knockout of protein phosphatase 1 in Leishmania major reveals its role during RNA polymerase II transcription termination.

The genomes of kinetoplastids are organized into polycistronic transcription units that are flanked by a modified DNA base (base J, beta-D-glucosyl-hydroxymethyluracil). Previous work established a role of base J in promoting RNA polymerase II (Pol II) termination in Leishmania major and Trypanosoma brucei. We recently identified a PJW/PP1 complex in Leishmania containing a J-binding protein (JBP3), PP1 phosphatase 1, PP1 interactive-regulatory protein (PNUTS) and Wdr82. Analyses suggested the complex regulates transcription termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of proteins, including Pol II, by PP1. However, we never addressed the role of PP1, the sole catalytic component, in Pol II transcription termination. We now demonstrate that deletion of the PP1 component of the PJW/PP1 complex in L. major, PP1-8e, leads to readthrough transcription at the 3'-end of polycistronic gene arrays. We show PP1-8e has in vitro phosphatase activity that is lost upon mutation of a key catalytic residue and associates with PNUTS via the conserved RVxF motif. Additionally, purified PJW complex with associated PP1-8e, but not complex lacking PP1-8e, led to dephosphorylation of Pol II, suggesting a direct role of PNUTS/PP1 holoenzymes in regulating transcription termination via dephosphorylating Pol II in the nucleus.

C3/CD11b-Mediated Leishmania major Internalization by Neutrophils Induces Intraphagosomal NOX2-Mediated Respiratory Burst but Fails to Eliminate Parasites and Induces a State of Stalled Apoptosis.

Recruited neutrophils are among the first phagocytic cells to interact with the phagosomal pathogen Leishmania following inoculation into the mammalian dermis. Analysis of Leishmania-infected neutrophils has revealed alterations in neutrophil viability, suggesting that the parasite can both induce or inhibit apoptosis. In this study, we demonstrate that entry of Leishmania major into murine neutrophils is dependent on the neutrophil surface receptor CD11b (CR3/Mac-1) and is enhanced by parasite opsonization with C3. Infected neutrophils underwent robust NADPH oxidase isoform 2 (NOX2)-dependent respiratory burst based on detection of reactive oxygen species within the phagolysosome but largely failed to eliminate the metacyclic promastigote life cycle stage of the parasite. Infected neutrophils displayed an "apoptotic" phosphatidylserine (PS)-positive phenotype, which was induced by both live and fixed parasites but not latex beads, suggesting that PS expression was parasite specific but does not require active infection. In addition, neutrophils from parasite/neutrophil coculture had increased viability, decreased caspase 3, 8, and 9 gene expression, and reduced protein levels of both the pro and cleaved forms of the classical apoptosis-inducing executioner caspase, Caspase 3. Our data suggest that CD11b-mediated Leishmania internalization initiates respiratory burst and PS externalization, followed by a reduction in both the production and cleavage of caspase 3, resulting in a phenotypic state of "stalled apoptosis."

In silico evolutionary and structural analysis of cAMP response proteins (CARPs) from Leishmania major.

With unidentified chemical triggers and novel-effectors, cAMP signaling is broadly noncanonical in kinetoplastida parasites. Though novel protein kinase A regulatory subunits (PKAR) have been identified earlier, cAMP Response Proteins (CARPs) have been identified as a unique and definite cAMP effector of trypanosomatids. CARP1-CARP4 emerged as critical regulatory components of cAMP signaling pathway in Trypanosoma with evidences that CARP3 can directly interact with a flagellar adenylate cyclase (AC). CARP-mediated regulations, identified so far, reflects the mechanistic diversity of cAMP signaling. Albeit the function of the orthologous is not yet delineated, in kinetoplastids like Leishmania, presence of CARP1, 2 and 4 orthologues suggests existence of conserved effector mechanisms. Targeting CARP orthologues in Leishmania, a comprehensive evolutionary analysis of CARPs have been aimed in this study which revealed phylogenetic relationship, codon adaptation and structural heterogeneity among the orthologues, warranting functional analysis in future to explore their involvement in infectivity.

The BRCT Domain from the Homologue of the Oncogene PES1 in Leishmania major (LmjPES) Promotes Malignancy and Drug Resistance in Mammalian Cells.

Around 15% of cancer cases are attributable to infectious agents. Epidemiological studies suggest that an association between leishmaniasis and cancer does exist. Recently, the homologue of PES1 in Leishmania major (LmjPES) was described to be involved in parasite infectivity. Mammalian PES1 protein has been implicated in cellular processes like cell cycle regulation. Its BRCT domain has been identified as a key factor in DNA damage-responsive checkpoints. This work aimed to elucidate the hypothetical oncogenic implication of BRCT domain from LmjPES in host cells. We generated a lentivirus carrying this BRCT domain sequence (lentiBRCT) and a lentivirus expressing the luciferase protein (lentiLuc), as control. Then, HEK293T and NIH/3T3 mammalian cells were infected with these lentiviruses. We observed that the expression of BRCT domain from LmjPES conferred to mammal cells in vitro a greater replication rate and higher survival. In in vivo experiments, we observed faster tumor growth in mice inoculated with lentiBRCT respect to lentiLuc HEK293T infected cells. Moreover, the lentiBRCT infected cells were less sensitive to the genotoxic drugs. Accordingly, gene expression profiling analysis revealed that BRCT domain from LmjPES protein altered the expression of proliferation- (DTX3L, CPA4, BHLHE41, BMP2, DHRS2, S100A1 and PARP9), survival- (BMP2 and CARD9) and chemoresistance-related genes (DPYD, Dok3, DTX3L, PARP9 and DHRS2). Altogether, our results reinforced the idea that in eukaryotes, horizontal gene transfer might be also achieved by parasitism like Leishmania infection driving therefore to some crucial biological changes such as proliferation and drug resistance.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins using Leishmania major.

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major, offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

New Insights on Heme Uptake in Leishmania spp.

The protozoan parasite Leishmania, responsible for leishmaniasis, is one of the few aerobic organisms that cannot synthesize the essential molecule heme. Therefore, it has developed specialized pathways to scavenge it from its host. In recent years, some proteins involved in the import of heme, such as LHR1 and LFLVCRB, have been identified, but relevant aspects regarding the process remain unknown. Here, we characterized the kinetics of the uptake of the heme analogue Zn(II) Mesoporphyrin IX (ZnMP) in Leishmania major promastigotes as a model of a parasite causing cutaneous leishmaniasis with special focus on the force that drives the process. We found that ZnMP uptake is an active, inducible, and pH-dependent process that does not require a plasma membrane proton gradient but requires the presence of the monovalent cations Na+ and/or K+. In addition, we demonstrated that this parasite can efflux this porphyrin against a concentration gradient. We also found that ZnMP uptake differs among different dermotropic or viscerotropic Leishmania species and does not correlate with LHR1 or LFLVCRB expression levels. Finally, we showed that these transporters have only partially overlapping functions. Altogether, these findings contribute to a deeper understanding of an important process in the biology of this parasite.

Evolutionary aspect of Miltefosine transporter proteins in Leishmania major.

Transporter proteins, P-glycoprotein (P-gp) and P4ATPase-CDC50, are responsible for the transport of Miltefosine drug across cell membrane of a protozoan parasite Leishmania major. Mutations or change in activity of these proteins may lead to emergence of resistance in the parasite. Owing to the structural and functional importance of these transporter proteins, we have tried to decipher the evolutionary divergence of these Miltefosine transporter proteins across different forms of life including Protists, Fungi, Plants and Animals. We retrieved 96, 207, and 189 sequences of P-gp, P4ATPase and CDC50 proteins respectively, across diverse variety of organisms for the conserved analysis. Phylogenetic trees were constructed for these three transporter proteins based on Bayesian posterior probability inference. The evolutionary analysis concluded that these proteins remain highly conserved throughout the species diversity but still substantial differences in the proteins for host (Homo sapiens) and parasite (L. major) were observed which have led in targeting these Miltefosine transporter proteins in a parasite specific manner. The functional and structural components observed in terms of pattern resulting from the variability in the phylogenetic tree are outlined.

Crystal structure of the ternary complex of Leishmania major pteridine reductase 1 with the cofactor NADP+/NADPH and the substrate folic acid.

Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydro-state substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process.

Effect of hybridization on lipophosphoglycan expression in Leishmania major.

Leishmania major is the causative agent of cutaneous leishmaniasis. It is one of the most studied Leishmania species not only during vector interaction but also in the vertebrate host. Lipophosphoglycan (LPG) is the Leishmania multifunctional virulence factor during host-parasite interaction, whose polymorphisms are involved in the immunopathology of leishmaniasis. Although natural hybrids occur in nature, hybridization of L. major strains in the laboratory was successfully demonstrated. However, LPG expression in the hybrids remains unknown. LPGs from parental (Friedlin, Fn and Seidman, Sd) and hybrids (FnSd3, FnSd4A, FnSd4B, and FnSd6F) were extracted, purified, and their repeat units analyzed by immunoblotting and fluorophore-assisted carbohydrate electrophoresis. Parental strains have distinct profiles in LPG expression, and a mixed profile was observed for all hybrids. Variable levels of NO production by macrophages were detected after LPG exposure (parental and hybrids) and were strain specific.

Thermal shift assays of marine-derived fungal metabolites from Aspergillus fischeri MMERU 23 against Leishmania major pteridine reductase 1 and molecular dynamics studies.

Marine-derived fungi are a promising source of bioactive molecules, especially species from extreme habitats. Although several secondary metabolites such as meroterpenoids and alkaloids have been isolated from cultures of Aspergillus fischeri, obtained from terrestrial habitats, there is no report on compounds isolated from marine-derived strains. Many metabolites isolated from marine-derived fungi exhibited a myriad of biological activities. Marine natural products have shown to be an important source of bioactive compounds and can assist in the discovery of molecules with affinity against validated targets from exclusive strains of parasites of medical importance such as pteridine reductase 1 (PTR1), from Leishmania major, which is essential for cell growth. Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which make the development of new drugs urgent. The previously described aszonalenin (ASL), aszonapyrone A (ASP), acetylaszonalenin (ACZ), and helvolic acid (HAC) were isolated from the ethyl acetate extract of the culture of a marine sponge-associated A. fischeri MMERU 23 and their affinities against PTR1 were determined by ThermoFluor®. Among the tested compounds, only ACZ showed dose-dependent affinity against PTR1. Moreover, complementary molecular dynamics studies (t = 100 000 ps) have showed that this molecule performs hydrogen bonds with key residues at the active site for more than 60% of the productive trajectory time. The results indicate that ACZ could be a promising PTR1 inhibitor and a potential candidate for development of antileishmanial drug.Communicated by Ramaswamy H. Sarma.

The role of Leishmania GP63 in the modulation of innate inflammatory response to Leishmania major infection.

Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L. major WT (L. majorWT), L. major GP63 knockout (L. majorKO), or L. major GP63 rescue (L. majorR) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L. majorKO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection.

Discovery and Validation of Lmj_04_BRCT Domain, a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis.

Since many of the currently available antileishmanial treatments exhibit toxicity, low effectiveness, and resistance, search and validation of new therapeutic targets allowing the development of innovative drugs have become a worldwide priority. This work presents a structure-based drug discovery strategy to validate the Lmj_04_BRCT domain as a novel therapeutic target in Leishmania spp. The structure of this domain was explored using homology modeling, virtual screening, and molecular dynamics studies. Candidate compounds were validated in vitro using promastigotes of Leishmania major, L. amazonensis, and L. infantum, as well as primary mouse macrophages infected with L. major. The novel inhibitor CPE2 emerged as the most active of a group of compounds against Leishmania, being able to significantly reduce the viability of promastigotes. CPE2 was also active against the intracellular forms of the parasites and significantly reduced parasite burden in murine macrophages without exhibiting toxicity in host cells. Furthermore, L. major promastigotes treated with CPE2 showed significant lower expression levels of several genes (α-tubulin, Cyclin CYCA, and Yip1) related to proliferation and treatment resistance. Our in silico and in vitro studies suggest that the Lmj_04_BRCT domain and its here disclosed inhibitors are new potential therapeutic options against leishmaniasis.