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Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

Leishmaniasis has been considered as emerging and re-emerging disease, and its increasing global incidence has raised concerns. The great clinical diversity of the disease is mainly determined by the species. In several American countries, tegumentary leishmaniasis (TL) is associated with both Leishmania amazonensis and L. braziliensis, while visceral leishmaniasis (VL) is associated with L. (L.) infantum. The major molecules that determine the most diverse biological variations are proteins. In the present study, through a DIGE approach, we identified differentially abundant proteins among the species mentioned above. We observed a variety of proteins with differential abundance among the studied species; and the biological networks predicted for each species showed that many of these proteins interacted with each other. The prominent proteins included the heat shock proteins (HSPs) and the protein network involved in oxide reduction process in L. amazonensis, the protein network of ribosomes in L. braziliensis, and the proteins involved in energy metabolism in L. infantum. The important proteins, as revealed by the PPI network results, enrichment categories, and exclusive proteins analysis, were arginase, HSPs, and trypanothione reductase in L. amazonensis; enolase, peroxidoxin, and tryparedoxin1 in L. braziliensis; and succinyl-CoA ligase [GDP -forming] beta-chain and transaldolase in L. infantum.

Leishmaniasis is a major public health problem, causing diseases ranging from self-healing skin lesions to life-threatening chronic infections. Understanding how Leishmania parasites evade the host defense system is crucial for understanding the different manifestations of the disease and for improving diagnostic tools and drug development. We performed high-resolution proteome profiling of Leishmania spp. across three species during macrophage infection and identified distinct temporal expression patterns. Clustering analysis revealed unique protein expression profiles for each Leishmania species, whereas pairwise enrichment analysis revealed specific up- and downregulation patterns at different infection stages. Our results confirmed known virulence factors and highlighted new ones, demonstrating how our dataset could be used. We validated the dataset by showing that deletion of putative L. mexicana virulence factors resulted in reduced stage differentiation capacity and infectivity.

Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major , localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9–mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (Sb III ), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in Sb III IC 50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance.

Leishmania is a vacuolar pathogen that replicates within parasitophorous vacuoles inside host phagocytes. To promote its replication, Leishmania relies on a panoply of strategies to acquire macromolecules such as lipids from host macrophages. In this study, we have evaluated the role of VAPA, an endoplasmic reticulum-resident membrane protein involved in inter-organellar lipid transport, in macrophages infected with L. amazonensis . Following infection of bone marrow-derived macrophages with L. amazonensis metacyclic promastigotes, we observed that VAPA gradually associates with communal parasitophorous vacuoles. Knockdown of VAPA prevented the replication of L. amazonensis , which was accompanied by an impaired parasitophorous vacuole expansion. Using fluorescent ceramide, we established that VAPA is required for the transport of sphingolipids to the parasitophorous vacuoles and for its acquisition by L. amazonensis amastigotes. Proximity-ligation assays revealed that L. amazonensis hijacks VAPA by disrupting its interactions with the host cell lipid transfer proteins CERT and ORP1L. Finally, we found that VAPA is essential for the transfer of the Leishmania virulence glycolipid lipophosphoglycan from the parasitophorous vacuoles to the host cell endoplasmic reticulum. We propose that VAPA contributes to the ability of L. amazonensis to colonize macrophages by mediating bi-directional transfer of lipids essential for parasite replication and virulence between the parasitophorous vacuoles and the host cell endoplasmic reticulum.

Leishmaniasis is a global health issue, especially in tropical and subtropical areas, with treatment challenges due to the development of resistance to current drugs. This has prompted the search for new antileishmanial compounds. Endoperoxides, due to parasites’ reliance on external iron and susceptibility to oxidative stress, are promising antileishmanial compounds. This study evaluated two sterol endoperoxides—ergosterol endoperoxide (ErgoEP) and dehydrocholesterol endoperoxide (DHCholEP)—for their antileishmanial activity and mechanism in vitro. Cell viability assays with Leishmania donovani and Leishmania tarentolae promastigotes showed IC50 values in the low micromolar range (from 2.0 to 4.5 µM, respectively) with low toxicity to murine and J774A.1 macrophages. Electron paramagnetic resonance spectroscopy confirmed radical generation in the presence of low-molecular-weight iron compounds. However, this did not trigger the antileishmanial effect, as neither N-acetylcysteine nor pyridoxal isonicotinoyl hydrazone altered activity. Mitochondrial function(s) and superoxide production in Leishmania remained unaffected. Both endoperoxides significantly inhibited synthesis of 5-dehydroepisterol, the major sterol in Leishmania tarentolae, suggesting targeting of the sterol biosynthesis pathway. Their limited toxicity to mammalian macrophages makes ergosterol and dehydrocholesterol endoperoxides promising candidates for future antileishmanial drug development.

Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. predominantly secretes the , -farnesol ( , -FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including , and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added , -farnesol on the proliferative ability of and to identify its presence in the lipid secretome of the parasite. The study involved the addition of exogenous , -farnesol to evaluate its interference with the proliferation of promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of , -farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of , -farnesol and related products derived from the precursor, farnesyl pyrophosphate. analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the genome. Exogenously added , -farnesol was found to interfere with the proliferation of promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of , -farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production. The findings collectively offer the first insights into the mechanism of action of farnesol on . While , -farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous , -farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in . Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against infections.

Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania . We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.

Drug repositioning has been considered a promising approach to discover novel treatments against neglected diseases. Among the major protozoan diseases, leishmaniasis remains a public health threat with few therapeutic alternatives, affecting 12 million people in 98 countries. In this study, we report the in vitro antileishmanial activity of the imidazole drugs clotrimazole, and for the first time in literature, econazole and bifonazole and their potential action to affect the regulation of reactive oxygen species (ROS) of the parasites. The lethal action of the imidazoles was investigated using spectrofluorimetric techniques to detect ROS content, plasma membrane permeability, and mitochondrial membrane potential. The imidazoles showed activity against L. (L.) infantum chagasi promastigotes with IC 50 values in a range of 2–8 μM; econazole was also effective against Leishmania intracellular amastigotes, with an IC 50 value of 11 μM, a similar in vitro effectiveness to miltefosine. Leishmania promastigotes rapidly up-regulated the ROS release after incubation with the imidazoles, but econazole showed a marked increase in ROS content of approximately 1,900 % higher than untreated parasites. When using SYTOX ® Green as a fluorescent probe, the imidazoles demonstrated considerable interference in plasma membrane permeability at the early time of incubation; econazole resulted in the higher influx of SYTOX ® Green at 60 min. Despite cellular alterations, no depolarization could be observed to the mitochondrial membrane potential of Leishmania until 60 min. The lethal action of econazole involved strong permeabilization of plasma membrane of promastigotes, with an overloaded ROS content that contributed to the death of parasites. Affecting the ROS regulation of Leishmania via small molecules would be an interesting strategy for new drugs.