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Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

LeishGEM is a genome-wide functional annotation community resource for Leishmania mexicana, where deletion mutant growth in vitro and in vivo is measured and protein localisation is determined by endogenous tagging and LOPIT-DC (localisation of organelle proteins by isotope tagging with differential centrifugation) spatial proteomics. Data are being made available pre-publication via http://leishgem.org which allows data-driven identification of the mechanisms for Leishmania parasitism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation., Competing Interests: Declaration of Competing Interest The authors Rebecca J. Burge, Katie H. Jameson, Vincent Geoghegan, Adam Dowle, Jeremy C. Mottram and Anthony J. Wilkinson of the manuscript ‘Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana’ being submitted for publication in Molecular and Biochemical Parasitology have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Importance: In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania . Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania . Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania , shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania , thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology., Competing Interests: The authors declare no conflict of interest.

Introduction: Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans , trans -farnesol ( t , t -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 Candida albicans , 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 trans , trans -farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite., Methods: The study involved the addition of exogenous trans , trans -farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans , trans -farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans , trans -farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome., Results: Exogenously added trans , trans -farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans , trans -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 Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production., Discussion: The findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis . While trans , trans -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 trans , trans -farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania . Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pinheiro, Andrade-Neto, Mantuano-Barradas, Pereira, Barbosa, de Oliveira, Menna-Barreto, Cunha-Júnior and Torres-Santos.)

Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro . Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT 10 -plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pacakova, Harant, Volf and Lestinova.)

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug., Competing Interests: The authors have declared that no competing interests exist.

The protozoan parasite Leishmania (L.) amazonensis infects and replicates inside host macrophages due to subversion of the innate host cell response. In the present study, we demonstrate that TLR3 is required for the intracellular growth of L. (L.) amazonensis . We observed restricted intracellular infection of TLR3 -/- mouse macrophages, reduced levels of IFN1β and IL-10, and increased levels of IL-12 upon L. (L.) amazonensis infection, compared with their wild-type counterparts. Accordingly, in vivo infection of TLR3 -/- mice with L. (L.) amazonensis displayed a significant reduction in lesion size. Leishmania (L.) amazonensis infection induced TLR3 proteolytic cleavage, which is a process required for TLR3 signaling. The chemical inhibition of TLR3 cleavage or infection by CPB-deficient mutant L. (L.) mexicana resulted in reduced parasite load and restricted the expression of IFN1β and IL-10. Furthermore, we show that the dsRNA sensor molecule PKR (dsRNA-activated protein kinase) cooperates with TLR3 signaling to potentiate the expression of IL-10 and IFN1β and parasite survival. Altogether, our results show that TLR3 signaling is engaged during L. (L.) amazonensis infection and this component of innate immunity modulates the host cell response.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania -macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania -macrophage interaction over time that extends beyond the analysis of single-gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gonçalves, Pulido, Mathias, Enes, Carvalho, de Melo Resende, Polak and Ruiz.)

Kinesins are motor proteins present in organisms from protists to mammals playing important roles in cell division, intracellular organisation and flagellum formation and maintenance. Leishmania mexicana is a protozoan parasite of the order Kinetoplastida causing human cutaneous leishmaniasis. Kinetoplastida genome sequence analyses revealed a large number of kinesins showing sequence and structure homology to eukaryotic kinesins. Here, we investigate the L. mexicana kinesin LmxKIN29 (LmxM.29.0350), also called DEATH kinesin. The activated MAP kinase LmxMPK3, a kinase affecting flagellum length in Leishmania, is able to phosphorylate recombinant full length LmxKIN29 at serine 554. Insect promastigote LmxKIN29 Leishmania null mutants showed no obvious phenotype. However, in mouse infection experiments, the null mutants were unable to cause the disease, whereas LmxKIN29 add-backs and single allele knockouts caused footpad lesions. Localisation using promastigotes expressing GFP-tagged LmxKIN29 revealed that the kinesin is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells the GFP-fusion protein was found at the anterior and posterior ends of the cells indicating a role in cytokinesis. The inability to cause lesions in infected animals and the amino acid sequence divergence from mammalian kinesins suggests that LmxKIN29 is a potential drug target against leishmaniasis., (© 2022. The Author(s).)

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of 20 Leishmania deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania . We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania . This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCE Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania , RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania . Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania , thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.

Background: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis., Methodology/principal Findings: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana., Conclusion/significance: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity., Competing Interests: The authors have declared that no competing interests exist.

The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O 2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply., (Copyright © 2021. Published by Elsevier Inc.)

Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania , a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana ( Lmx PP2C). Recombinant Lmx PP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn +2 ). Lmx PP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti- Lmx PP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized Lmx PP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, Lmx PP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms., Competing Interests: The procedure presently used to purify and quantify the expression of PP2C in L. mexicana has been patented in Mexico (MX/a/2020/011271) by AE-M and MA-G. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Escalona-Montaño, Zuñiga-Fabián, Cabrera, Mondragón-Flores, Gómez-Sandoval, Rojas-Bernabé, González-Canto, Gutiérrez-Kobeh, Pérez-Montfort, Becker and Aguirre-García.)

Leishmania are sandfly-transmitted protists that induce granulomatous lesions in their mammalian host. Although infected host cells in these tissues can exist in different activation states, the extent to which intracellular parasites stages also exist in different growth or physiological states remains poorly defined. Here, we have mapped the spatial distribution of metabolically quiescent and active subpopulations of Leishmania mexicana in dermal granulomas in susceptible BALB/c mice, using in vivo heavy water labeling and ultra high-resolution imaging mass spectrometry. Quantitation of the rate of turnover of parasite and host-specific lipids at high spatial resolution, suggested that the granuloma core comprised mixed populations of metabolically active and quiescent parasites. Unexpectedly, a significant population of metabolically quiescent parasites was also identified in the surrounding collagen-rich, dermal mesothelium. Mesothelium-like tissues harboring quiescent parasites progressively replaced macrophage-rich granuloma tissues following treatment with the first-line drug, miltefosine. In contrast to the granulomatous tissue, neither the mesothelium nor newly deposited tissue sequestered miltefosine. These studies suggest that the presence of quiescent parasites in acute granulomatous tissues, together with the lack of miltefosine accumulation in cured lesion tissue, may contribute to drug failure and nonsterile cure. IMPORTANCE Many microbial pathogens switch between different growth and physiological states in vivo in order to adapt to local nutrient levels and host microbicidal responses. Heterogeneity in microbial growth and metabolism may also contribute to nongenetic mechanisms of drug resistance and drug failure. In this study, we have developed a new approach for measuring spatial heterogeneity in microbial metabolism in vivo using a combination of heavy water ( 2 H 2 O) labeling and imaging mass spectrometry. Using this approach, we show that lesions contain a patchwork of metabolically distinct parasite populations, while the underlying dermal tissues contain a large population of metabolically quiescent parasites. Quiescent parasites also dominate drug-depleted tissues in healed animals, providing an explanation for failure of some first line drugs to completely eradicate parasites. This approach is broadly applicable to study the metabolic and growth dynamics in other host-pathogen interactions., (Copyright © 2021 Kloehn et al.)

Aim: Compounds that block enzyme activity can kill pathogens and help develop effective and safe drugs for Chagas disease and leishmaniasis. Materials & methods: A library of nonpeptidic nitrile-based compounds was synthesized and had their inhibitory affinity tested against cruzain, Leishmania mexicana cysteine protease B and cathepsin L. Isothermal titration calorimetry experiments and molecular simulations were performed for selected compounds to obtain thermodynamic fingerprints and identify main interactions and putative modes of binding with cruzain. Results: The derivatives provided increased affinity against all enzymes compared with the lead, and thermodynamic and computational studies showed improved thermodynamic properties and a possible different mode of binding. Conclusion: Our studies culminated in 1b, a compound 60-fold more potent in cruzain than its lead that also showed entropic and enthalpic contributions favorable to Gibbs binding energy.

Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma membrane. The exosomes from Leishmania parasites contain an array of parasite molecules such as virulence factors and survival messengers, capable of modulating the host immune response and thereby favoring the infection of the host. We here show that exosomes of L. mexicana amastigotes (aExo) contain the virulence proteins gp63 and PP2C. The incubation of aExo with bone marrow-derived macrophages (BMMs) infected with L. mexicana led to their internalization and were found to colocalize with the cellular tetraspanin CD63. Furthermore, aExo inhibited nitric oxide production of infected BMMs, permitting enhanced intracellular parasite survival. Expressions of antigen-presenting (major histocompatibility complex class I, MHC-I, and CD1d) and costimulatory (CD86 and PD-L1) molecules were modulated in a dose-dependent fashion. Whereas MHC-I, CD86 and PD-L1 expressions were diminished by exosomes, CD1d was enhanced. We conclude that aExo of L. mexicana are capable of decreasing microbicidal mechanisms of infected macrophages by inhibiting nitric oxide production, thereby enabling parasite survival. They also hamper the cellular immune response by diminishing MHC-I and CD86 on an important antigen-presenting cell, which potentially interferes with CD8 T cell activation. The enhanced CD1d expression in combination with reduction of PD-L1 on BMMs point to a potential shift of the activation route towards lipid presentations, yet the effectivity of this immune activation is not evident, since in the absence of costimulatory molecules, cellular anergy and tolerance would be expected., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Laura Enedina Soto-Serna et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kelly et al.)

There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania -infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania -infected macrophages., (Copyright © 2020 Riezk et al.)

1,2,3-triazolium salts are poorly understood regarding their antileishmanial activity. Hence, as an effort to identify novel chemical scaffolds as antileishmanial agents, a series of 1,2,3-triazolium salts (TS) and corresponding 1,2,3-triazole (T) precursors including new epoxide derivatives were synthesized and assayed against Leishmania amazonensis promastigote and intracellular amastigote forms. Among them, the compound TS-6 exhibited promising activity on promastigotes (IC 50  = 3.61 μM) and intracellular amastigotes (IC 50  = 7.61 μM) of L. amazonensis, superior to miltefosine (IC 50  > 10.0 μM), used as reference drug. In addition, TS-6 showed negligible cytotoxicity on murine peritoneal macrophages with a SI of about 10. Studies on the mode of action of TS-6 indicate mitochondrial dysfunction through an increase in 'total' and mitochondrial-ROS as well as depolarization of mitochondrial membrane potential of L. amazonensis promastigotes. In silico physicochemical studies indicate that the TS-6 could potentially be used as an oral drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This protocol describes the use of heavy water ( 2 H 2 O) labeling to determine the growth rate and metabolic state of Leishmania parasites in culture and in infected animals. In vitro labeling studies are undertaken by cultivating defined parasite developmental stages in standard medium supplemented with 5% 2 H 2 O, resulting in the incorporation of deuterium ( 2 H) into a range of metabolic precursors used in macromolecule (DNA, RNA, protein, lipid, and glycan) synthesis. The rate of turnover of different parasite macromolecules can subsequently be determined by analysis of deuterium enrichment in the different constituents of these macromolecules by gas chromatography-mass spectrometry (GC-MS). To measure the growth rate and physiological state of parasite stages in lesion tissue, infected mice were provided with 9% 2 H 2 O in their drinking water for various periods of time and 2 H-enrichment in the macromolecular constituents of isolated lesion-derived parasite stages determined by GC-MS. This protocol provides quantitative information on key cellular processes, such as replication (DNA turnover), transcription (RNA turnover), translation (protein turnover), membrane biogenesis (lipid turnover), and central carbon metabolism (glycan turnover) that define the growth state and phenome of different parasite stages in vitro and in vivo. This approach can be used to assess the impact of host immune responses on parasite growth and physiology (using different Leishmania strains/species, mouse lines), characterize different parasite populations during chronic and acute infections, and assess parasite responses to drug treatments. It is also broadly applicable to other microbial pathogens.

In the search for new compounds with antileishmanial activity, we synthesized a triazole hybrid analogue of the neolignans grandisin and machilin G (LASQUIM 25), which was previously found highly active against both promastigotes and intracellular amastigote forms of Leishmania amazonensis . In this work, we investigated the leishmanicidal effects of LASQUIM 25 to identify the mechanisms involved in the cell death of L. amazonensis promastigotes. Transmission electron microscopy (TEM) analysis showed marked effects of LASQUIM 25 (IC 50 = 7.2 µM) on the morphology of promastigote forms, notably on mitochondria. The direct action of the triazole derivative on the parasite was noticed over time from 2 h to 48 h, and cells displayed several ultrastructural alterations characteristic of apoptotic cells. Also, flow cytometric analysis (FACS) after TMRE staining detected changes in mitochondrial membrane potential after LASQUIM 25 treatment (64.83% labeling versus 83.38% labeling in nontreated cells). On the other hand, FACS after PI staining in 24 h-treatment showed a slight alteration in the integrity of the cell membrane, a necrotic event (16.76% necrotic cells versus 3.19% staining in live parasites). An abnormal secretion of lipids was observed, suggesting an exocytic activity. Another striking finding was the presence of autophagy-related lysosome-like vacuoles, suggesting an autophagic cell death that may arise as consequence of mitochondrial stress. Taken together, these results suggest that LASQUIM 25 leishmanicidal mechanisms involve some degree of mitochondrial dysregulation, already evidenced by the treatment with the IC 50 of this compound. This effect may be due to the presence of a methylenedioxy group originated from machilin G, whose toxicity has been associated with the capacity to generate electrophilic intermediates.

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.

The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis , are poorly understood. In L. amazonensis , reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages. Tight spatial and temporal regulation of H 2 O 2 is key to this signaling mechanism, suggesting a role for ascorbate-dependent peroxidase (APX), which degrades mitochondrial H 2 O 2 Earlier studies showed that APX -null L. major parasites are viable, accumulate higher levels of H 2 O 2 , generate a greater yield of infective metacyclic promastigotes, and have increased virulence. In contrast, we found that in L. amazonensis , the ROS-inducible APX is essential for survival of all life cycle stages. APX -null promastigotes could not be generated, and parasites carrying a single APX allele were impaired in their ability to infect macrophages and induce cutaneous lesions in mice. Similar to what was reported for L. major , APX depletion in L. amazonensis enhanced differentiation of metacyclic promastigotes and amastigotes, but the parasites failed to replicate after infecting macrophages. APX expression restored APX single-knockout infectivity, while expression of catalytically inactive APX drastically reduced virulence. APX overexpression in wild-type promastigotes reduced metacyclogenesis, but enhanced intracellular survival following macrophage infection or inoculation into mice. Collectively, our data support a role for APX-regulated mitochondrial H 2 O 2 in promoting differentiation of virulent forms in both L. major and L. amazonensis Our results also uncover a unique requirement for APX-mediated control of ROS levels for survival and successful intracellular replication of L. amazonensis ., (Copyright © 2019 American Society for Microbiology.)

Leishmaniasis, caused by protozoa belonging to the genus Leishmania, is an important public health problem found in >90 countries and with still limited options for treatment. Development of new anti-leishmanial drugs is an urgent need and the identification of new active compounds is a limiting factor that can be accelerated through large scale drug screening. This requires multiple steps and can be expensive and time consuming. Here, we propose an alternative approach for the colorimetric assessment of anti-Leishmania drug activity that can be easily scaled up. L. amazonensis and L. infantum cell lines were generated having the β-galactosidase (β-gal) gene integrated into their chromosomal 18S rRNA (ssu) locus. Both cell lines expressed high levels of β-gal and had their growth easily monitored and quantified colorimetrically. These two cell lines were then evaluated as tools to assess drug susceptibility and their use was validated through in vitro assays with Amphotericin B, which is routinely used against leishmaniasis. β-gal expression was also confirmed through flow-cytometry, another method of phenotypic detection. With these recombinant parasites, an alternative in vitro model of drug screening against cutaneous and visceral leishmaniasis is now available., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Isolated growth hormone (GH) deficiency (IGHD) affects approximately 1 in 4,000 to 1 in 10,000 individuals worldwide. We have previously described a large cohort of subjects with IGHD due to a homozygous mutation in the GH releasing hormone (GHRH) receptor gene. These subjects exhibit throughout the life very low levels of GH and its principal mediator, the Insulin Growth Factor-I (IGF-I). The facilitating role of IGF-I in the infection of mouse macrophages by different Leishmania strains is well-known. Nevertheless, the role of IGF-I in Leishmania infection of human macrophages has not been studied. This study aimed to evaluate the behavior of Leishmania infection in vitro in macrophages from untreated IGHD subjects. To this end, blood samples were collected from 14 IGHD individuals and 14 age and sex-matched healthy controls. Monocytes were isolated and derived into macrophages and infected with a strain of Leishmania amazonensis . In addition, IGF-I was added to culture medium to evaluate its effect on the infection. Cytokines were measured in the culture supernatants. We found that macrophages from IGHD subjects were less prone to Leishmania infection compared to GH sufficient controls. Both inflammatory and anti-inflammatory cytokines increase only in the supernatants of the control macrophages. Addition of IGF-I to the culture medium increased infection rates. In conclusion, we demonstrated that IGF-I is crucial for Leishmania infection of human macrophages.

To identify new agents for the treatment of American cutaneous leishmaniasis, a series of eight 1,4-bis(substituted benzalhydrazino)phthalazines was evaluated against Leishmania braziliensis and Leishmania mexicana parasites. These compounds represent a disubstituted version of the 1-chloro-4-(monoaryl/heteroarylhydranizyl)phthalazine that exhibited a significant response against L. braziliensis according to our previous findings. Two disubstituted phthalazines 3b and 3f were identified as potential antileishmanial agents against L. braziliensis parasites, exhibiting a submicromolar IC 50 response of 2.37 and 7.90 µM on the promastigote form, and of 1.82 and 4.56 µM against intracellular amastigotes, respectively. In particular, compound 3b showed interesting responses against amastigote isolates from reference, glucantime-resistant and clinical human strains, which were by far superior to the biological response found for the glucantime drug. With regard to the toxicity results, both 3b and 3f exhibited moderate LD 50 values against murine macrophages (BMDM), with good selectivity indexes on promastigotes and intracellular amastigotes of L. braziliensis. A comparison of biological response was established between the monosubstituted and disubstituted versions of these benzalhydrazino-phthalazines. Easy synthetic procedure and significant response against amastigote strains including against resistant lines made compound 3b a potential candidate for further pharmacokinetic and in vivo experiments as antileishmanial agent, and as a platform for further structural optimization. Mechanism-of-action studies and molecular docking simulations discarded to inhibition of superoxide dismutase as possible mode of action., (© 2019 Deutsche Pharmazeutische Gesellschaft.)

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4 + T cell and MΦ activation status in vitro and in vivo . L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4 + T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4 + T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

Intracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector, which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting, but overlooked finding, is that other cell types and even non-phagocytic cells have been found to be infected by Leishmania spp. Nevertheless, the mechanisms by which Leishmania invades such cells had not been previously studied. Here, we show that L. amazonensis can induce their own entry into fibroblasts independently of actin cytoskeleton activity, and, thus, through a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions, such as Ca 2+ signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm 2 . By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 μM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm 2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.

Cysteine redox state has been identified as one of the key biological influences behind protein structure and/or function. Altered protein redox state has been shown to cause significant physiological changes and can leave proteins with changed sensitivity to oxidative stress. Protein redox-state changes are recognized as an important mediator of disease, cellular abnormalities, and environmental changes, and therefore their characterization is of interest. Isotopic or isobaric labeling followed by sample multiplexing and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) allows relative comparison of protein expression levels or of protein redox states between several samples. Combining analysis of protein expression level and redox state into one analysis would add an extra dimension and permit the normalization of protein redox changes with protein abundance. To achieve this, we have developed a quantitation workflow that uses commercially available cysteine-reactive tandem mass tags (iodoTMT) to differentially label cysteine residues, and we have applied it to two Leishmania mexicana cell lines that have previously shown different responses to oxidative stress. The individually labeled samples have been pooled in different combinations to create multiple sixplex samples in order to study the relationship between cysteine oxidation and overall protein expression, as well as providing information about protein oxidation levels in each cell line. The results highlight 11 proteins that are differentially expressed between the two cell lines and/or have significant redox changes. This advanced multiplexing method effectively demonstrates the flexibility of tandem mass tags and how they can be used to maximize the amount of information that can be acquired.

Leishmania mexicana is an intracellular parasite that causes cutaneous leishmaniasis (CL) in some countries, including Mexico. Recently, we identified the elongation factor-1α (EF-1α) of L. mexicana by immunoproteomic analysis. In Leishmania donovani, this molecule has been reported as a virulence factor involved in downregulation of macrophages by no-canonical function when EF-1α interacts with protein tyrosine phosphatase-1 (SHP-1). However, in L. mexicana the key role of EF-1α in host-parasite relationship has not been elucidated, by this reason we started with cloning and recombinant expression of this antigen. A sequence of 1350 bp corresponding to EF-1α (EF-Lm) full-length gene was amplified from genomic DNA of L. mexicana (GenBank: MG256973); this gene contains only one nucleotide change: C464T, compared with L. mexicana reference sequence (GenBank: FR799570.1). The gene cloned (EF-Lm) codes for a protein of 449 residues. It was expressed in Escherichia coli and purified as 63 kDa sumo-fusion protein, which was recognized in the sera of patients diagnosed with CL. Our results show that EF-Lm is immunogenic during infection, and the rEF-Lm could be used for further analyses in the host-parasite relationship.

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation., (© 2018 The Authors.)

The available chemotherapeutic drugs for the treatment of leishmaniasis present problems relating to efficacy, emergence of parasite resistance, and adverse effects and cost. Azole antifungal drugs have been repurposed for this proposition but the clinical response has been variable. In this sense, this study assessed the leishmanicidal and immunomodulatory activities of azoles-derived imidazolium salts (IS), being the ionic imidazole-derived equivalents: 1-n-butyl-3-methylimidazolium chloride (C 4 MImCl), 1-n-decyl-3-methylimidazolium chloride (C 10 MImCl), 1-n-hexadecyl-3-methylimidazolium chloride (C 16 MImCl), 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C 16 MImMeS), 1-n-hexadecyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (C 16 MImNTf 2 ) and 1-methyl-3-n-octadecylimidazolium chloride (C 18 MImCl). Promastigotes of Leishmania amazonensis were incubated with IS at concentrations ranging from 0.1 to 100 μM, and the parasite survival was monitored. The effects of IS on reactive oxygen species (ROS) production and mitochondrial membrane potential of promastigotes, as well as on cytotoxicity against peripheral blood mononuclear cells (PBMC) and human erythrocytes were determined. Besides, the activities of IS against amastigotes and nitric oxide production were also evaluated. The IS inhibited parasite growth and showed potent leishmanicidal activity against promastigotes of L. amazonensis. In addition, IS induced mitochondrial dysfunction and ROS production in parasites, and presented low cytotoxicity against PBMC and human erythrocytes. Furthermore, at very low concentration (0.5 μM), C 18 MImCl, C 16 MImMeS, C 16 MImCl, C 10 MImCl and C 16 MImNTf 2 were able to kill intramacrophage parasites at levels of 91.3, 100, 94.4, 95.3 and 35.6%, respectively. These results indicate that IS are promising candidates for the development of drugs against L. amazonensis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3 s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3 s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources., (© 2018 John Wiley & Sons Ltd.)

Cyclopropane fatty acid synthase (CFAS) catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated fatty acid, generating a cyclopropane fatty acid (CFA). The gene encoding CFAS is present in many bacteria and several Leishmania spp. including Leishmania mexicana, Leishmania infantum and Leishmania braziliensis. In this study, we characterised the CFAS-null and -overexpression mutants in L. mexicana, the causative agent for cutaneous leishmaniasis in Mexico and central America. Our data indicate that L. mexicana CFAS modifies the fatty acid chain of plasmenylethanolamine (PME), the dominant class of ethanolamine glycerophospholipids in Leishmania, generating CFA-PME. While the endogenous level of CFA-PME is extremely low in wild type L. mexicana, overexpression of CFAS results in a significant increase. CFAS-null mutants (cfas - ) exhibit altered cell shape, increased sensitivity to acidic pH, and aberrant growth in serum-free media. In addition, the CFAS protein is preferentially expressed during the proliferative stage of L. mexicana and is required for the cell membrane targeting of lipophosphoglycan. Finally, the maturation and localization of CFAS protein are dependent upon the downstream sequence of the CFAS coding region. Without the downstream sequence, the mis-localised CFAS protein cannot fully rescue the defects of cfas - . Our data suggest that CFA modification of phospholipids can significantly affect the parasite's response to certain adverse conditions. These findings are distinct from the roles of CFAS in L. infantum, highlighting the functional divergence in lipid modification among Leishmania spp., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel's ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG's ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages.

Background: Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes., Methodology/principal Findings: A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis., Conclusions/significance: All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [ 3 H]-uridine but not of [ 3 H]-inosine. Conversely, the NT2-like genes mediated uptake of [ 3 H]-inosine but not [ 3 H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse-chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low-density lipoproteins indicated that retention of this source of cholesterol is increased in parasite-containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann-Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites' membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA-encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol-dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Targeted regulation of protein levels is an important tool to investigate the role of proteins essential for cell function and development. In recent years, methods based on the Escherichia coli dihydrofolate reductase destabilization domain (ecDHFR DD) have been established and used in various cell types. ecDHFR DD destabilizes the fused protein of interest and causes its degradation by proteasomes, unless it is stabilized by a specific ligand, trimethoprim. In this work we developed an inducible protein stabilization system in Leishmania mexicana based on ecDHFR DD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown., Methodology/principal Findings: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems., Conclusions/significance: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.

Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania ., (Copyright © 2017 American Society for Microbiology.)

Leishmania parasites multiply and develop in the gut of a sand fly vector in order to be transmitted to a vertebrate host. During this process they encounter and exploit various nutrients, including sugars, and amino and fatty acids. We have previously generated a mutant Leishmania line that is deficient in glucose transport and which displays some biologically important phenotypic changes such as reduced growth in axenic culture, reduced biosynthesis of hexose-containing virulence factors, increased sensitivity to oxidative stress, and dramatically reduced parasite burden in both insect vector and macrophage host cells. Here we report the generation and integration of proteomic and metabolomic approaches to identify molecular changes that may explain these phenotypes. Our data suggest changes in pathways of glycoconjugate production and redox homeostasis, which likely represent adaptations to the loss of sugar uptake capacity and explain the reduced virulence of this mutant in sand flies and mammals. Our data contribute to understanding the mechanisms of metabolic adaptation in Leishmania and illustrate the power of integrated proteomic and metabolomic approaches to relate biochemistry to phenotype., Biological Significance: This paper reports the application of comparative proteomic and metabolomic approaches to reveal the molecular basis for important phenotypic changes Leishmania parasites that are deficient in glucose uptake. Leishmania cause a very significant disease burden across the world and there are few effective drugs available for control. This work shows that proteomics and metabolomics can produce complementary data that advance understanding of parasite metabolism and highlight potential new targets for chemotherapy., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania and includes cutaneous, mucocutaneous and visceral clinical forms. The drugs currently available for leishmaniasis treatment are pentavalent antimonials, amphotericin B and miltefosine, which present high toxicity, elevated cost and development of parasite resistance. The natural products constitute an important source of substances with leishmanicidal potential. Here we evaluated in vitro the anti-Leishmania amazonensis activity of crude extracts of branches, leaves and fruits of Guatteria latifolia. The branch extract (GCE) exhibited promising leishmanicidal activity against promastigotes (IC 50 51.7 μg/ml), and was submitted to fractionation guided by in vitro assays. Among the seven subfractions obtained, GF1 and GF2 were the most actives against promastigotes with IC 50 25.6 and 16 μg/ml, respectively. Since GCE, GF1 and GF2 were not toxic for macrophages, next, we tested their effect on intracellular amastigotes, and the IC 50 values obtained were, respectively 30.5, 10.4 and 7.4 μg/ml, after 24 h treatment. The selectivity index for GCE, GF1 and GF2 were >6.5, >19.2 and > 27, respectively. Additionally, GCE, GF1 and GF2 affected the division pattern of the promastigotes by increasing 6.7, 9.4 and 7-fold the cells in Sub-G0/G1 phase, and decreasing 1.6, 2.5 and 1.8-fold the cells in G0/G1 phase, respectively. To assess the GCE and GFs capacity to modulate microbicidal mechanisms of macrophages, nitric oxide (NO) and TNF-α production were tested. Our results indicated that at the IC 50s GCE, GF1 and GF2 decreased NO production of infected macrophages stimulated with IFN-γ and LPS, besides, only GF1 decreased the production of TNF-α. Our data warrant further studies of GCE, GF1 and GF2 to identify active compounds against Leishmania parasites., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H 2 O 2 ) for 1h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3' G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The number of protein folds in nature is limited, thus is not surprising that proteins with the same fold are able to exert different functions. The cysteine protease inhibitors that adopt an immunoglobulin- like fold (Ig-ICPs) are inhibitors encoded in bacteria and protozoan parasites. Structural studies indicate that these inhibitors resemble the structure of archetypical proteins with an Ig fold, like antibodies, cadherins or cell receptors. The structure of Ig-ICPs from four different protozoan parasites clearly shows the presence of three loops that form part of a protein-ligand interaction surface that resembles the antigen binding sites of antibodies. Thus, Ig-ICPs bind to different cysteine proteases using a tripartite mechanism in which their BC, DE and FG loops are responsible for the main interactions with the target cysteine protease. Ig-ICPs from different protozoan parasites regulate the enzymatic activity of host or parasite's proteases and thus regulate virulence and pathogenesis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (c w50 ) and the cell membrane (c m50 ) and the membrane-aqueous medium partition coefficient of MT (K). With a c w50 of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had c w50 values of 2.4-3.1μM. The estimated c m50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays., (Copyright © 2016 Elsevier B.V. All rights reserved.)