The protein map of the protozoan parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum during growth phase transition and temperature stress.

Leishmania parasites cause a spectrum of diseases termed leishmaniasis, which manifests in two main clinical forms, cutaneous and visceral leishmaniasis. Leishmania promastigotes transit from proliferative exponential to quiescent stationary phases inside the insect vector, a relevant step that recapitulates early molecular events of metacyclogenesis. During the insect blood meal of the mammalian hosts, the released parasites interact initially with the skin, an event marked by temperature changes. Deep knowledge on the molecular events activated during Leishmania-host interactions in each step is crucial to develop better therapies and to understand the pathogenesis. In this study, the proteomes of Leishmania (Leishmania) amazonensis (La), Leishmania (Viannia) braziliensis (Lb), and Leishmania (Leishmania) infantum (syn L. L. chagasi) (Lc) were analyzed using quantitative proteomics to uncover the proteome modulation in three different conditions related to growth phases and temperature shifts: 1) exponential phase (Exp); 2) stationary phase (Sta25) and; 3) stationary phase subjected to heat stress (Sta34). Functional validations were performed using orthogonal techniques, focusing on α-tubulin, gp63 and heat shock proteins (HSPs). Species-specific and condition-specific modulation highlights the plasticity of the Leishmania proteome, showing that pathways related to metabolism and cytoskeleton are significantly modulated from exponential to stationary growth phases, while protein folding, unfolded protein binding, signaling and microtubule-based movement were differentially altered during temperature shifts. This study provides an in-depth proteome analysis of three Leishmania spp., and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts. SIGNIFICANCE: Leishmaniasis disease manifests in two main clinical forms according to the infecting Leishmania species and host immune responses, cutaneous and visceral leishmaniasis. In Brazil, cutaneous leishmaniasis (CL) is associated with L. braziliensis and L. amazonensis, while visceral leishmaniasis, also called kala-azar, is caused by L. infantum. Leishmania parasites remodel their proteomes during growth phase transition and changes in their mileu imposed by the host, including temperature. In this study, we performed a quantitative mass spectrometry-based proteomics to compare the proteome of three New world Leishmania species, L. amazonensis (La), L. braziliensis (Lb) and L. infantum (syn L. chagasi) (Lc) in three conditions: a) exponential phase at 25 °C (Exp); b) stationary phase at 25 °C (Sta25) and; c) stationary phase subjected to temperature stress at 34 °C (Sta34). This study provides an in-depth proteome analysis of three Leishmania spp. with varying pathophysiological outcomes, and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts.

Evasion of the complement system by Leishmania through the uptake of C4bBP, a complement regulatory protein, and probably by the action of GP63 on C4b molecules deposited on parasite surface.

The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis.

Enriched PUFA environment of Leishmania infantum promastigotes promotes the accumulation of lipid mediators and favors parasite infectivity towards J774 murine macrophages.

Leishmania parasites are the causative agents of visceral or cutaneous leishmaniasis in humans and of canine leishmaniosis. The macrophage is the predilected host cell of Leishmania in which the promastigote stage is transformed into amastigote. We previously showed changes in the fatty acid composition (FA) of lipids in two strains of Leishmania donovani upon differentiation of promastigote to amastigote, including increased proportions of arachidonic acid (AA) and to a less extent of docosahexaenoic acid (DHA). Here, we carried out supplementation with AA or DHA on two Leishmania infantum strains, a visceral (MON-1) and a cutaneous (MON-24), to evaluate the role of these FA in parasite/macrophage interactions. The proportions of AA or DHA in total lipids were significantly increased in promastigotes cultured in AA- or DHA-supplemented media compared to controls. The content of FA-derived oxygenated metabolites was enhanced in supplemented strains, generating especially epoxyeicosatrienoic acids (11,12- and 14,15-EET) and hydroxyeicosatetraenoic acids (5- and 8- HETE) from AA, and hydroxydocosahexaenoic acids (14- and 17-HDoHE) from DHA. For both MON-1 and MON-24, AA-supplemented promastigotes showed higher infectivity towards J774 macrophages as evidenced by higher intracellular amastigote numbers. Higher infectivity was observed after DHA supplementation for MON-24 but not MON-1 strain. ROS production by macrophages increased upon parasite infection, but only minor change was observed between control and supplemented parasites. We propose that under high AA or DHA environment that is associated with AA or DHA enrichment of promastigote lipids, FA derivatives can accumulate in the parasite, thereby modulating parasite infectivity towards host macrophages.

Lethal action of Licarin A derivatives in Leishmania (L.) infantum: Imbalance of calcium and bioenergetic metabolism.

Natural metabolites present an extraordinary chemo-diversity and have been used as the inspiration for new drugs. Considering the need for new treatments against the neglected parasitic disease leishmaniasis, three semi-synthetic derivatives of natural neolignane licarin A were prepared: O-acetyl (1a), O-allyl (1b), and 5-allyl (1c). Using an ex vivo assay, compounds 1a, 1b, and 1c showed activity against the intracellular amastigotes of Leishmania (L.) infantum, with IC50 values of 9, 13, and 10 µM, respectively. Despite no induction of hemolytic activity, only compound 1b resulted in mammalian cytotoxicity (CC50 = 64 µM). The most potent compounds (1a and 1c) resulted in selectivity indexes >18. The mechanism of action of compound 1c was evaluated by fluorescent/luminescent based techniques and MALDI-TOF/MS. After a short incubation period, increased levels of the cytosolic calcium were observed in the parasites, with alkalinization of the acidocalcisomes. Compound 1c also induced mitochondrial hyperpolarization, resulting in decreased levels of ATP without altering the reactive oxygen species (ROS). Neither plasma membrane damages nor DNA fragmentation were observed after the treatment, but a reduction in the cellular proliferation was detected. Using MALDI-TOF/MS, mass spectral alterations of promastigote proteins were observed when compared to untreated and miltefosine-treated groups. This chemically modified neolignan induced lethal alterations of the bioenergetic and protein metabolism of Leishmania. Future PKPD and animal efficacy studies are needed to optimize this promising natural-derived compound.

The Astonishing Large Family of HSP40/DnaJ Proteins Existing in Leishmania.

Abrupt environmental changes are faced by Leishmania parasites during transmission from a poikilothermic insect vector to a warm-blooded host. Adaptation to harsh environmental conditions, such as nutrient deprivation, hypoxia, oxidative stress and heat shock needs to be accomplished by rapid reconfiguration of gene expression and remodeling of protein interaction networks. Chaperones play a central role in the maintenance of cellular homeostasis, and they are responsible for crucial tasks such as correct folding of nascent proteins, protein translocation across different subcellular compartments, avoiding protein aggregates and elimination of damaged proteins. Nearly one percent of the gene content in the Leishmania genome corresponds to members of the HSP40 family, a group of proteins that assist HSP70s in a variety of cellular functions. Despite their expected relevance in the parasite biology and infectivity, little is known about their functions or partnership with the different Leishmania HSP70s. Here, we summarize the structural features of the 72 HSP40 proteins encoded in the Leishmania infantum genome and their classification into four categories. A review of proteomic data, together with orthology analyses, allow us to postulate cellular locations and possible functional roles for some of them. A detailed study of the members of this family would provide valuable information and opportunities for drug discovery and improvement of current treatments against leishmaniasis.

A zymographic study of metalloproteinase activities in whole cell extracts and extracellular secretions of Leishmania (L.) , L. major and L. infantum from Iran.

Leishmaniosis encompasses a group of diseases that is transmitted by sand flies and caused by different species of Leishmania. The skin is the initial organ to be infected by the Leishmania in cutaneous, mucocutaneous and visceral forms of leishmaniosis. The matrix metalloproteinases (MMPs) are capable of degrading all kinds of extracellular matrix (ECM) proteins. The aim of this study was to investigate the protease activity through zymography in cell extracts and extracellular secretions of L. major, L. tropica and L. infantum as three prevalent Leishmania spp. in Iran. The three Leishmania spp. were cultured in RPMI-1640 medium supplemented with fetal calf serum. Promastigotes and axenic amastigotes were harvested and lysed at various phases, and extracellular secretions and cell extracts were collected. Leishmania spp. were proved by targeting kDNA gene. Enzymes were characterized according to gelatin zymography and sensitivity to distinct proteinase inhibitors. We observed proteinase bands with molecular weights (MWs) between 66 to 180 kDa in cellular extracts of axenic amastigotes of L. infantum, L. tropica, and L. major, and from 66 to 92 kDa in extracellular secretions of L. infantum. No proteinase activities were observed in extracellular secretions of axenic amastigotes and in cellular extracts of promastigotes in logarithmic and stationary phases of L. major and L. tropica. Using specific inhibitors, we determined that these proteolytic activities are due to metalloproteases. Our study demonstrated that amastigotes of all three Leishmania spp. have distinct amounts of proteinase activities and therefore can cause various types of lesions and outcomes of the disease.

Pyridazino-pyrrolo-quinoxalinium salts as highly potent and selective leishmanicidal agents targeting trypanothione reductase.

Fifteen pyridazino-pyrrolo-quinoxalinium salts were synthesized and tested for their antiprotozoal activity against Leishmania infantum amastigotes. Eleven of them turned out to be leishmanicidal, with EC50 values in the nanomolar range, and displayed low toxicity against the human THP-1 cell line. Selectivity indices for these compounds range from 10 to more than 1000. Compounds 3b and 3f behave as potent inhibitors of the oxidoreductase activity of the essential enzyme trypanothione disulfide reductase (TryR). Interestingly, binding of 3f is not affected by high trypanothione concentrations, as revealed by the noncompetitive pattern of inhibition observed when tested in the presence of increasing concentrations of this substrate. Furthermore, when analyzed at varying NADPH concentrations, the characteristic pattern of hyperbolic uncompetitive inhibition supports the view that binding of NADPH to TryR is a prerequisite for inhibitor-protein association. Similar to other TryR uncompetitive inhibitors for NADPH, 3f is responsible for TryR-dependent reduction of cytochrome c in a reaction that is typically inhibited by superoxide dismutase.

The Inflammatory Effects of Dietary Lipids Regulate Growth of Parasites during Visceral Leishmaniasis.

Visceral leishmaniasis is a potentially fatal disease caused by the protozoon Leishmania donovani or L. infantum (Li). Although previous studies revealed that high lipid intake reduces parasite burdens in Leishmania donovani-infected mice, the specific contributions of dietary lipids to Li-associated pathogenesis are not known. To address this, we evaluated parasite growth, liver pathology, and transcriptomic signatures in Li-infected BALB/c mice fed either a control, high-fat, high-cholesterol, or high-fat-high-cholesterol diet. Using quantitative PCR (qPCR), we observed significantly reduced liver parasite burdens in mice fed the high-fat-high-cholesterol diet compared to mice fed the control diet. In contrast to the liver, parasite expansion occurred earlier in the spleens of mice fed the experimental diets. Histological examination revealed an intense inflammatory cell infiltrate in livers predominantly composed of neutrophils caused by the high-fat-high-cholesterol diet specifically. After 8 weeks of infection (12 weeks of diet), Illumina microarrays revealed significantly increased expression of transcripts belonging to immune- and angiogenesis-related pathways in livers of both uninfected and Li-infected mice fed the high-fat-high-cholesterol diet. These data suggest that increased fat and cholesterol intake prior to Li infection leads to a hepatic inflammatory environment and thus reduces the parasite burden in the liver. Defining inflammatory signatures as well as pathology in the liver may reveal opportunities to modify the therapeutic approach to Li infection. IMPORTANCE Leishmaniasis is a spectrum of diseases caused by Leishmania species protozoa that is most common in warm climates, coinciding with impoverished regions. Visceral leishmaniasis is a potentially fatal disease in which parasites infect reticuloendothelial organs and cause progressive wasting and immunocompromise. The distribution and demographics of visceral leishmaniasis have changed over recent years, coinciding with modernizing societies and the increased availability of Western diets rich in lipid content. We report here that increased dietary fat and cholesterol intake affected disease pathogenesis by increasing inflammation and reducing localized parasite burdens in the liver. These diet-induced changes in disease pathogenesis might explain in part the changing epidemiology of visceral leishmaniasis. A relationship between diet and inflammatory responses may occur in leishmaniasis and other microbial or immune-mediated diseases, possibly revealing opportunities to modify the therapeutic approach to microbial infections.

Analysis of the mechanisms of action of isopentenyl caffeate against Leishmania.

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC50 of 1.56 and 1.71 µM, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 µM, while for L. chagasi the values were 2.48, 1.84 and 1.60 µM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis.

Expression analysis of centrin gene in promastigote and amastigote forms of leishmania infantum iranian isolates: a promising target for live attenuated vaccine development against canine leishmaniasis.

BACKGROUND: Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran. RESULTS: All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05). CONCLUSIONS: Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum.

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.

Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01 ±â€¯1.080 µg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ±â€¯1.211 µg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.

The Fraction of the Snake Venom, Its Leishmanicidal Effect, and the Stimulation of an Anti-Leishmania Response in Infected Macrophages.

BACKGROUND AND AIMS: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOVF11) on Leishmania infantum and its broad mode of action. METHODS: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue), macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme- linked immunosorbent assay (ELISA) on L. infantum promastigote and amastigote stages were used. IC50 values of L. infantum stages, CC50 value, and apoptosis were also analyzed. RESULTS: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent manner compared to the untreated control group. Interleukin (IL)-12, TNF-α, and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity, which displayed a significant reduction (p < 0.001). CONCLUSION: NNOV-F11 possessed a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions, which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic-like mechanisms, and inhibited L-ARG activity, which collectively led to the parasite death. Further studies are crucial to assess the effect of the NNOV-F11 on animal models or clinical settings.

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines.

BACKGROUND: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing. METHODS: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search. RESULTS: The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation. CONCLUSIONS: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Study of the differentially abundant proteins among Leishmania amazonensis, L. braziliensis, and L. infantum.

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.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.

Pseudoirreversible slow-binding inhibition of trypanothione reductase by a protein-protein interaction disruptor.

BACKGROUND AND PURPOSE: Peptide P4 was described as a dimerization disruptor of trypanothione reductase (TryR), a homodimeric enzyme essential for survival of trypanosomatids. Determination of the true inhibitory constant (Ki ) for P4 was not achieved because reaction rates continuously decreased with time, even when substrate concentration was kept constant. The aim of this study was to find a suitable kinetic model that could allow characterization of the complex pattern of TryR inhibition caused by P4. EXPERIMENTAL APPROACH: After showing the slow-binding and pseudoirreversible activity of P4 against Leishmania infantum trypanothione reductase (Li-TryR), analysis of the curvatures of the reaction progress curves at different inhibitor concentrations allowed us to define the apparent inhibitory constants (Kiapp ) at five different substrate concentrations. Analysis of the changes in Kiapp values allowed precise definition of the type of inhibition. KEY RESULTS: Li-TryR inhibition by P4 requires two sequential steps that involve rapid generation of a reversible enzyme-inhibitor complex followed by a pseudoirreversible slow inactivation of the enzyme. Recovery of enzyme activity after inhibitor dissociation is barely detectable. P4 is a non-competitive pseudoirreversible inhibitor of Li- TryR that displays an overall inhibition constant (Ki* ) smaller than 0.02 µM. CONCLUSION AND IMPLICATIONS: Li-TryRdimer disruption by peptide P4 is a pseudoirreversible time-dependent process which is non-competitive with respect to the oxidized trypanothione (TS2 ) substrate. Therefore, unlike reversible Li-TryR competitive inhibitors, enzyme inhibition by P4 is not affected by the TS2 accumulation observed during oxidant processes such as the oxidative burst in host macrophages.

The AAA + ATPase valosin-containing protein (VCP)/p97/Cdc48 interaction network in Leishmania.

Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to the fucose-mannose ligand of Leishmania infantum combined with glycyrrhizin.

BACKGROUND: The fucose-mannose ligand (FML) of Leishmania infantum is a complex glycoprotein which does not elicit adequate immunogenicity in humans. In recent years, adjuvant compounds derived from plants have been used for improving the immunogenicity of vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known immunomodulatory activities. In the present study, we investigated the effects of co-treatment with FML and GL on the production of cytokines and nitric oxide (NO) by macrophages, in vitro. METHODS: Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 µg/ml) of L. infantum and various concentrations of GL (1 µg/ml, 10 µg/ml and 20 µg/ml). After 48 h of treatment, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 were measured by sandwich ELISA and NO concentration by Griess reaction. RESULTS: Our results indicate that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α and IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone. CONCLUSIONS: Therefore, we concluded that GL can improve the immunostimulatory effect of FML on macrophages and leads to their polarization towards an M1-like phenotype.