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Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5–6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer’s disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease. [ABSTRACT FROM AUTHOR]

Background: Accurate and accessible diagnosis is key for the control of visceral leishmaniasis (VL). Yet, current diagnostic tests for VL have severe limitations: they are invasive or not suitable as point of care (POC) test or their performance is suboptimal in East Africa. We analysed the antigens in the VL serodiagnostics development pipeline to identify shortcomings and to propose strategies in the development of an alternative POC test for VL in East Africa. Objectives: The objective of this study was to identify and to analyse all antigens for VL serodiagnosis that have been published before 2018 in order to identify candidates and gaps in the pipeline for a new POC test in East Africa. Methods: A systematic literature search was performed on PubMed for original research articles on Leishmania-specific antigens for antibody detection of VL in humans. From each article, the following information was extracted: the antigen name, test format and characteristics, its reported sensitivity and specificity and study cohort specifications. Results: One hundred and seven articles containing information about 96 tests based on 89 different antigens were included in this study. Eighty six of these tests, comprising 80 antigens, were evaluated in phase I and II studies only. Only 20 antigens, all of which are native, contain a carbohydrate and/or lipid moiety. Twenty-four antigens, of which 7 are non-native, are composed of antigen mixtures. Nineteen tests, comprising 18 antigens, have been evaluated on East African specimens, of which only 2 (rK28 based immunochromatographic test and intact promastigote based indirect fluorescent antibody technique) consistently showed sensitivities above 94 and specificities above 97% in a phase III study and one in a phase II study (dot blot with SLA). Only rK28 is a non-native mixture of antigens which we consider suitable for further evaluation and implementation. Conclusions: The development pipeline for an alternative serodiagnostic test for VL is almost empty. Most antigens are not sufficiently evaluated. Non-protein antigens and antigen mixtures are being neglected. We propose to expand the evaluation of existing antigen candidates and to investigate the diagnostic potential of defined non-native carbohydrate and lipid antigens for VL serodiagnosis in East Africa. [ABSTRACT FROM AUTHOR]

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from exoskeleton Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 μM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 μM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1–4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants’ extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction. [ABSTRACT FROM AUTHOR]

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV. [ABSTRACT FROM AUTHOR]

In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L. tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L. tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L. tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L. tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species. [ABSTRACT FROM AUTHOR]

Background: In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. Methods: Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360μl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. Results: Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10−9 m/s and 1.23 x 10−7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. Conclusions: Perfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year and more than 1 billion people at risk of contracting this disease in endemic areas. The innate immune system plays a central role in controlling L. major infection by initiating a signaling cascade that results in production of pro-inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation. The degradation of CXCL1 is not dependent on host factors as L. major can directly degrade recombinant CXCL1 in a cell-free system. Using mass spectrometry, we discovered that the L. major protease cleaves at the C-terminal end of murine CXCL1. Finally, our data suggest that L. major metalloproteases are involved in the direct cleavage and degradation of CXCL1, and a synthetic peptide spanning the CXCL1 cleavage site can be used to inhibit L. major metalloprotease activity. In conclusion, our study has identified an immune evasion strategy employed by L. major to evade innate immune responses in mice, likely reservoirs in the endemic areas, and further highlights that targeting these L. major metalloproteases may be important in controlling infection within the reservoir population and transmittance of the disease. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. [ABSTRACT FROM AUTHOR]

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. [ABSTRACT FROM AUTHOR]

Background: Leishmania parasites are transmitted to vertebrate hosts by phlebotomine sandflies and, in humans, may cause tegumentary or visceral leishmaniasis. The role of PKR (dsRNA activated kinase) and Toll-like receptor 3 (TLR3) activation in the control of Leishmania infection highlights the importance of the engagement of RNA sensors, which are usually involved in the antiviral cell response, in the fate of parasitism by Leishmania. We tested the hypothesis that Phlebovirus, a subgroup of the Bunyaviridae, transmitted by sandflies, would interfere with Leishmania infection. Methodology/Principal findings: We tested two Phlebovirus isolates, Icoaraci and Pacui, from the rodents Nectomys sp. and Oryzomys sp., respectively, both natural sylvatic reservoir of Leishmania (Leishmania) amazonensis from the Amazon region. Phlebovirus coinfection with L. (L.) amazonensis in murine macrophages led to increased intracellular growth of L. (L.) amazonensis. Further studies with Icoaraci coinfection revealed the requirement of the PKR/IFN1 axis on the exacerbation of the parasite infection. L. (L.) amazonensis and Phlebovirus coinfection potentiated PKR activation and synergistically induced the expression of IFNβ and IL-10. Importantly, in vivo coinfection of C57BL/6 mice corroborated the in vitro data. The exacerbation effect of RNA virus on parasite infection may be specific because coinfection with dengue virus (DENV2) exerted the opposite effect on parasite load. Conclusions: Altogether, our data suggest that coinfections with specific RNA viruses shared by vectors or reservoirs of Leishmania may enhance and sustain the activation of host cellular RNA sensors, resulting in aggravation of the parasite infection. The present work highlights new perspectives for the investigation of antiviral pathways as important modulators of protozoan infections. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. Methods/Principle findings: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2–3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfβ1–4[Galpβ1–6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfβ1–4[Galpβ1–6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins’ O-glycans on the former but not the latter parasite clone. Conclusion/Significance: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies. [ABSTRACT FROM AUTHOR]

Background: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. Methodology: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). Principal findings: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 μg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 μg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. Conclusions: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial. [ABSTRACT FROM AUTHOR]

Background: Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression–profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly–and culture-derived promastigotes. Findings: Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly–derived promastigotes have been found. Sand fly–derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly–derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut. Conclusions: Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly–derived promastigotes so far. [ABSTRACT FROM AUTHOR]

Background: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a CLA as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. Methodology: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. Results: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. Conclusion: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. [ABSTRACT FROM AUTHOR]

Most treatments of leishmaniasis require hospitalization and present side effects or parasite resistance; innovations in drug formulation/reposition can overcome these barriers and must be pursued to increase therapeutic alternatives. Therefore, we tested polymyxin B (polB) potential to kill Leishmania amazonensis, adsorbed or not in PBCA nanoparticles (PBCAnp), which could augment polB internalization in infected macrophages. PBCAnps were fabricated by anionic polymerization and analyzed by Dynamic Light Scattering (size, ζ potential), Nanoparticle Tracking Analysis (size/concentration), vertical diffusion cell (release rate), drug incorporation (indirect method, protein determination) and in vitro cell viability. Nanoparticles coated with polB (PBCAnp-polB) presented an adequate size of 261.5 ± 25.9 nm, low PDI and ζ of 1.79 ± 0.17 mV (stable for 45 days, at least). The 50% drug release from PBCAnp-polB was 6–7 times slower than the free polB, which favors a prolonged and desired release profile. Concerning in vitro evaluations, polB alone reduced in vitro amastigote infection of macrophages (10 μg/mL) without complete parasite elimination, even at higher concentrations. This behavior limits its future application to adjuvant leishmanicidal therapy or antimicrobial coating of carriers. The nanocarrier PBCAnp also presented leishmanicidal effect and surpassed polB activity; however, no antimicrobial activity was detected. PolB maintained its activity against E. coli, Pseudomonas and Klebsiella, adding antimicrobial properties to the nanoparticles. Thus, this coated drug delivery system, described for the first time, demonstrated antileishmanial and antimicrobial properties. The bactericidal feature helps with concomitant prevention/treatment of secondary infections that worst ulcers induced by cutaneous L. amazonensis, ultimately ending in disfiguring or disabling lesions. [ABSTRACT FROM AUTHOR]

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection. [ABSTRACT FROM AUTHOR]

Background: Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of Δ22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase. Methodology: In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants. Conclusion: We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting β-sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways. [ABSTRACT FROM AUTHOR]

Here we described a new trypanosomatid species, Phytomonas lipae, parasitizing the dock bug Coreus marginatus based on axenic culture and in vivo material. Using light and electron microscopy we characterized the development of this flagellate in the intestine, hemolymph and salivary glands of its insect host. The intestinal promastigotes of Phytomonas lipae do not divide and occur only in the anterior part of the midgut. From there they pass into hemolymph, increasing in size, and then to salivary glands, where they actively proliferate without attachment to the host's epithelium and form infective endomastigotes. We conducted molecular phylogenetic analyses based on 18s rRNA, gGAPDH and HSP83 gene sequences, of which the third marker performed the best in terms of resolving phylogenetic relationships within the genus Phytomonas. Our inference demonstrated rather early origin of the lineage comprising the new species, right after that of P. oxycareni, which represents the earliest known branch within the Phytomonas clade. This allowed us to compare the development of P. lipae and three other Phytomonas spp. in their insect hosts and reconstruct the vectorial part of the life cycle of their common ancestor. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL. [ABSTRACT FROM AUTHOR]

Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein. [ABSTRACT FROM AUTHOR]

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3–5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. Methods/Principle findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. Conclusion/Significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications. [ABSTRACT FROM AUTHOR]

Leishmania parasites lack pathways for de novo purine biosynthesis. The depletion of purines induces differentiation into virulent metacyclic forms. In vitro, the parasites can survive prolonged periods of purine withdrawal changing their morphology to long and slender cells with an extended flagellum, and decreasing their translation rates. Reduced translation leads to the appearance of discrete granules that contain LeishIF4E-3, one of the six eIF4E paralogs encoded by the Leishmania genome. We hypothesize that each is responsible for a different function during the life cycle. LeishIF4E-3 is a weak cap-binding protein paralog, but its involvement in translation under normal conditions cannot be excluded. However, in response to nutritional stress, LeishIF4E-3 concentrates in specific cytoplasmic granules. LeishIF4E-3 granulation can be induced by the independent elimination of purines, amino acids and glucose. As these granules contain mature mRNAs, we propose that these bodies store inactive transcripts until recovery from stress occurs. In attempt to examine the content of the nutritional stress-induced granules, they were concentrated over sucrose gradients and further pulled-down by targeting in vivo tagged LeishIF4E-3. Proteomic analysis highlighted granule enrichment with multiple ribosomal proteins, suggesting that ribosome particles are abundant in these foci, as expected in case of translation inhibition. RNA-binding proteins, RNA helicases and metabolic enzymes were also enriched in the granules, whereas no degradation enzymes or P-body markers were detected. The starvation-induced LeishIF4E-3-containing granules, therefore, appear to store stalled ribosomes and ribosomal subunits, along with their associated mRNAs. Following nutritional stress, LeishIF4E-3 becomes phosphorylated at position S75, located in its less-conserved N-terminal extension. The ability of the S75A mutant to form granules was reduced, indicating that cellular signaling regulates LeishIF4E-3 function. [ABSTRACT FROM AUTHOR]

Leishmaniasis, as a major health problem in tropical and sub-tropical areas in the world, needs novel, safe, nontoxic and plausible therapeutic solutions for its control. As a part of innate immune system, natural antimicrobial peptides have a potential to be used as new generation of antibiotics especially after persistent resistance of conventional antimicrobial agents. Brevinin 2R, a member of Defensin families of host defense peptides, showed promising effects against bacterial and fungal infections as well as cancerous cell lines. In the current research, the anti-leishmanial effect of Brevinin 2R and its lauric acid conjugate was investigated against Leishmania major (L. major) parasite. The data revealed that, conjugation of fatty acid to Brevinin 2R, strengthen its effect on L. major promastigotes in respect to toxicity and hemolytic effect. These peptides showed anitleishmanial activity through cell membrane disruption and changes in the electrical and mitochondrial membrane potential. No signs of apoptosis induction or caspase activation were detected. Despite its hemolytic and cytotoxic effect in in vitro conditions, lauric acid- Brevinin 2R (L- Brevinin 2R) did not show site specific adverse reactions in animal model. Treatment course with L- Brevinin 2R in the L. major infected mice exhibited decreased parasite load in the lymph nodes adjacent to the infected site despite cytokine production profile and footpad swelling data. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a disease caused by the protozoan parasite, Leishmania. The disease remains a global threat to public health requiring effective chemotherapy for control and treatment. In this study, the effect of some selected phenolic compounds on Leishmania donovani was investigated. The compounds were screened for their anti-leishmanial activities against promastigote and intracellular amastigote forms of Leishmania donovani. Methodology/Principal findings: The dose dependent effect and cytotoxicity of the compounds were determined by the MTT assay. Flow cytometry was used to determine the effect of the compounds on the cell cycle. Parasite morphological analysis was done by microscopy and growth kinetic studies were conducted by culturing cells and counting at 24 hours intervals over 120 hours. The cellular levels of iron in promastigotes treated with compounds was determined by atomic absorption spectroscopy and the effect of compounds on the expression of iron dependent enzymes was investigated using RT-qPCR. The IC50 of the compounds ranged from 16.34 μM to 124 μM compared to amphotericin B and deferoxamine controls. Rosmarinic acid and apigenin were the most effective against the promastigote and the intracellular amastigote forms. Selectivity indexes (SI) of rosmarinic acid and apigenin were 15.03 and 10.45 respectively for promastigotes while the SI of 12.78 and 5.20 respectively was obtained for intracellular amastigotes. Morphologically, 70% of rosmarinic acid treated promastigotes showed rounded morphology similar to the deferoxamine control. About 30% of cells treated with apigenin showed distorted cell membrane. Rosmarinic acid and apigenin induced cell arrest in the G0/G1 phase in promastigotes. Elevated intracellular iron levels were observed in promastigotes when parasites were treated with rosmarinic acid and this correlated with the level of expression of iron dependent genes. Conclusions/Significance: The data suggests that rosmarinic acid exerts its anti-leishmanial effect via iron chelation resulting in variable morphological changes and cell cycle arrest. [ABSTRACT FROM AUTHOR]

Leishmania infantum causes zoonotic visceral leishmaniasis (ZVL) in the Mediterranean basin and South America. The parasite has been shown to co-infect HIV patients and an outbreak in central Spain was reported in the last decade. Therfore, ZVL is a public health problem, dogs being the parasite's reservoir. We have developed a DNA vaccine based on the L. infantum activated protein kinase A receptor (LACK) using different plasmid vectors and vaccinia virus strains as vehicles. Recently, we have generated an antibiotic resistance marker-free plasmid vector called pPAL. Homologous pPAL-LACK prime-boost vaccination protects Beagle dogs as well as a heterologous plasmid-virus regime. For both reasons, pPAL improves safety. IL12 was described to trigger Th1 response through IFN-γ production in infected dogs, being a good candidate for cytokine therapy in conventional treatment-unresponsive dogs. Herein, we report a complete protection study in dogs through inoculation of genes encoding for the p35 and p40 subunits which compose canine IL12 in combination with the LACK gene. A homologous plasmid-plasmid regime using independent pPAL constructs for each gene was inoculated in a 15-day interval. The infectious challenge using L. infantum promastigotes was successful. The outcome was pPAL-LACK vaccine protection suppression by IL12 administration. The important implications of this finding are discussed in the manuscript. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi is the obligate intracellular parasite that causes Chagas disease. The pathogenesis of this disease is a multifactorial complex process that involves a large number of molecules and particles, including the extracellular vesicles. The presence of EVs of T. cruzi was first described in 1979 and, since then, research regarding these particles has been increasing. Some of the functions described for these EVs include the increase in heart parasitism and the immunomodulation and evasion of the host immune response. Also, EVs may be involved in parasite adhesion to host cells and host cell invasion. Methodology/Principal findings: EVs (exosomes) of the Pan4 strain of T. cruzi were isolated by differential centrifugation, and measured and quantified by TEM, NTA and DLS. The effect of EVs in increasing the parasitization of Vero cells was evaluated and the ED50 was calculated. Changes in cell permeability induced by EVs were evaluated in Vero and HL-1 cardiomyocyte cells using cell viability techniques such as trypan blue and MTT assays, and by confocal microscopy. The intracellular mobilization of Ca2+ and the disruption of the actin cytoskeleton induced by EVs over Vero cells were followed-up in time using confocal microscopy. To evaluate the effect of EVs over the cell cycle, cell cycle analyses using flow cytometry and Western blotting of the phosphorylated and non-phosphorylated protein of Retinoblastoma were performed. Conclusion/Significance: The incubation of cells with EVs of trypomastigotes of the Pan4 strain of T. cruzi induce a number of changes in the host cells that include a change in cell permeability and higher intracellular levels of Ca2+ that can alter the dynamics of the actin cytoskeleton and arrest the cell cycle at G0/G1 prior to the DNA synthesis necessary to complete mitosis. These changes aid the invasion of host cells and augment the percentage of cell parasitization. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis is a neglected parasitic disease with no vaccine available and its pharmacological treatment is reduced to a limited number of unsafe drugs. The scarce readiness of new antileishmanial drugs is even more alarming when relapses appear or the occurrence of hard-to-treat resistant strains is detected. In addition, there is a gap between the initial and late stages of drug development, which greatly delays the selection of leads for subsequent studies. Methodology/Principal findings: In order to address these issues, we have generated a red-shifted luminescent Leishmania infantum strain that enables long-term monitoring of parasite burden in individual animals with an in vivo limit of detection of 106 intracellular amastigotes 48 h postinfection. For this purpose, we have injected intravenously different infective doses (104—5x108) of metacyclic parasites in susceptible mouse models and the disease was monitored from initial times to 21 weeks postinfection. The emission of light from the target organs demonstrated the sequential parasite colonization of liver, spleen and bone marrow. When miltefosine was used as proof-of-concept, spleen weight parasite burden and bioluminescence values decreased significantly. Conclusions: In vivo bioimaging using a red-shifted modified Leishmania infantum strain allows the appraisal of acute and chronic stage of infection, being a powerful tool for accelerating drug development against visceral leishmaniasis during both stages and helping to bridge the gap between early discovery process and subsequent drug development. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host. [ABSTRACT FROM AUTHOR]

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3’ untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi. [ABSTRACT FROM AUTHOR]

Background: The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections, such as Trypanosoma cruzi in the chronic phase of Chagas disease. We evaluated the utility of the digital droplet (dd)PCR platform in the detection of T. cruzi infection. Methodology/Principal findings: We imported a validated qPCR assay targeting the T. cruzi satellite tandem repeat (TcSTR) region to the ddPCR platform. Following optimization, we tested and repeated a standard curve of TcI epimastigotes to characterise the analytical performance of the assay on the ddPCR platform. We compared this to published qPCR performance data, and the performance of the qPCR assay in our own testing. We subsequently tested a panel of 192 previously characterized DNA specimens, extracted from the blood of individuals with and without T. cruzi infection. The assay performed well on the ddPCR platform, showing a limit of detection of 5 copies/μL or 1 parasite/mL. This was higher than the published limit of detection for qPCR, which was 0.46 parasites/mL. The ddPCR platform was not significantly more accurate than qPCR at any concentration tested. However, the clinical sensitivity and specificity of the assay were both 100% with perfect agreement between qPCR and ddPCR positive and negative result calling in clinical specimens. An average of 9,286 copies of TcSTR were detected per parasite. Conclusions/Significance: The use of the ddPCR platform to run this assay was comparable, but not superior in terms of performance, to the qPCR platform. [ABSTRACT FROM AUTHOR]

Background: To overcome the current problems in leishmaniasis chemotherapy, natural products have become an interesting alternative over the past few decades. Flavonoids have been studied as promising family of compounds for leishmaniasis treatment. 2’-Hydroxyflavanone (2HF) is a flavanone, a class of flavonoid that has shown promising results in cancer studies. In this study, we demonstrated the effects of 2HF in vitro and in vivo against wild-type and antimony-resistant Leishmania amazonensis promastigotes. Methodology/Principal findings: 2HF was effective against promastigotes and the intracellular amastigote form, decreasing the infection index in macrophages infected with wild-type and antimony-resistant promastigotes, but it was not toxic to macrophages. In silico analysis indicated 2HF as a good oral candidate for leishmaniasis treatment. In vivo, 2HF was able to reduce the lesion size and parasite load in a murine model of cutaneous leishmaniasis using wild-type and antimony-resistant promastigotes, demonstrating no cross-resistance with antimonials. Conclusions/Significance: Taken together, these results suggest 2HF as a potential candidate for leishmaniasis chemotherapy for cutaneous leishmaniasis caused by both wild-type and antimony-resistant Leishmania species by oral administration. Furthermore, studies should be conducted to determine the ideal dose and therapeutic regimen. [ABSTRACT FROM AUTHOR]

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts. [ABSTRACT FROM AUTHOR]

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL. [ABSTRACT FROM AUTHOR]

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite. [ABSTRACT FROM AUTHOR]

Leishmania species are sand fly-transmitted protozoan parasites that cause leishmaniasis, neglected tropical diseases that affect millions of people. Leishmania amastigotes must overcome a variety of host defenses, including reactive oxygen species (ROS) produced by the NADPH oxidase. Leishmania species encode three superoxide dismutases (SODs): the mitochondrial SODA and two glycosomal SODs (SODB1 and SODB2). SODs are metalloenzymes that function in antioxidant defense by converting superoxide to oxygen and hydrogen peroxide. Here, we investigated a role for SODB1 in Leishmania infection of macrophages and virulence in mice. We found that a single allele deletion of SODB1 (SODB1/⊗sodb1) had minimal effects on the replication of axenically-grown L. major promastigotes or differentiation to infective metacyclic promastigotes. Disruption of a single SODB1 allele also did not affect L. donovani differentiation to amastigotes induced axenically, or the replication of axenically-grown L. donovani promastigotes and amastigotes. In contrast, the persistence of SODB1/⊗sodb1 L. major in WT macrophages was impaired, and the development of cutaneous lesions in SODB1/⊗sodb1 L. major-infected C57BL/6 and BALB/c mice was strongly reduced. The reduced disease severity in mice was associated with reduced burdens of SODB1/⊗sodb1 L. major parasites in the foot at late, but not early times post-inoculation, as well as an impaired capacity to disseminate from the site of inoculation. Collectively, these data suggest that SODB1 is critical for L. major persistence in macrophages and virulence in mice. [ABSTRACT FROM AUTHOR]

A young, healthy traveler returning to the United States presented with fever, night sweats, splenomegaly, and pancytopenia. Bone marrow biopsy revealed leishmaniasis (Leishmania infantum), likely acquired in southern France. Although many cases of endemic visceral leishmaniasis (VL) have been reported in Europe, this is a rare case of imported VL in a healthy traveler returning from Europe to the US. Despite successful initial treatment with liposomal amphotericin B (LamB), relapse occurred. Treatments for VL in immunocompetent individuals are highly effective, but relapse can occur. There is more extensive experience in endemic areas with treating relapse that may be lacking in North America. This case alerts physicians in the US that immunocompetent adults can acquire VL during brief visits to endemic areas in Europe. It is important that travelers be counseled on preventive measures. Patients should be monitored after treatment for relapse. [ABSTRACT FROM AUTHOR]

All cellular processes, including those involved in normal cell metabolism to those responsible for cell proliferation or death, are finely controlled by cell signaling pathways, whose core proteins constitute the family of phosphatidylinositol 3-kinase-related kinases (PIKKs). Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3 related (ATR) are two important PIKK proteins that act in response to DNA damage, phosphorylating a large number of proteins to exert control over genomic integrity. The genus Leishmania belongs to a group of early divergent eukaryotes in evolution and has a highly plastic genome, probably owing to the existence of signaling pathways designed to maintain genomic integrity. The objective of this study was to evaluate the use of specific human inhibitors of ATR and ATM in Leishmania major. Bioinformatic analyses revealed the existence of the putative PIKK genes ATR and ATM, in addition to mTOR and DNA-PKcs in Leishmania spp. Moreover, it was possible to suggest that the inhibitors VE-821 and KU-55933 have binding affinity for the catalytic sites of putative L. major ATR and ATM, respectively. Promastigotes of L. major exposed to these inhibitors show slight growth impairment and minor changes in cell cycle and morphology. It is noteworthy that treatment of promastigotes with inhibitors VE-821 and KU-55933 enhanced the oxidative damage caused by hydrogen peroxide. These inhibitors could significantly reduce the number of surviving L. major cells following H2O2 exposure whilst also decreasing their evaluated IC50 to H2O2 to less than half of that observed for non-treated cells. These results suggest that the use of specific inhibitors of ATR and ATM in Leishmania interferes in the signaling pathways of this parasite, which can impair its tolerance to DNA damage and affect its genome integrity. ATR and ATM could constitute novel targets for drug development and/or repositioning for treatment of leishmaniases. [ABSTRACT FROM AUTHOR]

Blastocrithidia papi is a unique trypanosomatid in that its life cycle is synchronized with that of its host, and includes an obligate stage of development in Malpighian tubules (MTs). This occurs in firebugs, which exited the winter diapause. In the short period, preceding the mating of overwintered insects, the flagellates penetrate MTs of the host, multiply attached to the epithelial surface with their flagella, and start forming cyst-like amastigotes (CLAs) in large agglomerates. By the moment of oviposition, a large number of CLAs are already available in the rectum. They are discharged on the eggs' surface with feces, used for transmission of bugs' symbiotic bacteria, which are compulsorily engulfed by the newly hatched nymphs along with the CLAs. The obligate development of B. papi in MTs is definitely linked to the life cycle synchronization. The absence of peristalsis allow the trypanosomatids to accumulate and form dense CLA-forming subpopulations, whereas the lack of peritrophic structures facilitates the extensive discharge of CLAs directly into the hindgut lumen. The massive release of CLAs associated with oviposition is indispensable for maximization of the infection efficiency at the most favorable time point. [ABSTRACT FROM AUTHOR]

Background: Drugs currently used for the treatment of Chagas’ disease, nifurtimox and benznidazole, have a limited effectiveness and toxic side effects. With the aim of finding new therapeutic approaches, in vitro and in vivo anti-Trypanosoma cruzi activity of vitamin C alone and combined with benznidazole were investigated. Methodology/Principal findings: The trypanocidal activity on epimastigote and trypomastigote forms was evaluated by counting parasites in a Neubauer chamber after treatment with the compounds. For the amastigote stage, transgenic parasites expressing β-galactosidase were used and quantified by measuring the β-galactosidase activity. The cytotoxicity of compounds was tested on Vero cells. The redox state of the parasite was evaluated by determining the reduced thiol levels (spectrophotometric assay) and the intracellular oxidative state (by flow cytometry). The in vivo trypanocidal activity was evaluated on a murine model of Chagas’ disease. The trypanocidal activity of vitamin C and benznidazole was similar for the three parasite forms. When combining both drugs, vitamin C did not induce any change in the antiparasitic activity of benznidazole on trypomastigotes; however, on mammal cells, vitamin C diminished the cytotoxicity degree of benznidazole. Two mechanisms of action may be postulated for vitamin C: a lethal pro-oxidant effect on the parasite when used alone, and an antioxidant effect, when combined with benznidazole. A similar behavior was observed on infected mice; i.e., parasite counts in infected mice treated with vitamin C were lower than that of the control group. Animals treated with benznidazole presented lower parasitemia levels, as compared with those treated with vitamin C alone. Again, vitamin C did not cause any effect on the antiparasitic profile of benznidazole. Even though a combined treatment was employed, the antioxidant effect of vitamin C on the host was evidenced; a 100% survival was observed and the weight loss occurring during the acute phase of the infection was reduced. Conclusions/Significance: Based on these results, the combination of vitamin C with benznidazole could be considered as an alternative treatment for Chagas’ disease. These preliminary results encourage further research to improve the treatment of Chagas’ disease. [ABSTRACT FROM AUTHOR]

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and overexpression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species. [ABSTRACT FROM AUTHOR]

The increase in the global land temperature, expected under predictions of climate change, can directly affect the transmission of some infectious diseases, including Chagas disease, an anthropozoonosis caused by Trypanosoma cruzi and transmitted by arthropod vectors of the subfamily Triatominae. This work seeks to study the effects of temperature on the development of the life cycle, fertility and fecundity of the insect vector Rhodnius prolixus and on the metacyclogenesis of T. cruzi. All of the variables were subjected to 3 temperatures: 26°C, 28°C and 30°C. Hatching time was evaluated, along with time to fifth instar, time to adult, fecundity studied using the e-value, and egg viability during the first 3 reproductive cycles. In addition, the amounts of metacyclic trypomastigotes of the TcI and TcII DTUs in R. prolixus were evaluated from days 2 to 20 at two-day intervals and from weeks 6 to 8 post-infection. Decreases were observed in time to hatching (15–10 days on average) and in time to fifth instar (70–60 days on average) and transition to adult (100–85 days on average). No significant differences in egg viability were observed in any of the reproductive cycles evaluated, but an increase in fecundity was observed at 30°C during the third reproductive cycle. At 30°C, there was also an increase in the number of infective forms and a decrease in the time at which metacyclic trypomastigotes were detected in the rectal ampulla of the insects for both TcI and TcII. According to these results, the expected temperature increase under climate change would cause an increase in the number of insects and a greater probability of infection of the parasite, which affects the transmission of Chagas disease. [ABSTRACT FROM AUTHOR]

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages. [ABSTRACT FROM AUTHOR]

Background: Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation. Methodology: Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection. Findings: Our studies in RAW macrophages showed that induction of host UPR against L.donovani infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate. Conclusion: Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection. [ABSTRACT FROM AUTHOR]

The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models. [ABSTRACT FROM AUTHOR]

Chagas’ disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments. [ABSTRACT FROM AUTHOR]

Immune activating cytokines Interferon (IFN)-γ and Tumor necrosis factor (TNF)-α are known to activate macrophages for killing of Leishmania parasite. IFN-γ provides therapeutic potential while TNF-α has been recognized to mediate protection in visceral model of infection. In the present study we investigated whether combination of IFN-γ and TNF-α has better therapeutic strength than individually using one of these cytokines in Visceral Leishmaniasis (VL) patients. We performed combined blockade of IFN-γ and TNF-α in VL splenic biopsies and demonstrated it’s impact on number of viable amastigotes and cytokine production. Additionally, selective depletion of splenic cell subsets was performed to establish the cellular sources of IFN-γ and TNF-α. Treatment of splenic aspirate cells with combination of anti-IFN-γ and anti-TNF-α monoclonal antibodies for 72 hours enabled no direct additive impact of these cytokines on parasite replication and IL-10 secretion, but IL-4 production was induced. Further assessment of splenic biopsies put forward CD4+ T cells as a source of IFN-γ whereas CD14+ cells contribute towards TNF-α production. Overall our results suggest, the interplay of pro-inflammatory cytokines IFN-γ derived from CD4+T lymphocytes and TNF-α from CD14+ cells has no direct additive impact on parasite replication but induces IL-4 production. Our data does not support direct targeting of IFN-γ and TNF-α for combination therapy but targeting these cytokines as an adjuvant in patients with exaggerated tissue inflammatory responses can have favourable patient outcome. [ABSTRACT FROM AUTHOR]