GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the ß- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Using meta-analysis and machine learning to investigate the transcriptional response of immune cells to Leishmania infection.

BACKGROUND: Leishmaniasis is a parasitic disease caused by the Leishmania protozoan affecting millions of people worldwide, especially in tropical and subtropical regions. The immune response involves the activation of various cells to eliminate the infection. Understanding the complex interplay between Leishmania and the host immune system is crucial for developing effective treatments against this disease. METHODS: This study collected extensive transcriptomic data from macrophages, dendritic, and NK cells exposed to Leishmania spp. Our objective was to determine the Leishmania-responsive genes in immune system cells by applying meta-analysis and feature selection algorithms, followed by co-expression analysis. RESULTS: As a result of meta-analysis, we discovered 703 differentially expressed genes (DEGs), primarily associated with the immune system and cellular metabolic processes. In addition, we have substantiated the significance of transcription factor families, such as bZIP and C2H2 ZF, in response to Leishmania infection. Furthermore, the feature selection techniques revealed the potential of two genes, namely G0S2 and CXCL8, as biomarkers and therapeutic targets for Leishmania infection. Lastly, our co-expression analysis has unveiled seven hub genes, including PFKFB3, DIAPH1, BSG, BIRC3, GOT2, EIF3H, and ATF3, chiefly related to signaling pathways. CONCLUSIONS: These findings provide valuable insights into the molecular mechanisms underlying the response of immune system cells to Leishmania infection and offer novel potential targets for the therapeutic goals.

Case Report: Simple Nodular Cutaneous Leishmaniasis Caused by Autochthonous Leishmania (Mundinia) orientalis in an 18-Month-Old Girl: The First Pediatric Case in Thailand and Literature Review.

We report an autochthonous case of simple, localized cutaneous leishmaniasis in a healthy 18-month-old girl from southern Thailand. The patient presented with a solitary chronic cutaneous nodular lesion on her left cheek for approximately 1 year. Histopathological dissection of the cheek skin biopsy demonstrated remarkably nodular and interstitial infiltrates of lymphocytes and histiocytes full of intracellular oval-shaped amastigotes, consistent with cutaneous leishmaniasis. The Leishmania promastigotes were also cultured successfully from the lesion biopsy and were designated with the WHO code MHOM/TH/2021/CULE5. Using internal transcribed spacer 1-specific polymerase chain reaction, the parasite DNA was demonstrated in both saliva and lesion biopsy. Based on the BLASTn and phylogenetic analysis, the parasite was identified as Leishmania orientalis, clustered in the Mundinia subgenus. The patient responded well to a 6-week course of oral itraconazole, without recurrence. To our knowledge, this is the fourth case of autochthonous leishmaniasis resulting from L. orientalis and the youngest patient of leishmaniasis ever reported in Thailand. More importantly, we also demonstrate the clinical course of the lesion according to the timeline before and after treatment, which can help physicians better understand and provide an accurate diagnosis with appropriate treatment of this emerging parasitic disease.

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay.

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

The ChaC family of γ-glutamyl cyclotransferases is required for Leishmania to switch to a slow growth state and for long-term survival of the parasite.

The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC2a and LmChaC2b) in the unicellular pathogen Leishmania. Activity measurements suggest that these proteins catalyze degradation of GSH but no other γ-glutamyl peptides. Recombinant LmChaC2a protein shows ∼17-fold lower catalytic efficiency (kcat ∼ 0.9 s-1) than LmChaC2b (kcat ∼ 15 s-1), although they showed comparable Km values (∼1.75 mM for LmChaC2a and ∼2.0 mM for LmChaC2b) toward GSH. qRT-PCR and Western blot analyses suggest that the LmChaC2a protein was found to be constitutively expressed, whereas LmChaC2b was regulated by sulfur stress. To investigate its precise physiological function in Leishmania, we generated overexpressed, knockout, and complement cell lines. Flow cytometric analyses show the presence of a higher intracellular GSH concentration and lower intracellular ROS level, indicative of a more reductive environment in null mutants. We found LmChaC2-expressing cells grow in GSH-containing sulfur-limited media, while the null mutants failed to grow, suggesting that LmChaC2 is crucial for cell growth with GSH as the only sulfur source. Null mutants, although reach the stationary phase rapidly, display impaired long-term survival, indicating that LmChaC2-mediated GSH degradation is necessary for prolonged survival. In vivo studies suggest that LmChaC2-dependent controlled GSH degradation promotes chronic infection by the parasite. Altogether, these data indicate that LmChaC2 plays an important role in GSH homeostasis in Leishmania.

Leishmania infection-derived extracellular vesicles drive transcription of genes involved in M2 polarization.

Although it is known that the composition of extracellular vesicles (EVs) is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs (LiEVs) containing parasite-derived molecules. In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages, is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that significantly increase numbers as the infection progresses. To evaluate the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10, and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha, and IFN-gamma and not M2 signature molecules. Finally, in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers, although they exhibit differences in their spatial distribution in infected tissues. In conclusion, EVs produced by macrophages during Leishmania infection lead to the gene expression consistent with M2 polarization. In contrast, the EVs produced during S. Typhimurium infection stimulated the transcription of genes associated with M1 polarization.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 µM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Clinical-therapeutic follow-up of patients with American cutaneous leishmaniasis caused by different Leishmania spp. in Brazil.

American cutaneous leishmaniasis (ACL) may present different clinical manifestations, immune and therapeutic responses, depending on the Leishmania species, as well as inoculum size and factors inherent to the affected individual. Thus, the aim of this study was to carry out clinical-therapeutic follow-up of Brazilian patients with ACL caused by different Leishmania species. Between 2015 and 2018, patients with ACL from Amazonas and Pernambuco states (Brazil) were submitted to blood collection before and after treatment. The qPCR technique was used to quantify the parasite load. To identify the Leishmania species, one of the following techniques was employed: a conventional PCR performed from biopsy or blood DNA, followed by sequencing; or Multilocus Enzyme Electrophoresis from Leishmania isolated from biopsy/aspirated lesion. A total of 10.8% (23/213) of the patients included in positive cases were followed-up. All 23 patients were clinically and epidemiologically compatible with ACL and were also positive in parasitological tests (86.96%), molecular tests (73.91%) or both (60.87%). Seventeen samples collected before treatment and 11 collected after treatment were positive in the qPCR assay, with a mean parasite load (MPL) of 38.33 fg/µL and 11.81 fg/µL, respectively. Eight samples were positive in both collections. Thirteen patients (56.52%) were clinically cured (wound healing). Ten patients (43.47%) were not clinically cured at the time of return with the attending physician. Identification of Leishmania species was carried out in samples from nine patients, and six were identified as L. (Viannia) braziliensis, 2 as L (Viannia) guyanensis and 1 as L (Leishmania) amazonensis. One patient infected with L. guyanensis and other with L. braziliensis were not clinically cured and increased the mean parasite load after treatment. The data obtained from the followed-up patients and the relationship between clinical evolution and the infecting species demonstrate the need to understand its etiology to define the effective therapeutic protocol.

Serological and molecular detection of Leishmania species in dog peripheral blood from Bobo-Dioulasso city, a confirmation of canine leishmaniasis enzootic area for Burkina Faso.

Canine leishmaniasis is increasingly reported worldwide and represent a threat to both animal and human health. In a previous pilot study conducted in Bobo-Dioulasso, the second town of Burkina Faso, we reported five cases of canine leishmaniasis. With the perspective of a One Health action plan, and in the context of increasing urbanization, this study aimed to provide new information on Leishmania spp in dogs in this city. A cross-sectional survey was carried out from May to August 2018 in six districts of the city in order to record clinical and biological data from domestic dogs randomly selected per district. Blood samples were collected into EDTA tubes (4-5 mL), treated and stored at -20 °C until further analyses. The infection status of the dogs was performed by serological tests using plasma, and real time-PCR (RT-PCR) to detect Leishmania parasites using buffy coats. Nested PCR was used for typing the Leishmania species in dogs which were found to be RT-PCR positive. A total of 147 dogs were examined clinically and sampled for blood collection, including 53.7% females and 46.3% of males with a median age of 3 years. The seroincidence of Leishmania parasites within this dog population was 4.76% (95% CI:2.26-9.72). The incidence of Leishmania was 10.88% (95% CI: 6.73-17.11) by RT-PCR which was significantly more sensitive (p = 0,047) and a fair concordance was observed between both tests (Kappa = 0.39, p < 0.001). The characterization of Leishmania species revealed that L. major was circulating in this domestic dog population. Our results confirmed the persistence of zoonotic circulation of Leishmania parasites such as L. major currently in Bobo-Dioulasso city and highlight the need for targeted interventions in order to control transmission of leishmaniasis in this region.

Polyamine Metabolism in Leishmania Parasites: A Promising Therapeutic Target.

Parasites of the genus Leishmania cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is urgent because no vaccine is available, and treatment options are limited due to a lack of specificity and the emergence of drug resistance. Polyamines are metabolites that play a central role in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania. Numerous studies using a variety of inhibitors as well as gene deletion mutants have elucidated the pathway and routes of transport, revealing unique aspects of polyamine metabolism in Leishmania parasites. These studies have also shed light on the significance of polyamines for parasite proliferation, infectivity, and host-parasite interactions. This comprehensive review article focuses on the main polyamine biosynthetic enzymes: ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase, and it emphasizes recent discoveries that advance these enzymes as potential therapeutic targets against Leishmania parasites.

Characterization of Leishmania (L.) amazonensis oligopeptidase B and its role in macrophage infection.

Leishmania spp. are parasitic protozoa that cause leishmaniasis, a disease endemic in 98 countries. Leishmania promastigotes are transmitted by the vector and differentiate into amastigotes within phagocytic cells of the vertebrate host. To survive in multiple and hostile environments, the parasite has several virulence factors. Oligopeptidase B (OPB) is a serine peptidase present in prokaryotes, some eukaryotes and some higher plants. It has been considered a virulence factor in trypanosomatids, but only a few studies, performed with Old World species, analysed its role in Leishmania virulence or infectivity.L. (L.) amazonensis is an important agent of cutaneous leishmaniasis in Brazil. The L. (L.) amazonensis OPB encoding gene has been sequenced and analysed in silico but has never been expressed. In this work, we produced recombinant L. (L.) amazonensis OPB and showed that its pH preferences, Km and inhibition patterns are similar to those reported for L. (L.) major and L. (L.) donovani OPBs. Since Leishmania is known to secrete OPB, we performed in vitro infection assays using the recombinant enzyme. Our results showed that active OPB increased in vitro infection by L. (L.) amazonensis when present before and throughout infection. Our findings suggest that OPB is relevant to L. (L.) amazonensis infection, and that potential drugs acting through OPB will probably be effective for Old and New World Leishmania species. OPB inhibitors may eventually be explored for leishmaniasis chemotherapy.

Production of Functional Plant Legumain Proteases Using the Leishmania tarentolae Expression System.

Plant proteases of the legumain-type are key players in many processes along the plant life cycle. In particular, legumains are especially important in plant programmed cell death and the processing and maturation of seed storage proteins within the vacuole. Plant legumains are therefore synonymously called vacuolar processing enzymes (VPEs). Because of their dual protease and cyclase activities, plant legumains are of great interest to biotechnological applications, e.g., for the development of cyclic peptides for drug design. Despite this high interest by the scientific community, the recombinant expression of plant legumains proved challenging due to several posttranslational modifications, including (1) the formation of structurally critical disulfide bonds, (2) activation via pH-dependent proteolytic processing, and (3) stabilization by varying degrees of glycosylation. Recently we could show that LEXSY is a robust expression system for the production of plant legumains. Here we provide a general protocol for the recombinant expression of plant legumains in Leishmania cells. We further included detailed procedures for legumain purification, activation and subsequent activity assays and additionally note specific considerations with regard to isoform specific activation intermediates. This protocol serves as a universal strategy for different legumain isoforms from different source organisms.

Comparison of immunohistochemical and qPCR methods from granulomatous dermatitis lesions for detection of leishmania in dogs living in endemic areas: a preliminary study.

BACKGROUND: In canine leishmaniosis (CanL) endemic areas, pathologists often receive skin biopsies for testing with histopathologic findings suggestive-but not conclusive for a definitive diagnosis-of CanL lesions. I the absence of data on the infective status of animals, the diagnosis can therefore be challenging. The aim of this retrospective study was to evaluate the ability of immunohistochemistry (IHC) and quantitative PCR (qPCR) methods to detect Leishmania infection in skin biopsies with a histopathologic diagnosis of lymphoplasmacytic/histiocytic and/or granulomatous dermatitis and to correlate the pattern, depth and severity of the histopathologic lesions with the parasite load detected by qPCR and IHC. METHODS: Thirty formalin-fixed, paraffin-embedded skin samples were evaluated by hematoxylin-eosin (H&E) staining, IHC, conventional PCR (cPCR) and qPCR. The severity, pattern and depth of the dermal inflammation and parasite load were graded. RESULTS: Leishmania was detected by H&E staining in 8/30 sections (26.66%) and by IHC in 14/30 samples (46.66%). Parasite DNA was detected in 14/30 samples (46.66%) by cPCR and in 21/30 samples (70%) by qPCR, with an extremely variable parasite load (1.32-62.700 copies). The level of agreement was fair between H&E staining and cPCR (κ = 0.32), and moderate between H&E staining and IHC (κ = 0.58). The level of agreement between IHC and cPCR was good (κ = 0.65); between IHC and qPCR, moderate (κ = 0.41); and between cPCR and qPCR, fair (κ = 0.28). A significant association was found between the severity of dermal inflammation and the parasitic skin load by IHC, although with weak linear correlation. CONCLUSIONS: Our study underlines the difficulty of obtaining a definitive diagnosis of CanL cutaneous lesions, even with the most accurate diagnostic tests currently available. Based on our results, no single test is suitable on its own for the diagnosis of cutaneous lesions caused by Leishmania. However, in the presence of a moderate/severe lymphoplasmacytic/histiocytic and/or granulomatous dermatitis, we suggest performing IHC, as in our study this technique proved to be the method with the highest discriminatory power to estimate the role of the parasite in skin lesions. In mild lesions, IHC loses its discriminatory power and should be effectively combined with techniques such as qPCR.

Culture of Cutaneous Leishmania from Skin Biopsy Specimens.

This protocol describes the culture of Leishmania parasites from skin biopsy samples of patients with cutaneous lesions. The use of antibiotics to prevent bacterial contamination of these cultures increases the ability of researchers to collect isolates for various research purposes, including genetic analysis and in vitro and in vivo experiments. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Culture of Leishmania from skin biopsy specimens.

In silico analysis of Leishmania proteomes and protein-protein interaction network: Prioritizing therapeutic targets and drugs for repurposing to treat leishmaniasis.

Leishmaniasis is a serious world health problem and its current therapies have several limitations demanding to develop novel therapeutics for this disease. The present study aims to prioritize novel broad-spectrum targets using proteomics and protein-protein interaction network (PPIN) data for 11 Leishmania species. Proteome comparison and host non-homology analysis resulted in 3605 pathogen-specific conserved core proteins. Gene ontology analysis indicated their involvement in major molecular functions like DNA binding, transportation, dioxygenase, and catalytic activity. PPIN analysis of these core proteins identified eight hub proteins (viz., vesicle-trafficking protein (LBRM2903_190011800), ribosomal proteins S17 (LBRM2903_34004790) and L2 (LBRM2903_080008100), eukaryotic translation initiation factor 3 (LBRM2903_350086700), replication factor A (LBRM2903_150008000), U3 small nucleolar RNA-associated protein (LBRM2903_340025600), exonuclease (LBRM2903_200021800), and mitochondrial RNA ligase (LBRM2903_200074100)). Among the hub proteins, six were classified as drug targets and two as vaccine candidates. Further, druggability analysis indicated three hub proteins, namely eukaryotic translation initiation factor 3, ribosomal proteins S17 and L2 as druggable. Their three-dimensional structures were modelled and docked with the identified ligands (2-methylthio-N6-isopentenyl-adenosine-5'-monophosphate, artenimol and omacetaxine mepesuccinate). These ligands could be experimentally validated (in vitro and in vivo) and repurposed for the development of novel antileishmanial agents.

Molecular survey of Leishmania spp. in skin samples of capybaras (Hydrochoerus hydrochaeris) from different areas of Brazil / Investigação molecular de Leishmania spp. em amostras de pele de capivaras (Hydrochoerus hydrochaeris) de diferentes regiões do Brasil

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the genus Leishmania, with some species of rodents being incriminated as reservoirs. The capybara is the largest extant rodent species in the world and is widely distributed in South America. The occurrence of infection by Leishmania spp. was investigated in capybaras captured in Brazil during 2015­2019 from established populations in five highly anthropic areas of the state of São Paulo and two natural areas of the states of Mato Grosso and Mato Grosso do Sul. A total of 186 individuals were captured and subjected to abdominal skin biopsy. All skin samples were Leishmania kDNA-negative, suggesting that capybaras have no role in the transmission cycles of Leishmania species in the studied areas despite the well-known role of other rodents in the life cycle of Leishmania spp.(AU)

As leishmanioses compreendem um espectro de doenças causadas por protozoários do gênero Leishmania e algumas espécies de roedores são incriminadas como reservatórios de Leishmania spp. As capivaras compreendem a maior espécie de roedores existentes e são amplamente distribuídas na América do Sul. Para investigar a ocorrência de infecção por Leishmania spp. em capivaras, durante os anos de 2015-2019 capivaras foram capturadas em cinco áreas antrópicas do estado de São Paulo e em duas áreas naturais dos estados do Mato Grosso e do Mato Grosso do Sul, todos esses ambientes com populações de capivaras estabelecidas. Um total de 186 indivíduos foram capturados e submetidos à biópsia de pele abdominal. Todas as amostras de pele foram negativas para o alvo kDNA, assim, os dados sugerem que nas áreas estudadas as capivaras não têm papel no ciclo de transmissão de espécies de Leishmania spp., apesar do papel bem conhecido de outros roedores no ciclo de vida de Leishmania spp.(AU)

In Silico Design of Recombinant Chimera T Cell Peptide Epitope Vaccines for Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. Systemic VL is fatal if untreated and there are no prophylactic human vaccines available. Several studies suggest that Th1 cell-mediated immunity plays a major role in protecting against VL. In this chapter we describe a method for designing recombinant chimera vaccines in silico based on the prediction of T cell epitopes within protein antigens identified as potential protective immunogens. Development of a recombinant chimera protein (RCP) vaccine using T cell epitope peptides identified from four Leishmania proteins is used as an exemplar of this method.

Genome instability drives epistatic adaptation in the human pathogen Leishmania.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.