Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy.

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.

Aqueous ozone therapy improves the standard treatment of leishmaniasis lesions in animals leading to local and systemic alterations.

The current treatment of leishmaniasis presents some problems, such as cell toxicity, parenteral route, and time of treatment. Ozone emerges as an option to accelerate the standard treatment due to the immunomodulatory, antioxidant, and wound healing activity reported in the literature. This work aimed to evaluate the efficacy of aqueous ozone as an adjuvant to the standard treatment of cutaneous lesions caused by Leishmania amazonensis in an experimental model. For in vivo experiments, mice were randomly distributed in 6 groups, which were infected with L. amazonensis and treated in five different schedules using the standard treatment with Glucantime® with or without aqueous ozone. After the last day of treatment, the animals were euthanized and were analyzed: the thickness of lesions; collagen deposition, the parasitic burden of the lesions; blood leukocyte number; NO; and cytokine dosages and arginase activity from peritoneal macrophages. All treated groups showed a decrease in the lesion, but with a significative deposition of collagen in lesions with local ozone treatment. The parasite burden showed that ozone enhanced the leishmanicidal activity of the reference drug. The reduction of NO production and blood leukocyte count and increases in the arginase activity showed an immunomodulatory activity of ozone in the treated animals. Thus, ozone therapy has been shown to work as an adjuvant in the treatment of Leishmania lesions, enhancing leishmanicidal and wound healing activity of standard treatment.

Molecular Method Confirms Canine Leishmania Infection Detected by Serological Methods in Non-Endemic Area of Brazil.

In Brazil, visceral leishmaniasis (VL) is expanding and becoming urbanized, especially in non-endemic areas such as the State of Rio Grande do Sul. Considering that infected dogs are the main reservoir for zoonotic VL, this study evaluated the prevalence of canine visceral leishmaniasis (CVL) in the metropolitan area of Porto Alegre, a new area of expansion of VL in Brazil. Serum and plasma from 405 asymptomatic dogs from the municipalities of Canoas (n=107), São Leopoldo (n=216), and Novo Hamburgo (n=82) were tested for CVL using immunochromatographic (DPP®) and ELISA EIE® assays (2 assays officially adopted by the Brazilian government for the diagnosis of CVL) and real-time PCR to confirm the results. There was no agreement among serological and real-time PCR results, indicating that the Leishmania infection in asymptomatic animals with low parasite load, confirmed by negative parasitological tests (smears and parasite culture), need to be evaluated by molecular methods. The prevalence of LVC in the metropolitan region of Porto Alegre, confirmed by real-time PCR was 4% (5.6% in Canoas and 4.6% in São Leopoldo). The use of molecular method is essential for accurate diagnosis of CVL, especially in asymptomatic dogs in non-endemic areas.

Experimental Infection of Lutzomyia (Nyssomyia) whitmani (Diptera: Psychodidae: Phlebotominae) With Leishmania (Viannia) braziliensis and Leishmania (L.) amazonensis, Etiological Agents of American Tugumentary Leishmaniasis.

Leishmania (L.) amazonensis (Lainson & Shaw, 1972) and Leishmania (Viannia) braziliensis (Vianna, 1911) are the principal causative agents of American tegumentary leishmaniasis (ATL) in Brazil. L. amazonensis also causes diffuse cutaneous leishmaniasis (DCL) vectored principally by Lutzomyia flaviscutellata and secondarily by Lutzomyia whitmani (Antunes & Coutinho, 1939). The latter is the most common phlebotomine in the state of Maranhão, and it is the focal species for potential ATL transmission. For this reason, we tested the ability of L. whitmani to become infected with Lutzomyia parasites. Phlebotomines were derived from a colony maintained in the laboratorial conditions. The first generation, uninfected females were offered a bloodmeal with mice infected with the strains of both parasites. We found that L. whitmani can become infected with both parasite species, with infection rates of 65.2% (L. braziliensis) and 47.4% (L. amazonensis). We conclude that in Maranhão, L. whitmani is likely an important vector in the transmission of ATL and may function as a vector of DCL. This possibility should be further investigated.

Canine Leishmaniosis Associated with Acute Pleural Effusion and Sudden Death in a Dog.

A two-year-old female crossbreed dog, previously a stray with no known owner, was adopted and subsequently spayed. The dog exhibited weight loss over a period of two months and died suddenly during a leashed walk. Upon necropsy, enlargement of the submandibular, prescapular, and popliteal lymph nodes was noted. The intrathoracic cavity contained a substantial volume of yellowish-white fluid. Lymph nodes in the mediastinal and ventral thoracic centers were also enlarged, hemorrhagic, and friable. Microscopic examination revealed significant architectural changes in the lymph nodes, characterized by a pronounced cellular infiltrate consisting of lymphocytes and histiocytes, along with macrophages containing intracytoplasmic Leishmania amastigotes. Immunohistochemical analysis of the lymph nodes confirmed positive staining for Leishmania amastigotes. This case represents the first report of canine leishmaniasis associated with acute pleural effusion and sudden death.

Human visceral leishmaniasis and polymorphisms in interleukin-coding genes: a systematic review.

Visceral leishmaniasis (VL) is a neglected disease that is typical of tropical and subtropical parts of the world and is caused by the trypanosomatid Leishmania donovani complex. This disease is a multifactorial condition that involves parasitic, environmental, and immunogenetic characteristics. Genetic changes in genes encoding cytokines may be associated with changes in their expression and, consequently, with the development of clinical resistance or susceptibility to the disease. This systematic review and meta-analysis aimed to assess whether single nucleotide polymorphisms (SNPs) in interleukin genes influence the clinical consequences of visceral leishmaniasis infection. To this end, we carried out a systematic review and meta-analysis with structured searches in the EMBASE, PubMed, Scopus, SciELO, and Web of Science databases without time restrictions. Two independent reviewers examined the studies, performed data extraction, and assessed quality by assigning scores. If there were any discrepancies, a third reviewer with more experience was consulted. After the screening process, 28 articles were included in the systematic review and 9 in the final analysis of the meta-analysis. Statistical analyses were carried out using various genetic models. The odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated to estimate the associations. Overall, the main clinical outcomes were classified as not associated or associated when they presented susceptibility, resistance, risk, or protective factors for the development of the disease. Associations between IFN-γ +874T/A polymorphisms in the dominant model (OR 1.64, 95% CI 1.13-2.38, I2 = 0%, p < 0.01) and heterozygous model (OR 1.72, 95% CI 1.15-2.57, I2 = 0%, p < 0.01) and IL-18 -137G/C in the recessive model (OR 1.33, 95% CI 1.02-1.71, I2 = 9%, p = 0.03) and VL were observed. For the IL-10 gene SNPs, there was no significant association. Our findings suggest that SNPs in the IFN-γ and IL-18 genes may be associated with the risk of developing VL.

Zinc and Manganese Imbalances in BALB/c Mice Experimentally Infected with Leishmania (Leishmania) amazonensis.

PURPOSE: The clinical progression of Leishmania (Leishmania) amazonensis infection depends on multiple factors, including immunological status of the host and their genotypic interaction. Several immunological processes depend directly on minerals for an efficient performance. Therefore, this study used an experimental model to investigate the alterations of trace metals in L. amazonensis infection associate with clinical outcome, parasite load, and histopathological lesions, and the effect of CD4 + T cells depletion on these parameters. METHODS: A total of 28 BALB/c mice were divided into 4 groups: 1-non-infected; 2-treated with anti-CD4 antibody; 3-infected with L. amazonensis; and 4-treated with anti-CD4 antibody and infected with L. amazonensis. After 24 weeks post-infection, levels of calcium (Ca), iron (Fe), magnesium (Mg), manganese (Mn), Cu, and Zn were determined by inductively coupled plasma optical emission spectroscopy using tissue samples of the spleen, liver, and kidneys. Additionally, parasite burdens were determined in the infected footpad (inoculation site) and samples of inguinal lymph node, spleen, liver, and kidneys were submitted to histopathological analysis. RESULTS: Despite no significant difference was observed between groups 3 and 4, L. amazonensis-infected mice had a significant reduction of Zn (65.68-68.32%) and Mn (65.98 to 82.17%) levels. Presence of L. amazonensis amastigotes was also detected in the inguinal lymph node, spleen, and liver samples in all infected animals. CONCLUSION: The results showed that significant alterations in micro-elements levels occur in BALB/c mice experimentally infected with L. amazonensis and may increase the susceptibility of individuals to the infection.

Antiprotozoal compounds from Mikania periplocifolia (Asteraceae).

Chagas disease, African trypanosomiasis and Leishmaniasis are neglected parasitic diseases which affect millions of people worldwide. In a previous work, we report the antiprotozoal activity of the dichloromethane extract of Mikania periplocifolia Hook. & Arn. (Asteraceae). The aim of this work was to isolate and identify the bioactive compounds present in the extract. The fractionation of the dichloromethane extract has led to the isolation of the sesquiterpene lactone miscandenin and the flavonoid onopordin, together with the sesquiterpene lactones mikanolide, dihydromikanolide and deoxymikanolide, which have previously shown antiprotozoal activity. Miscandenin and onopordin were assayed in vitro against Trypanosoma cruzi, T. brucei and Leishmania braziliensis. Miscandenin was active against T. cruzi trypomastigotes and amastigotes with IC50 values of 9.1 and 7.7 µg/ml, respectively. This sesquiterpene lactone and the flavonoid onopordin showed activity against T. brucei trypomastigotes (IC50 = 0.16 and 0.37 µg/ml) and L. braziliensis promastigotes (IC50 = 0.6 and 1.2 µg/ml), respectively. The CC50 values on mammalian cells were 37.9 and 53.4 µg/ml for miscandenin and onopordin, respectively. Besides, the pharmacokinetic and physicochemical properties of miscandenin were assessed in silico, showing a good drug-likeness profile. Our results highlight this compound as a promising candidate for further preclinical studies in the search of new drugs for the treatment of trypanosomiasis and leishmaniasis.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Surveillance and genotype characterization of zoonotic trypanosomatidae in Didelphis marsupialis in two endemic sites of rural Panama.

Didelphis marsupialis has been reported as a competent reservoir for trypanosomatid parasites infections. The aim of this study was to measure Trypanosoma cruzi, T. rangeli, and Leishmania spp. infection rates and to characterize discrete typing units (DTUs) of T. cruzi in D. marsupialis from two Chagas disease endemic sites in Panama. Blood from 57 wild-caught D. marsupialis were examined from two rural communities, Las Pavas (N = 18) and Trinidad de las Minas (N = 39). Twenty-two (38.60%) opossums were positive for flagellates by general hemoculture. T. cruzi infection was confirmed by positive hemoculture and/or kDNA based PCR performed in 31/57 (54.39%) blood samples from opossums. T. rangeli infection was confirmed by hemoculture and/or TrF/R2-Primer PCR assay applied on 12/57 (21.05%) blood samples. Nine (15.79%) D. marsupialis harbored T. cruzi/T. rangeli coinfections. All opossums tested negative for Leishmania spp. by PCR assays based on kDNA and HSP70 gene amplification. There was a significant association between T. cruzi infection and site (Fisher exact test, p = 0.02), with a higher proportion of T. cruzi infected opossums in Las Pavas (77.78%, n = 14/18) compared to Trinidad de las Minas (43.59%, n = 17/39). A significant association was found between habitat type and T. cruzi infection in opossums across both communities, (X2 = 6.91, p = 0.01, df = 1), with a higher proportion of T. cruzi infection in opossums captured in forest remnants (76%, 19/25) compared to peridomestic areas (37.5%, 12/32). T. rangeli detection, but not T. cruzi detection, may be improved by culture followed by PCR. TcI was the only DTU detected in 22 T. cruzi samples using conventional and real-time PCR. Eight T. rangeli positive samples were characterized as KP1(-)/lineage C. Trypanosome infection data from this common synanthropic mammal provides important information for improved surveillance and management of Chagas disease in endemic regions of Panama.

Characterisation of the Leishmania donovani/L. infantum Hybrid Isolated from an Autochothonous Kala-Azar Patient: Preliminary Results of an In Vivo Model

Objective: In the present study, preliminary outcomes of the in vivo assessment of a Leishmania donovani/L. infantum hybrid isolated from a hospitalised patient with visceral leishmaniasis in Manisa and identified through analysis of the Leishmania-specific ITS-1, hsp70 and cpb gene regions are presented in comparison with reference strains of L. donovani and L. infantum. Methods: Three different study groups [(SG); n=16 mice each] and a control group (n=8 mice) were established with female Balb/C mice weighing 25-30 g. Reference L. donovani (MHOM/IN/1980/DD8), reference L. infantum (MHOM/TN/1980/IP1) and a L. donovani/L. infantum hybrid (MHOM/TR/2014/CBVL-LI/ LD), stored in liquid nitrogen, were thawed, cultured and incubated at 25 °C. A 15-µL dose of 1x108/mL promastigotes of three strains was applied to the tail veins of mice in the SG. After the mice were sacrificed, the liver and spleen tissues were removed and stored for immunological, immunohistochemical and pathological analyses. Results: The presence of infection in the liver and spleen tissues of mice was detected both by a specific enzyme-linked immunosorbent assay test and from the recovery of Leishmania promastigotes from liver and spleen tissues in NNN medium. However, Leishmania amastigotes were not observed in the touch biopsy smears of livers or spleens in either of the SGs. In addition, no evidence of tissue damage was identified in the SGs after immunohistochemical staining (with antibodies against IL-9, CD-117, MBP, CD163, CD4, CD8 and CD31). Conclusion: The obtained results show that hybrid Leishmania and reference L. donovani and L. infantum strains reached the liver and spleens of Balb/C mice in SGs but were of no pathological consequence. Yet, these three Leishmania isolates caused skin lesions when applied subcutaneously in Balb/C mice in another study. The findings presented in this study will be reassessed upon completion of the project, once the final results are obtained.

Leishmania-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

Zoonotic parasites infecting free-living armadillos from Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild-caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen's disease). Forty-three free-living armadillos from Pantanal and seven road-killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti-T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

Exploring TERRA during Leishmania major developmental cycle and continuous in vitro passages.

Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3'-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5' upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRA-R loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite.

Detection of Natural Infection by Leishmania sp. in Bats (Chiroptera, Mammalia) that Inhabit the City of Corrientes, Argentina.

Leishmaniasis is a zoonotic disease of importance in Argentina. It is important to be acquainted with its possible natural hosts and reservoirs. Bats have several reservoir attributes, which means they could fulfill these roles. The aim of this work was to investigate the presence of Leishmania sp. in bats that inhabit the city of Corrientes. Bats were captured and identified. From the captured specimens, spleen samples were taken and Leishmania sp. detection techniques were applied, including both optical microscopy and PCR. It was possible to capture 95 bat specimens belonging to 10 species. Using cytological preparations, a prevalence of 1.29% was detected and using PCR a prevalence of 32% was found. Our results confirm the presence of natural infection by Leishmania sp. in bats of the city of Corrientes which reveals the possibility of these animal species fulfilling a reservoir role for this disease.

La leishmaniose est une zoonose importante en Argentine. La connaissance de ses réservoirs possibles est essentielle. Les chauves-souris ont plusieurs attributs pour remplir ce rôle. L'objectif de ce travail était d'étudier la présence de Leishmania sp. chez les chauves-souris qui habitent la ville de Corrientes. Des captures de chauves-souris ont été faites et celles-ci ont été identifiées. Des échantillons de rates ont été prélevés sur ces spécimens pour rechercher Leishmania sp. en utilisant la microscopie optique et la PCR. Il a été possible de capturer 95 spécimens de chauves-souris appartenant à dix espèces. En utilisant des préparations cytologiques, une prévalence de 1,29 % a été détectée et en utilisant la PCR, une prévalence de 32 % a été trouvée. Nos résultats confirment la présence d'une infection naturelle par Leishmania sp. chez les chauves-souris de la ville de Corrientes, ce qui révèle la possibilité que ces espèces animales jouent un rôle de réservoir pour cette maladie.

Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus.

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.

BACKGROUND: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. METHODS AND RESULTS: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3'-5' Exonuclease I digestion, and unfold telomeric G-quadruplex. CONCLUSIONS: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.

Trypanosomatids in Small Mammals of an Agroecosystem in Central Brazil: Another Piece in the Puzzle of Parasite Transmission in an Anthropogenic Landscape.

We surveyed infection by Trypanosoma spp. and Leishmania spp. in small wild mammals from Cumari, Goiás State aiming to investigate the diversity of trypanosomatid in a modified landscape of the Brazilian Cerrado (and possible infection overlapping with canids from the same area). Blood, skin, spleen, and liver samples were collected for parasitological, serological, and molecular assays. Gracilinanus agilis was the most abundant species (N = 70; 48.6%) and it was the only one with patent parasitemia. Characterization by mini-exon and 18SrDNA targets were achieved in 7/10 hemocultures with positive fresh blood examination, which confirmed the T. cruzi infection by Discrete Typing Units (DTU) TcI in single (N = 2) and mixed infections with other DTUs (N = 5). T. rangeli and T. dionisii were detected in skin fragments from Didelphis albiventris and Oecomys cleberi, respectively. G. agilis were found to be infected by L. braziliensis and L. guyanensis, while Leishmania sp. DNA was detected in the liver of Oligoryzomys nigripes and Calomys expulsus. Subpatent infection by T. cruzi and Leishmania sp. was serologically detected in 15% and 9% of the small mammal fauna, respectively. Small mammals from Cumari are included in T. cruzi and Leshmania spp. transmission cycles, showing a higher diversity of trypanosomatid species and/or genotypes than that observed in canids of the same agroecosystem.

Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites.

Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through inhibition of the respiratory burst, suggesting that they act as virulence factors. Extracellular acid phosphatase activity in Leishmania (L.) donovani has been associated with the degree of promastigote virulence/infectivity. The levels of acid ecto-phosphatase activity in different Leishmania sp or even strains of the same species vary and this has been linked to their virulence. It may also be related to their ability to survive and multiply in the insect host. Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane, however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result. Therefore, measuring phosphatase activity at the surface of live cells in acidic pH range is the only accurate way to measure acid ecto-phosphatase activity. This assay is performed at 25 °C or 37 °C for 30 min using as substrate the generic phosphatase substrate p-nitrophenyl phosphate (pNPP), in a citrate buffer, with or without sodium tartrate (L(+)-tartaric acid), as histidine acid phosphatases are classified according to their sensitivity to tartate inhibition. The steps of the protocol consist of pelleting cells in suspension, in this case Leishmania promastigotes, washing twice with HEPES buffer, resuspending the cells in the substrate reaction mixture and terminating the reaction by the addition of 0.5 N NaOH. The cells are removed by centrifugation and the absorbance of the reaction product (p-nitrophenolate=pNP) in the supernatant is measured at 405 nm. The enzymatic activity (A405 values) is normalized for the mean number of cells/ml used for each independent experiment.