Characterization of a protein with unknown function (LinJ.30.3360) in Leishmania amazonensis and Leishmania infantum.

Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania. In the Americas, the species Leishmania amazonensis is predominantly associated with American cutaneous leishmaniasis (ACL) while L. infantum is an agent of visceral leishmaniasis (VL). The genome sequences of Leishmania spp. have shown that each genome can contain about 8000 genes encoding proteins, more than half of which have an unknown function (''hypotheticals") at the time of publication. To understand the biology and genome of the organisms, it is important to discover the function of these "hypothetical" proteins; however, few studies have focused on their characterizations. Previously, LinJ.30.3360 (a protein with unknown function) was identified as immunogenic to canine serum with VL and a good antigen to diagnose the visceral form in dogs. Here, we show that the LinJ.30.3360 protein is conserved in L. infantum, L. tarantolae, L. donovani, L. major, L. mexicana, L. braziliensis, L. panamensis, Leptomonas pyrrhocoris, and Leptomonas seymouri. It has been annotated as a MORN (Membrane Occupation and Recognition Nexus) domain protein. However, since the function of this motif is unknown, functional inferences based on the primary sequence are not possible. The protein has a folded ß-leaf secondary structure, and phosphorylation was the only post-translational modification (PTM) found using prediction approach. Experiments have shown that it is located close to the flagellar pocket and presents similar abundance in both L. amazonensis and L. infantum. Furthermore, because it is a conserved protein in trypanosomatids but not in mammals and also because of its antigenicity, LinJ.30.3360 may constitute a potential drug target and/or vaccine for leishmaniasis.

Binding of Leishmania infantum Lipophosphoglycan to the Midgut Is Not Sufficient To Define Vector Competence in Lutzomyia longipalpis Sand Flies.

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors in mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild-type (WT), LPG-defective (Δlpg1 mutants), and add-back (Δlpg1 + LPG1) lines to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivoLe. infantum WT parasites attached to the Lu. longipalpis midgut in vitro, with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with the Le. infantum WT or Le. infantum Δlpg1 or Le. infantum Δlpg1 + LPG1 line of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to numbers similar to those seen with the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood-engorged midgut extracts led to mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpisIMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.

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

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

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

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

Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum / Leishmania donovani.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.

DNA aptamers targeting Leishmania infantum H3 protein as potential diagnostic tools.

Leishmaniasis is a disease caused by a parasite of the genus Leishmania that affects millions of people worldwide. These parasites are characterized by the presence of a DNA-containing granule, the kinetoplastid, located in the single mitochondrion at the base of the cell's flagellum. Interestingly, these flagellates do not condense chromatin during mitosis, possibly due to the specific molecular features of their histones. Although histones are extremely conserved proteins, kinetoplastid core histone sequences diverge significantly from those of higher eukaryotes. This divergence makes kinetoplastid core histones potential diagnostic and/or therapeutic targets. Aptamers are short single-stranded nucleic acids that are able to recognize target molecules with high affinity and specificity. Their binding capacity is a consequence of the particular three-dimensional structure acquired depending on their sequence. These molecules are currently used for detection, diagnosis and therapeutic purpose. Starting from a previously obtained ssDNA aptamer population against rLiH3 protein we have isolated two individual aptamers, AptLiH3#4 and AptLiH3#10. Next, we have performed ELONA, Western blot and slot blot assays to establish aptamer specificity and affinity for LiH3 histone. In addition, ELONA assays using peptides corresponding to overlapped sequences of LiH3 were made to map the aptamers:LiH3 interaction. Finally, different assays using aptamers were performed in order to evaluate the possibility of using these aptamers as sensing molecule to recognize the endogenous protein LiH3. Our results indicate that both aptamers have high affinity and specificity for the target and are able to detect the endogenous LiH3 histone protein in promastigotes lysates. In silico analysis reveals that these two aptamers have different potential secondary structure among them, however, both of them are able to recognize the same peptide sequences present in the protein. In conclusion, our findings indicate that these aptamers could be used for LiH3 histone detection and, in consequence, as potential biosensing molecules in a diagnostic tool for leishmaniasis.

In-Silico Identification of the Best Compound Against Leishmania infantum: High Throughput Screening of All FDA Approved Drugs

Objective: Current in-silico research was designed and administered for the screening of 20000 Food and Drug Administration-approved drug compounds with the goal of finding promising drugs against lipophosphoglycan (LPG) and γ-glutamylcysteine synthetase (γ-GCS) of Leishmania infantum. Methods: After the protein sequence of both targets was taken, the 3D structures of protein of interest were predicted and validated. Molecular docking was done among the two putative targets (LPG and γ-GCS) and approved compounds were selected using AutoDock 4.2 program to predict ligand-receptor interactions. Results: After docking experiment was done on 20000 drug compounds, a total number of seven ligands, two for γ-GCS receptor and five for LPG receptor, were assigned as novel, potent anti-leishmanial drugs based on their binding affinity and energy. Of those, five ligands possessed cytotoxic and anti-cancer characteristics and showed good binding capacity to LPG receptor with ΔGbinding up to 8.5 kcal/mol more negative; while two compounds showed good binding capacity to glutamyl receptor with ΔGbinding up to 7.8 kcal/mol more negative. Conclusion: The latest software-based methods are powerful tools for scanning and predicting new peptide templates specific to biological targets in organisms for new drug discovery. However, the use of in vitro and in vivo techniques is a requirement for better evaluation of the potential of projected ligands with the help of in-silico approaches, identifying molecular mechanism of action of the more active compounds is possible. This can help in defining the most likely molecular target, so that the subsequent optimization using in vitro and in vivo techniques can be undertaken.

Importance of L. Infantum H2B Recombinant Antigen for Serodiagnosis of Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) can lead to death in more than 95% of cases if left untreated. Accurate and early diagnosis has an important role in reducing mortality rate of this disease. OBJECTIVE: To express recombinant H2B antigen from an Iranian isolate of Leishmania Infantum and evaluate its efficacy in the diagnosis of VL. METHODS: The recombinant H2B antigen was produced in a prokaryotic system, and its efficacy for VL diagnosis was evaluated by ELISA. The serum samples from 80 VL patients, 100 individuals from endemic and non-endemic regions of VL, and 58 non-VL patients were collected. VL cases were confirmed based on the clinical sign, positive IFAT (>64), real time PCR, and response to treatment. RESULTS: The H2B gene sequence of the Iranian L. infantum isolate had about 4% diversity in comparison with the H2B gene of the L. infantum counterpart. ELISA, using the produced H2B recombinant antigen, showed sensitivity of 71.25% (95% CI: 60.05%-80.82%) and specificity of 69.62% (95% CI: 61.81%-76.68%) regarding VL diagnosis. CONCLUSION: Recombinant H2B antigen expressed in the prokaryotic system had suboptimal performance for the serological diagnosis of VL. It seems that the production and expression of recombinant H2B antigen in a eukaryotic system may enhance the performance of this antigen in the diagnosis of VL in Iran.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Large-scale cultivation of Leishmania infantum promastigotes in stirred bioreactor.

BACKGROUND & OBJECTIVES: Bioreactors are practical tools that are used for economical, time-conserving and large-scale production of biomass from cell cultivation. They provide optimal environmental conditions such as pH and temperature required for obtaining maximum amounts of biomass. However, there is no evidence in the literature on the large-scale cultivation of Leishmania infantum parasites in the bioreactor. Therefore, the present study was undertaken to develop a new approach for obtaining L. infantum biomass by using pH and temperature controllable stirred bioreactor and to compare parasitic growth kinetics with classical method within erlenmeyers. METHODS: In order to obtain parasite biomass, a newly developed pH and temperature controlled stirred bioreactor was used and its efficacy was compared with a graduated classical scale-up method. Growth kinetics of parasites within erlenmeyers and bioreactors were determined by evaluating promastigote numbers using haemocytometer. The graduated scale enlargement of culture was followed by T25 flask, T75 flask, and 1 L erlenmeyer, respectively. RESULTS: Obtained results showed a 10-fold increase in the number of promastigotes within the conventional culture performed in 700 ml medium, while parasite numbers increased approximately 15 times due to initial inoculation amounts in the bioreactor culture performed in the 3.5 l medium. Thus, there was 7.5 times more biomass collection in bioreactor compared to classical method. INTERPRETATION & CONCLUSION: It is postulated that constant culture pH and temperature in the bioreactor extends cultivation time. This could lead to significant increase in parasite numbers. Hence, pH and temperature controllable bioreactors provided acquisition of sufficient amounts of biomass in contrast to classical methods. Therefore, this type of bioreactors may substitute classical culture methods in the production of antigenic molecules for vaccine development.

The Unknown Nature of the Antigen in the Direct Agglutination Test for Visceral Leishmaniasis Hampers Development of Serodiagnostic Tests.

Current diagnostic tests for visceral leishmaniasis (VL) are either not adapted for use in resource-poor settings or are insufficiently accurate in Eastern Africa. Only the direct agglutination test (DAT), based on whole Leishmania promastigotes, is highly reliable in all endemic regions, but its implementation is hampered by the need for a cold chain, minimal laboratory conditions, and long incubation times. Integrating the DAT antigen(s) in an immunochromatographic rapid diagnostic test (RDT) would overcome these disadvantages. Unfortunately, the identity of the DAT antigen(s) involved in the agglutination reaction is unknown. For this study, we reviewed all publications that might shed some light on this issue. We conclude that the DAT antigen is a mixture of Leishmania-specific epitopes of protein, carbohydrate, and lipid nature. To develop an accurate RDT for VL diagnosis in Eastern Africa, we suggest to complement the classical protein antigen discovery with approaches to identify carbohydrate and lipid epitopes.

Using FT-IR spectroscopy for the identification of the T. cruzi, T. rangeli, and the L. chagasi species.

The cross-reaction in the diagnosis results is a serious problem, leading to an incorrect treatment and several injuries to patients. The Trypanosoma rangeli and Trypanosoma cruzi belong to the genus Trypanosoma, but the Trypanosoma rangeli is a non-pathogenic parasite to humans. While Trypanosoma cruzi is the etiological agent of Chagas' disease, which affects circa 2-3 million people and more than 6000 deaths annually in Brazil. The Leishmania chagasi causes infectious disease known as visceral leishmaniasis. This diseases have in common the crossed antigenic reaction promoted by serological tests and its differentiation is relevant for epidemiological studies and clinical practice. In this study the Fourier Transform Infrared (FT-IR) Spectroscopy was used to differentiate these microorganisms, which were cultivated and the spectra analyzed. Data analysis were performed by Gaussian curve fitting and multivariate statistical analysis. The cluster analysis have shown four specific regions to identify the microorganisms. The first three PCs of principal component analysis associated to linear discriminant were able to classify 95.6% of the parasites using cross-validation. The curve fitting method showed the quantitative differentiation among L. chagasi, T. cruzi, and T. rangeli species in the vibrational regions of polysaccharides, amide III, lipid esters, and fatty acid.

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes.

BACKGROUND: The Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa. METHODS: Using mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides. RESULTS: Metacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes. CONCLUSIONS: The data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

Serological diagnosis and prognostic of tegumentary and visceral leishmaniasis using a conserved Leishmania hypothetical protein.

New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor.

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.

Structure-based domain assignment in Leishmania infantum EndoG: characterization of a pH-dependent regulatory switch and a C-terminal extension that largely dictates DNA substrate preferences.

Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145-180 is indeed far more active than its wild-type counterpart at pH values >7.0. In addition, we discovered that (i) LiEndoG exists as a homodimer, (ii) replacement of Ser211 in the active-site SRGH motif with the canonical aspartate from the DRGH motif of other nucleases leads to a catalytically deficient enzyme, (iii) the activity of the S211D variant can be restored upon the concomitant replacement of Ala247 with Arg and (iv) a C-terminal extension is responsible for the observed preferential cleavage of single-stranded DNA (ssDNA) and ssDNA-dsDNA junctions. Taken together, our results support the view that LiEndoG is a multidomain molecular machine whose nuclease activity can be subtly modulated or even abrogated through architectural changes brought about by environmental conditions and interaction with other binding partners.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4+ and CD8+ T cells. Importantly, the activation of CD8+ T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated-infected mice revealed that protection was accompanied by significant increase of IFNγ and lower levels of IL-4 and IL-10 in protected mice when compared to control infected group. Conclusively, the above nanoformulations hold promise for future vaccination strategies against VL.

An in silico functional annotation and screening of potential drug targets derived from Leishmania spp. hypothetical proteins identified by immunoproteomics.

Leishmaniasis is a parasitic disease caused by the protozoan of the Leishmania genus. While no human vaccine is available, drugs such as pentavalent antimonials, pentamidine and amphotericin B are used for treat the patients. However, the high toxicity of these pharmaceutics, the emergence of parasite resistance and/or their high cost have showed to the urgent need of identify new targets to be employed in the improvement of the treatment against leishmaniasis. In a recent immunoproteomics approach performed in the Leishmania infantum species, 104 antigenic proteins were recognized by antibodies in sera of visceral leishmaniasis (VL) dogs. Some of them were later showed to be effective diagnostic markers and/or vaccine candidates against the disease. Between these proteins, 24 considered as hypothetical were identified in the promastigote and amastigote-like extracts of the parasites. The present study aimed to use bioinformatics tools to select new drug targets between these hypothetical proteins. Their cellular localization was predicted to be seven membrane proteins, as well as eight cytoplasmic, three nuclear, one mitochondrial and five proteins remained unclassified. Their functions were predicted as being two transport proteins, as well as five with metabolic activity, three as cell signaling and fourteen proteins remained unclassified. Ten hypothetical proteins were well-annotated and compared to their homology regarding to human proteins. Two proteins, a calpain-like and clavaminate synthase-like proteins were selected by using Docking analysis as being possible drug targets. In this sense, the present study showed the employ of new strategies to select possible drug candidates, according their localization and biological function in Leishmania parasites, aiming to treat against VL.

Morphological and immunohistochemical clues for the diagnosis of cutaneous leishmaniasis and the interpretation of CD1a status.

BACKGROUND: CD1a immunoexpression by amastigotes of Leishmania major and L tropica has been demonstrated. OBJECTIVE: We studied the CD1a and the langerin status of amastigotes in cases of L infantum. METHODS: We investigated 19 cases of cutaneous leishmaniasis. All cases were immunostained with CD1a, langerin, and CD68. We also studied 4 cases of visceral leishmaniasis. RESULTS: We found expression of CD1a by amastigotes in all of these 19 cases. CD1a(-) amastigotes are found in reticular areas of the dermis. The pattern of CD1a immunostaining of amastigotes is characteristic, with peripheral positivity, a negative nucleus in the center, and reinforcement of the kinetoplast in 1 pole. Leishmania amastigotes were langerin-negative. Visceral Leishmania amastigotes also express CD1a. LIMITATIONS: Our study was limited because it only included cases of infection by L infantum. CONCLUSIONS: (1) L infantum is CD1a(+), (2) the pattern of CD1a immunostaining of amastigotes is peculiar, (3) CD1a(-) amastigotes are found in reticular areas of the dermis, and (4) visceral Leishmania amastigotes also express CD1a.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.