ABSTRACT
BACKGROUND: The development of new therapeutic strategies to treat patients for leishmaniasis has become a priority. The antileishmanial activity of the strychnobiflavone flavonoid was recently demonstrated against Leishmania amazonensis and Leishmania infantum amastigotes and promastigotes. The biological effect of this molecule was identified due to its capacity to interfere in the parasite mitochondrial membrane; however, the underlying molecular mechanism remains unclear. METHODS AND RESULTS: In this study, a computational approach using bioinformatics was performed to screen biological targets of strychnobiflavone in L. infantum. Computational programs, such as the target fishing approach and molecular docking assays, were used. Results showed that the putative pathway targeted by strychnobiflavone in L. infantum is the methylglyoxal degradation superpathway, and one hydrolase-like protein was predicted to be the molecular target of this flavonoid in the parasites. CONCLUSION: In this context, this study provides the basis for understanding the mechanism of action of strychnobiflavone in L. infantum and presents a strategy based on bioinformatics programs to screen targets of other molecules with biological action against distinct pathogens.
ABSTRACT
In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4+ and CD8+ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.
Subject(s)
Antigens, Protozoan/immunology , Cross Reactions/immunology , Leishmania braziliensis/immunology , Leishmania mexicana/immunology , Leishmaniasis Vaccines/immunology , Leishmaniasis, Cutaneous/prevention & control , Peptide Initiation Factors/immunology , RNA-Binding Proteins/immunology , Animals , Antigens, Protozoan/chemistry , Cytokines/metabolism , Disease Models, Animal , Female , Host-Parasite Interactions/immunology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Mice , Parasite Load , Peptide Initiation Factors/chemistry , RNA-Binding Proteins/chemistry , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Eukaryotic Translation Initiation Factor 5AABSTRACT
The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).
