RESUMO
In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.
Assuntos
Moringa oleifera , Ligantes , N-Glicosil Hidrolases , Extratos Vegetais/análise , Folhas de Planta/química , Espectrometria de Massas em TandemRESUMO
Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ/IL-10 and TNF-α/IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis.
Assuntos
Proteção Cruzada , Epitopos , Leishmania braziliensis , Leishmania donovani , Leishmaniose Cutânea , Animais , Feminino , Camundongos , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Proteção Cruzada/imunologia , Citocinas/imunologia , Epitopos/imunologia , Leishmania braziliensis/imunologia , Leishmania donovani/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos Endogâmicos BALB CRESUMO
Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.
Assuntos
Antígenos de Protozoários/imunologia , Células Dendríticas/imunologia , Imunoterapia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/terapia , N-Glicosil Hidrolases/imunologia , Receptores CCR7/genética , Animais , Movimento Celular , Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Imunidade Celular , Leishmania donovani , Leishmaniose Visceral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores CCR7/imunologiaRESUMO
Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1ß, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α-IFN-γ-, CD3+CD4+IL-2+TNF-α+IFN-γ-, CD3+CD4+IL-2+TNF-α-IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = -0.428, P = 0.05) and liver sizes (R = -0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = -0.595, P = 0.005) and F2 (R = -0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α-IFN-γ-, CD3+CD8+IL-2+TNF-α+IFN-γ-, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4+ and CD8+ T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.
RESUMO
BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.
Assuntos
Humanos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , N-Glicosil Hidrolases/genética , Técnicas de Inativação de Genes , Genes BacterianosRESUMO
Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.