RESUMO
Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such as Leishmania protozoan parasites. Some strains of Leishmania are known to bear a viral endosymbiont termed Leishmania RNA virus 1 (LRV1). Recognition of LRV1 by the innate immune sensor Toll-like receptor-3 (TLR3) leads to conditions worsening the disease severity in mice. This process is governed by type I interferon (type I IFNs) arising downstream of TLR3 stimulation and favoring the formation of secondary metastatic lesions. The formation of these lesions is mediated by the inflammatory cytokine IL-17A and occurs in the absence, or low level of, protective cytokine IFN-γ. Here, we described that the presence of LRV1 led to the initial expression of iNOS and low production of NO that failed to control infection. We subsequently showed that LRV1-triggered type I IFN was essential but insufficient to induce robust iNOS induction, which requires strong activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Leishmania guyanensis carrying LRV1 (LgyLRV1+) parasites mitigated strong iNOS production by limiting NF-kB activation via the induction of tumor necrosis factor-alpha-induced protein 3 (TNFAIP3), also known as A20. Moreover, our data suggested that production of LRV1-induced iNOS could be correlated with parasite dissemination and metastasis via elevated secretion of IL-17A in the draining lymph nodes. Our findings support an additional strategy by which LRV1-bearing Leishmania guyanensis evaded killing by nitric oxide and suggest that low levels of LRV1-induced NO might contribute to parasite metastasis.
Assuntos
Leishmania guyanensis , Leishmania , Óxido Nítrico Sintase Tipo II , Animais , Citocinas , Humanos , Interleucina-17 , Leishmania guyanensis/virologia , Leishmaniavirus , Camundongos , NF-kappa B , Óxido Nítrico , Óxido Nítrico Sintase Tipo II/metabolismo , Receptor 3 Toll-LikeRESUMO
OBJECTIVES: The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. METHODS: The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. RESULTS: The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). DISCUSSION: Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.
Assuntos
Leishmania guyanensis/virologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniavirus/classificação , Pentamidina/uso terapêutico , Adulto , Feminino , Guiana Francesa , Variação Genética , Técnicas de Genotipagem , Humanos , Leishmaniavirus/genética , Leishmaniavirus/isolamento & purificação , Masculino , Filogenia , Estudos Retrospectivos , Análise de Sequência de RNA , Falha de Tratamento , Adulto JovemRESUMO
Cutaneous leishmaniasis is a neglected parasitic disease that manifests in infected individuals under different phenotypes, with a range of factors contributing to its broad clinical spectrum. One factor, Leishmania RNA Virus 1 (LRV1), has been described as an endosymbiont present in different species of Leishmania. LRV1 significantly worsens the lesion, exacerbating the immune response in both experimentally infected animals and infected individuals. Little is known about the composition and genetic diversity of these viruses. Here, we investigated the relationship between the genetic composition of LRV1 detected in strains of Leishmania (Viannia) braziliensis and L. (V.) guyanensis and the interaction between the endosymbiont and the parasitic species, analyzing an approximately 850 base pair region of the viral genome. We also included one LRV1 sequence detected in L. (V.) shawi, representing the first report of LRV1 in a species other than L. braziliensis and L. guyanensis. The results illustrate the genetic diversity of the LRV1 strains analyzed here, with smaller divergences detected among viral sequences from the same parasite species. Phylogenetic analyses showed that the LRV1 sequences are grouped according to the parasite species and possibly according to the population of the parasite in which the virus was detected, corroborating the hypothesis of joint evolution of the viruses with the speciation of Leishmania parasites.
Assuntos
Leishmania/virologia , Leishmaniavirus/genética , Coevolução Biológica/genética , Variação Genética/genética , Genoma Viral/genética , Leishmania braziliensis/virologia , Leishmania guyanensis/virologia , Leishmaniose/parasitologia , Filogenia , Análise de Sequência de DNA , América do Sul , Especificidade da EspécieRESUMO
Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.
Assuntos
Interações Hospedeiro-Parasita/imunologia , Leishmania guyanensis/patogenicidade , Leishmaniose Cutânea/patologia , Leishmaniavirus/patogenicidade , Mucosa/patologia , Animais , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interleucina-17/biossíntese , Interleucina-17/imunologia , Leishmania guyanensis/imunologia , Leishmania guyanensis/virologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniavirus/fisiologia , Camundongos , Mucosa/imunologia , Mucosa/parasitologia , Nasofaringe/imunologia , Nasofaringe/parasitologia , Nasofaringe/patologia , Índice de Gravidade de DoençaRESUMO
Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 µm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 µm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 µm 2CMA accumulated 2CMA-TP to 410 µm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.
Assuntos
Trifosfato de Adenosina , Leishmania guyanensis/virologia , Leishmaniavirus/enzimologia , RNA Polimerase Dependente de RNA , Proteínas Virais , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Relação Dose-Resposta a Droga , Leishmania guyanensis/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.
Assuntos
Interferon Tipo I/imunologia , Leishmania guyanensis , Leishmaniose Mucocutânea/imunologia , Leishmaniavirus/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Febre por Flebótomos/imunologia , Vírus da Febre do Flebótomo Napolitano/imunologia , Animais , Coinfecção , Interferon Tipo I/genética , Leishmania guyanensis/imunologia , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/genética , Leishmaniose Mucocutânea/patologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/patologia , Camundongos , Camundongos Knockout , Febre por Flebótomos/genética , Febre por Flebótomos/patologiaRESUMO
The endogenous double-stranded RNA (dsRNA) virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures in Leishmania braziliensis and Leishmania guyanensis infections. Thus, methods targeting LRV1 could have therapeutic benefit. Here we screened a panel of antivirals for parasite and LRV1 inhibition, focusing on nucleoside analogs to capitalize on the highly active salvage pathways of Leishmania, which are purine auxotrophs. Applying a capsid flow cytometry assay, we identified two 2'-C-methyladenosine analogs showing selective inhibition of LRV1. Treatment resulted in loss of LRV1 with first-order kinetics, as expected for random virus segregation, and elimination within six cell doublings, consistent with a measured LRV1 copy number of about 15. Viral loss was specific to antiviral nucleoside treatment and not induced by growth inhibitors, in contrast to fungal dsRNA viruses. Comparisons of drug-treated LRV1+ and LRV1- lines recapitulated LRV1-dependent pathology and parasite replication in mouse infections, and cytokine secretion in macrophage infections. Agents targeting Totiviridae have not been described previously, nor are there many examples of inhibitors acting against dsRNA viruses more generally. The compounds identified here provide a key proof-of-principle in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.
Assuntos
Antivirais/farmacologia , Leishmania braziliensis/virologia , Leishmania guyanensis/virologia , Leishmaniavirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Leishmaniose/parasitologia , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , Camundongos Endogâmicos C57BL , Nucleotídeos/farmacologiaRESUMO
Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities.
Assuntos
Proteínas do Capsídeo/imunologia , Leishmania guyanensis/virologia , Leishmaniose/prevenção & controle , Leishmaniavirus/imunologia , Animais , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Feminino , Humanos , Imunidade Celular , Leishmania guyanensis/genética , Leishmania guyanensis/imunologia , Leishmania guyanensis/fisiologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Leishmaniavirus/genética , Leishmaniavirus/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Simbiose , Linfócitos T/imunologia , VacinaçãoRESUMO
Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.
Assuntos
Antiprotozoários/farmacologia , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniavirus/efeitos dos fármacos , Oligorribonucleotídeos Antissenso/farmacologia , RNA de Cadeia Dupla/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Animais , Antiprotozoários/química , Antiprotozoários/metabolismo , Expressão Gênica , Sequências Repetidas Invertidas , Leishmania braziliensis/patogenicidade , Leishmania braziliensis/virologia , Leishmania guyanensis/patogenicidade , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/virologia , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , Macrófagos/parasitologia , Macrófagos/virologia , Camundongos , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/metabolismo , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Simbiose/genética , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Some strains of the protozoan parasite Leishmania guyanensis (L.g) harbor a viral endosymbiont called Leishmania RNA virus 1 (LRV1). LRV1 recognition by TLR-3 increases parasite burden and lesion swelling in vivo. However, the mechanisms by which anti-viral innate immune responses affect parasitic infection are largely unknown. Upon investigating the mammalian host's response to LRV1, we found that miR-155 was singularly and strongly upregulated in macrophages infected with LRV1+ L.g when compared to LRV1- L.g. LRV1-driven miR-155 expression was dependent on TLR-3/TRIF signaling. Furthermore, LRV1-induced TLR-3 activation promoted parasite persistence by enhancing macrophage survival through Akt activation in a manner partially dependent on miR-155. Pharmacological inhibition of Akt resulted in a decrease in LRV1-mediated macrophage survival and consequently decreased parasite persistence. Consistent with these data, miR-155-deficient mice showed a drastic decrease in LRV1-induced disease severity, and lesional macrophages from these mice displayed reduced levels of Akt phosphorylation.
Assuntos
Imunidade Inata , Leishmania guyanensis/virologia , Leishmaniavirus/imunologia , Macrófagos/parasitologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Leishmania guyanensis/patogenicidade , Leishmania guyanensis/fisiologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/patologia , Macrófagos/imunologia , Camundongos , Camundongos KnockoutRESUMO
Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.
Assuntos
Antiprotozoários/farmacologia , Leishmania guyanensis/efeitos dos fármacos , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniose Mucocutânea/virologia , Leishmaniavirus , Adulto , Antiprotozoários/uso terapêutico , Estudos de Coortes , Feminino , Humanos , Leishmaniose Mucocutânea/epidemiologia , Masculino , Pentamidina/farmacologia , Pentamidina/uso terapêutico , Recidiva , Falha de TratamentoRESUMO
Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB-/- mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.
Assuntos
Imunidade Inata , Leishmania guyanensis/virologia , Fator 88 de Diferenciação Mieloide/metabolismo , Vírus de RNA/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Suscetibilidade a Doenças , Subunidade p35 da Interleucina-12/deficiência , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/deficiência , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Leishmania guyanensis/fisiologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/patologia , Leishmaniose Mucocutânea/veterinária , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genéticaAssuntos
Imunidade Adaptativa/fisiologia , Leishmaniose Mucocutânea/classificação , Leishmaniose Mucocutânea/complicações , Infecções Oportunistas/complicações , Humanos , Inflamação/complicações , Inflamação/etiologia , Leishmania guyanensis/imunologia , Leishmania guyanensis/fisiologia , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/diagnóstico , Leishmaniose Mucocutânea/imunologia , Modelos Biológicos , Infecções Oportunistas/imunologia , Infecções por Vírus de RNA/complicações , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Vírus de RNA/fisiologiaRESUMO
Infection by the human protozoan parasite Leishmania can lead, depending primarily on the parasite species, to either cutaneous or mucocutaneous lesions, or fatal generalized visceral infection. In the New World, Leishmania (Viannia) species can cause mucocutaneous leishmaniasis (MCL). Clinical MCL involves a strong hyper-inflammatory response and parasitic dissemination (metastasis) from a primary lesion to distant sites, leading to destructive metastatic secondary lesions especially in the nasopharyngal areas. Recently, we reported that metastasizing, but not non-metastatic strains of Leishmania (Viannia) guyanensis, have high burden of a non-segmented dsRNA virus, Leishmania RNA Virus (LRV). Viral dsRNA is sensed by the host Toll-like Receptor 3 (TLR3) thereby inducing a pro-inflammatory response and exacerbating the disease. The presence of LRV in Leishmania opens new perspectives not only in basic understanding of the intimate relation between the parasite and LRV, but also in understanding the importance of the inflammatory response in MCL patients.
Assuntos
Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/imunologia , Vírus de RNA/fisiologia , Totiviridae/fisiologia , Animais , Citocinas/genética , Citocinas/imunologia , Humanos , Leishmania guyanensis/genética , Leishmania guyanensis/imunologia , Leishmaniose Mucocutânea/genética , Leishmaniose Mucocutânea/parasitologia , Vírus de RNA/genética , Vírus de RNA/imunologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Totiviridae/genética , Totiviridae/imunologiaAssuntos
Interações Hospedeiro-Patógeno/imunologia , Leishmania/imunologia , Leishmania/virologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/parasitologia , Vírus de RNA/fisiologia , Animais , Cricetinae , Humanos , Inflamação/imunologia , Inflamação/parasitologia , Mediadores da Inflamação/imunologia , Interferons/imunologia , Leishmania/isolamento & purificação , Leishmania/patogenicidade , Leishmania guyanensis/imunologia , Leishmania guyanensis/isolamento & purificação , Leishmania guyanensis/patogenicidade , Leishmania guyanensis/virologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Fagossomos/parasitologia , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , RNA Viral/imunologia , Receptores Toll-Like/deficiência , Receptores Toll-Like/imunologiaRESUMO
Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Leishmania guyanensis/patogenicidade , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/imunologia , Leishmaniavirus/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Mediadores da Inflamação/metabolismo , Leishmaniose Mucocutânea/parasitologia , Leishmaniavirus/fisiologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Parasitemia , Fagossomos/parasitologia , RNA de Cadeia Dupla/imunologia , RNA Viral/imunologia , Receptores Toll-Like/imunologiaRESUMO
In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonuclease activity was found to be at 37(O)C to 42(O)C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.
Assuntos
Capsídeo/enzimologia , Endorribonucleases/metabolismo , Leishmania guyanensis/virologia , Vírus de RNA/enzimologia , Substituição de Aminoácidos , Animais , Antibacterianos/farmacologia , Baculoviridae , Linhagem Celular , Cisteína/genética , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Temperatura Alta , Insetos , RNA/química , Vírus de RNA/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genética , Transdução GenéticaRESUMO
Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.
Assuntos
Leishmania braziliensis/virologia , Leishmania guyanensis/virologia , Leishmaniavirus/genética , RNA Viral/química , Animais , Leishmania braziliensis/genética , Leishmania guyanensis/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Biossíntese de Proteínas , Análise de Sequência de RNARESUMO
Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.