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1.
Cell Rep ; 33(4): 108317, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33113362

RESUMO

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

2.
Sci Rep ; 10(1): 18653, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122717

RESUMO

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFNγ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines' field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation.

3.
Nat Commun ; 11(1): 3461, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651371

RESUMO

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leishmania major/genética , Leishmania major/patogenicidade , Vacinas Atenuadas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Edição de Genes , Engenharia Genética , Humanos , Imunossupressão , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Psychodidae/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Trends Parasitol ; 36(6): 498-501, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32359871

RESUMO

Epigenetic manipulation of host cells by intracellular pathogens has become increasingly evident. Lecoeur et al. show us how Leishmania amazonensis inhibits macrophage inflammasomes by modifying histone H3 activation marks on NF-κB-associated gene promoters that increase the expression of inhibitors and downmodulates activators of this pathway.


Assuntos
Leishmania , Epigênese Genética , Histonas/metabolismo , Inflamassomos/metabolismo , Leishmania/metabolismo , Macrófagos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
5.
PLoS Negl Trop Dis ; 14(3): e0008014, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126078

RESUMO

BACKGROUND: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. CONCLUSIONS: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.


Assuntos
Expressão Gênica , Marcadores Genéticos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Trato Gastrointestinal/parasitologia , Perfilação da Expressão Gênica , Análise de Sequência de RNA
7.
Nat Microbiol ; 3(5): 548-555, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29556108

RESUMO

Sand flies, similar to most vectors, take multiple blood meals during their lifetime1-4. The effect of subsequent blood meals on pathogens developing in the vector and their impact on disease transmission have never been examined. Here, we show that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes, considered a terminally differentiated stage inside the vector 5 , to a leptomonad-like stage, the retroleptomonad promastigote. Reverse metacyclogenesis occurs after every subsequent blood meal where retroleptomonad promastigotes rapidly multiply and differentiate to metacyclic promastigotes enhancing sand fly infectiousness. Importantly, a subsequent blood meal amplifies the few Leishmania parasites acquired by feeding on infected hosts by 125-fold, and increases lesion frequency by fourfold, in twice-fed compared with single-fed flies. These findings place readily available blood sources as a critical element in transmission and propagation of vector-borne pathogens.


Assuntos
Leishmania/fisiologia , Leishmaniose/transmissão , Psychodidae/parasitologia , Animais , Cricetinae , Replicação do DNA , Insetos Vetores/parasitologia , Leishmania/patogenicidade , Proteínas de Protozoários , Psychodidae/patogenicidade
8.
Cell Host Microbe ; 23(1): 134-143.e6, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29290574

RESUMO

Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1ß (IL-1ß), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1ß in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1ß are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.


Assuntos
Microbioma Gastrointestinal/imunologia , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/transmissão , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Psychodidae/parasitologia , Animais , Antiparasitários/farmacologia , Cricetinae , Feminino , Mordeduras e Picadas de Insetos/parasitologia , Insetos Vetores/parasitologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia
9.
Trends Parasitol ; 33(10): 748-750, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28867329

RESUMO

Doehl et al. have combined empirical data with computer simulation to demonstrate that RAG-2 mice intravenously infected with Leishmania donovani form heterogeneous skin parasite patches that govern infectiousness to sand flies. This model provides a much-needed tool to explore the relevance of asymptomatic and symptomatic visceral leishmaniasis patients as infection reservoirs.


Assuntos
Insetos Vetores/parasitologia , Leishmaniose Visceral/transmissão , Psychodidae/parasitologia , Pele/parasitologia , Animais , Reservatórios de Doenças/parasitologia , Humanos , Leishmania donovani/fisiologia , Leishmaniose Visceral/parasitologia , Camundongos
10.
PLoS Pathog ; 13(7): e1006477, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28742144

RESUMO

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.


Assuntos
Reparo do DNA , Genoma de Protozoário , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Dano ao DNA/efeitos dos fármacos , Evolução Molecular , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos
11.
Sci Rep ; 7(1): 6156, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28733613

RESUMO

Protein kinases (PKs) are a class of druggable targets in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets.


Assuntos
Proteínas Quinases/genética , Análise de Sequência de RNA/métodos , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/parasitologia , Animais , Genes Essenciais , Camundongos , Proteínas de Protozoários/genética , Interferência de RNA , Transdução de Sinais , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genética , Tripanossomíase Africana/veterinária , Fatores de Virulência/genética
12.
mBio ; 8(1)2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28096483

RESUMO

The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission. IMPORTANCE: Leishmania infantum, a parasitic protozoan causing fatal visceral leishmaniasis, is transmitted to humans through the bite of the sand fly Lutzomyia longipalpis Development of the parasite to its virulent metacyclic state occurs in the sand fly gut. In this study, the microbiota within the Lu. longipalpis midgut was delineated by 16S ribosomal DNA (rDNA) sequencing, revealing a highly diverse community composition that lost diversity as parasites developed to their metacyclic state and increased in abundance in infected flies. Perturbing sand fly gut microbiota with an antibiotic cocktail, which alone had no effect on either the parasite or the fly, arrested both the development of virulent parasites and parasite expansion. These findings indicate the importance of bacterial commensals within the insect vector for the development of virulent pathogens, and raise the possibility that impairing the microbial composition within the vector might represent a novel approach to control of vector-borne diseases.


Assuntos
Vetores de Doenças , Microbioma Gastrointestinal , Leishmania infantum/fisiologia , Psychodidae/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Sobrevivência Celular , DNA Ribossômico/química , DNA Ribossômico/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
13.
PLoS Negl Trop Dis ; 6(10): e1850, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23071853

RESUMO

BACKGROUND: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.


Assuntos
Adenosina Trifosfatases/biossíntese , Evasão da Resposta Imune , Leishmania braziliensis/enzimologia , Leishmania braziliensis/patogenicidade , Leishmaniose Mucocutânea/patologia , Leishmaniose Mucocutânea/parasitologia , Fatores de Virulência/biossíntese , Nucleotídeos de Adenina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Hidrólise , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo
14.
Eur J Immunol ; 42(5): 1203-15, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22311598

RESUMO

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.


Assuntos
Antígenos CD40/imunologia , Células Dendríticas/imunologia , Leishmania/imunologia , Leishmaniose/imunologia , Receptor A2B de Adenosina/imunologia , Acetamidas/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Antígeno B7-2/biossíntese , Antígeno B7-2/imunologia , Células da Medula Óssea/imunologia , Antígenos CD40/biossíntese , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidases/biossíntese , Purinas/farmacologia , Suramina/farmacologia , Linfócitos T/imunologia , Tripanossomicidas/farmacologia
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