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1.
Infect Immun ; 82(5): 1850-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24566628

RESUMO

Tuberculosis remains a major worldwide epidemic because of its sole etiological agent, Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistant M. tuberculosis strains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves the ethA-ethR locus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of the ethA-ethR locus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthR in vivo has never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant of Mycobacterium bovis BCG. Our results indicate that absence of the ethA-ethR locus led to greater persistence of M. bovis BCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between the ethA-ethR KO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here that M. bovis BCG ethA-ethR is involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties and in vivo persistence. This study thus provides some experimental clues to the possible physiological role of ethA-ethR and proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence.


Assuntos
Aderência Bacteriana/fisiologia , Mycobacterium bovis/genética , Ácidos Micólicos/metabolismo , Oxirredutases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Parede Celular , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium bovis/metabolismo , Estresse Oxidativo , Oxirredutases/genética , Proteínas Repressoras/genética , Organismos Livres de Patógenos Específicos
2.
Infect Immun ; 80(8): 2771-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22645285

RESUMO

Urease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficient Mycobacterium tuberculosis strain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage on M. tuberculosis in macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity in M. tuberculosis nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that M. tuberculosis virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Nitrogênio/metabolismo , Tuberculose/microbiologia , Urease/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Mycobacterium tuberculosis/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fatores de Tempo , Urease/genética
3.
JCI Insight ; 2(24)2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29263304

RESUMO

Declining levels of maternal antibodies were shown to sensitize infants born to dengue-immune mothers to severe disease during primary infection, through the process of antibody-dependent enhancement of infection (ADE). With the recent approval for human use of Sanofi-Pasteur's chimeric dengue vaccine CYD-TDV and several vaccine candidates in clinical development, the scenario of infants born to vaccinated mothers has become a reality. This raises 2 questions: will declining levels of maternal vaccine-induced antibodies cause ADE; and, will maternal antibodies interfere with vaccination efficacy in the infant? To address these questions, the above scenario was modeled in mice. Type I IFN-deficient female mice were immunized with live attenuated DENV2 PDK53, the core component of the tetravalent DENVax candidate currently under clinical development. Pups born to PDK53-immunized dams acquired maternal antibodies that strongly neutralized parental strain 16681, but not the heterologous DENV2 strain D2Y98P-PP1, and instead caused ADE during primary infection with this strain. Furthermore, pups failed to seroconvert after PDK53 vaccination, owing to maternal antibody interference. However, a cross-protective multifunctional CD8+ T cell response did develop. Thus, our work advocates for the development of dengue vaccine candidates that induce protective CD8+ T cells despite the presence of enhancing, interfering maternal antibodies.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Dengue/imunologia , Vírus da Dengue/imunologia , Feminino , Imunidade Celular , Imunidade Materno-Adquirida , Masculino , Troca Materno-Fetal/imunologia , Camundongos da Linhagem 129 , Gravidez , Soroconversão
4.
PLoS Negl Trop Dis ; 10(3): e0004536, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27007501

RESUMO

Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals' death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates.


Assuntos
Anticorpos Antivirais/sangue , Dengue/imunologia , Interferon Tipo I/metabolismo , Hepatopatias/etiologia , Animais , Citocinas/metabolismo , Dengue/genética , Dengue/mortalidade , Vírus da Dengue/classificação , Feminino , Deleção de Genes , Imunoglobulina G/sangue , Interferon Tipo I/genética , Camundongos , Receptores de Interferon/genética , Receptores de Interferon/metabolismo
5.
Sci Rep ; 6: 37796, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27883085

RESUMO

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8+ T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4+ subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-ß mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat's immunity, opening up new perspectives of therapeutic interventions for humans.


Assuntos
Quirópteros/imunologia , Subpopulações de Linfócitos/imunologia , Animais , Medula Óssea/imunologia , Sistema Imunitário/imunologia , Interleucina-17/imunologia , Interleucinas/imunologia , Linfonodos/imunologia , Fenótipo , Fator de Crescimento Transformador beta/imunologia , Interleucina 22
6.
Immunobiology ; 219(1): 78-86, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24091276

RESUMO

The role of the CD137-CD137 ligand (CD137L) signaling pathway in T cell co-stimulation has been well established. Dysregulated CD137 or CD137L stimulation can lead to pathological conditions such as inflammatory diseases or cancer. However, the contribution of CD137-CD137L interaction to the control of infectious diseases has not been extensively studied, with the few available reports focusing mainly on viral infections. Here we investigated the role of the CD137-CD137L interactions during Mycobacterium tuberculosis infection. Using CD137L-deficient mice, we found that absence of the CD137L-mediated signaling pathway during M. tuberculosis infection resulted in delayed activation of CD4(+) T cells in the draining lymph nodes. This finding was supported by an in vitro mixed lymphocyte reaction assay that revealed impaired priming of T cells by CD137L-deficient dendritic cells upon mycobacterial infection. In addition, greater numbers of CD4(+) T cells and antigen presenting cells were measured in the lungs of CD137L-deficient mice. Strikingly, the lung cytokine production profile was profoundly altered in M. tuberculosis-infected CD137L-deficient mice with lower levels of TNF-α, IL-12 and IL-6 and elevated concentrations of IL-17 compared to their wild type counterparts. However and surprisingly, these tangible immunological disorders translated only into a mild and transient increase in the bacterial loads and a higher number of granulomatous lesions with impaired architecture in the lungs of the CD137L-deficient infected mice. Together, while our data support the engagement of the CD137L signaling pathway during M. tuberculosis infection, they underscore the functional redundancy and robustness of the host defense arsenal deployed against mycobacterial infection.


Assuntos
Ligante 4-1BB/imunologia , Mycobacterium tuberculosis/imunologia , Transdução de Sinais/imunologia , Tuberculose/imunologia , Ligante 4-1BB/deficiência , Ligante 4-1BB/genética , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Feminino , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Imuno-Histoquímica , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/microbiologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/fisiologia , Transdução de Sinais/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/microbiologia , Fatores de Tempo , Tuberculose/genética , Tuberculose/microbiologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
7.
Eur J Immunol ; 35(12): 3591-8, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16285011

RESUMO

Allergen-specific immunotherapy (SIT) leads to a long-term amelioration of IgE- and Th2-mediated allergic diseases. However, SIT efficiency is low, with years of treatment along with frequent allergic side effects. The goal of this study was to reduce the side effects by destroying IgE-binding epitopes, i.e. by heat-denaturation, while preserving the therapeutic effect. Mice were immunised with bee venom, birch pollen, grass pollen or cat hair allergens, or with ovalbumin. Heat-denatured allergens bound less IgE but enhanced Th1-dependent IgG2a production as measured by ELISA. The strong IgG2a antibody responses also prevented allergic anaphylaxis in mice, as measured by body temperature drop after a challenge with a high allergen dose. We found that optimal heat-denaturation of allergens left a small proportion in the native conformation to sufficiently stimulate B cells, while non-B cell-mediated effects were probably amplified. The enhanced immunogenicity of heat-denatured allergens is likely explained by enhanced antigen presentation to T cells due to the particulate nature of heat-denatured proteins. This enables Th1 skewing of the immune response with strong production of IgG2a in mice. Therefore, heat-denaturation represents probably the simplest way to enhance the efficiency of SIT while reducing its side effects.


Assuntos
Alérgenos/administração & dosagem , Alérgenos/imunologia , Dessensibilização Imunológica/métodos , Temperatura Alta , Alérgenos/efeitos adversos , Alérgenos/metabolismo , Animais , Sítios de Ligação de Anticorpos , Dessensibilização Imunológica/efeitos adversos , Feminino , Humanos , Imunoglobulina G/biossíntese , Isotipos de Imunoglobulinas/biossíntese , Camundongos , Camundongos Endogâmicos CBA , Desnaturação Proteica
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