RESUMO
Bacille Calmette-Guérin (BCG) is the vaccine against tuberculosis (TB), but has varied efficacy in different geographical locations. Recombinant strategies to genetically modify the organism to enhance the quality of the immune response have aimed at improving BCG efficacy. Here we describe such a strategy using rBCGΔureCâ·hly expressing defined latency-associated antigens and test this construct for long-term protection against an isolate of the Mycobacterium tuberculosis (Mtb) Beijing/W lineage. Expression of the antigens Rv2659c, Rv3407 and Rv1733c by rBCGΔureCâ·hly improved long-term efficacy in both lung and spleen at day 200 post-infection after intradermal vaccination of mice. Our data support expression of Mtb latency associated antigens by rBCG to improve protection against Mtb.
Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Antígenos , Antígenos de Bactérias/genética , Vacina BCG/administração & dosagem , Vacina BCG/genética , Proteínas de Transporte de Cátions , Contagem de Colônia Microbiana , Feminino , Injeções Intradérmicas , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Baço/imunologia , Baço/microbiologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Apoptosis and activation of macrophages play an important role in the host response to mycobacterial infection involving TNF-alpha as a critical autocrine mediator. The underlying mechanisms are still ill-defined. Here, we demonstrate elevated levels of methylglyoxal (MG), a small and reactive molecule that is usually a physiological product of various metabolic pathways, and advanced glycation end products (AGE) during mycobacterial infection of macrophages, leading to apoptosis and activation of macrophages. Moreover, we demonstrate abundant AGE in pulmonary lesions of tuberculosis (TB) patients. Global gene expression profiling of MG-treated macrophages revealed a diverse spectrum of functions induced by MG, including apoptosis and immune response. Our results not only provide first evidence for the involvement of MG and AGE in TB, but also form a basis for novel intervention strategies against infectious diseases in which MG and AGE play critical roles.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Mycobacterium/patogenicidade , Aldeído Pirúvico/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/genética , Expressão Gênica , Macrófagos Alveolares/microbiologia , Camundongos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/patologia , Mycobacterium bovis/patogenicidade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Mycobacterium tuberculosis contains multiple versions of the accA and accD genes that encode the alpha- and beta-subunits of at least three distinct multi-functional acyl-CoA carboxylase complexes. Because of its proposed involvement in pathogenic M. tuberculosis survival, the high-resolution crystal structure of the beta-subunit gene accD5 product has been determined and reveals a hexameric 356 kDa complex. Analysis of the active site properties of AccD5 and homology models of the other five M. tuberculosis AccD homologues reveals unexpected differences in their surface composition, providing a molecular rational key for a sorting mechanism governing correct acyl-CoA carboxylase holo complex assembly in M. tuberculosis.
Assuntos
Acil Coenzima A/metabolismo , Carboxil e Carbamoil Transferases/química , Carboxil e Carbamoil Transferases/metabolismo , Mycobacterium tuberculosis/enzimologia , Dobramento de Proteína , Sítios de Ligação , Carboxil e Carbamoil Transferases/genética , Cristalografia por Raios X , Modelos Moleculares , Mycobacterium tuberculosis/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Primers do DNA , Proteínas Recombinantes/química , Difração de Raios XRESUMO
The first tuberculosis vaccine candidates have reached clinical testing. Novel subunit vaccine candidates aimed at boosting previous BCG-prime vaccination and novel viable attenuated vaccine candidates aimed at substituting BCG have both completed the preclinical stage. Despite these achievements, rational vaccine design against tuberculosis has not come to an end. Novel findings in basic immunology and microbiology will advance further improvements in vaccine development. These include the potential role of crosspriming to induce more potent T-cell responses, the role of memory T cells and regulatory T cells in sustaining or curtailing optimal immune responses, respectively, as well as the involvement of cytokines in T-cell migration to nonimmunologic tissue sites and in the generation of memory. Knowledge about basic mechanisms underlying optimum protection will not only have a direct impact on future vaccine design against tuberculosis but also help in the formulation of a set of biomarkers with predictive value for vaccine efficacy assessment.
Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/imunologia , Humanos , Interleucina-15/imunologia , Interleucina-17/imunologia , Interleucina-23 , Subunidade p19 da Interleucina-23 , Interleucina-7/imunologia , Interleucinas/imunologiaRESUMO
Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4'-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.
Assuntos
Aciltransferases/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/enzimologia , Aciltransferases/química , Ácidos Decanoicos/química , Espectrometria de Massas , Modelos MolecularesRESUMO
The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.
Assuntos
Vacina BCG , Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Listeria monocytogenes/metabolismo , Tuberculose/prevenção & controle , Adulto , Animais , Apoptose , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Células Cultivadas , Criança , Proteínas Hemolisinas , Humanos , Concentração de Íons de Hidrogênio , Listeria monocytogenes/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Taxa de Sobrevida , Vacinas SintéticasRESUMO
Mycobacterium bovis bacille Calmette Guérin (BCG) is one of the most widely used live vaccines. Technologic advancement in genome manipulation enables the construction of recombinant BCG (rBCG) strains, which can be employed as highly immunogenic vaccines against tuberculosis with improved safety profile.