Vaccines against Intracellular Pathogens

Mycobacterium marinum, a relatively rapid-growing fish and human pathogen, has become an important model for the investigation of mycobacterial pathogenesis. M. marinum is closely related to the Mycobacterium tuberculosis complex and causes a disease in fish and amphibians with pathology similar to tuberculosis. We have developed an in vitro model for the study of M. marinum virulence mechanisms using the carp monocytic cell line CLC (carp leukocyte culture). We found that fish monocytes can differentiate between pathogenic and nonpathogenic mycobacterial species. Interestingly, M. marinum enters fish monocytes at a 40-to 60-foldhigher rate than Mycobacterium smegmatis. In addition, M. marinum survives and replicates in fish monocytes while M. smegmatis is killed. We also found that M. marinum inhibits lysosomal fusion in fish monocytes, indicating that these cells may be used to dissect the mechanisms of intracellular trafficking in mycobacteria. We conclude from these observations that monocytic cells from fish, a natural host for M. marinum, provide an extremely valuable model for the identification and characterization of mycobacterial virulence determinants in the laboratory.Tuberculosis, caused by Mycobacterium tuberculosis, is currently the number one cause of death worldwide from a single infectious agent (11,28). Genetic analysis of the molecular mechanisms of pathogenesis by M. tuberculosis is ongoing, but the few putative virulence determinants that have been identified are not well understood and specific inactivation of many of these genes by allelic exchange has not been accomplished. The fact that few specific mutations have been constructed in M. tuberculosis may be at least partially due to the fact that homologous recombination is more efficient in rapidly growing mycobacterial species (49, 50). Due to the low growth rate of M. tuberculosis, difficulty of manipulation, risk to research personnel, and cost of running a biosafety level three facility, model systems that would allow a better understanding of the causes of tuberculosis are of great interest (8,12). Recently, it has become apparent that Mycobacterium marinum has all of the necessary characteristics of an ideal model organism for genetic analysis of M. tuberculosis pathogenesis. M. marinum has a generation time of 4 h compared to 20 h for M. tuberculosis (17, 34), it is a biosafety level two organism, human disease caused by M. marinum presents almost exclusively as lesions on the extremities (4,18,20), and construction of specific mutations by homologous recombination is relatively straightforward (52, 53). For these reasons, there is heightened interest in M. marinum as a model for the study of mycobacterial pathogenesis (6,51,54,73).Since M. marinum is a natural pathogen of poikilothermic organisms (17), the fish (70) and frog (54) animal models should offer the opportunity to closely approximate natural mycobacterial infections in the laboratory. M. marinum is predominantly an aquatic organism that causes systemic tuberculous inf...

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“…Improved animal models would greatly facilitate studies focusing on mycobacterial pathogenesis and vaccines (9,35).…”

Section: Discussionmentioning

confidence: 99%

Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions

2001

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“…The class II haplotypes are indicated using the D-region haplotype nomenclature (16,42,47,53). The official allele names assigned by the Bovine Leukocyte Antigen Nomenclature Committee of the International Society for Animal Genetics were used for the class II alleles present in each haplotype (14,54 For analysis of the composition of peripheral blood at the initiation of culture, 10 ml of blood was collected in ACD and lysed in Tris-buffered NH 4 Cl to remove erythrocytes. The resultant cells were washed several times in phosphate-buffered saline (PBS) containing 20% ACD (PBS-ACD) to remove excess platelets and then used in FC as described elsewhere (18).…”

Section: Methodsmentioning

confidence: 99%

“…It causes significant economic loss to producers, especially the dairy industry, due to increase in forage consumption, decreased milk production, and early culling due to poor health of affected animals (reviewed in reference 51). The disease has been difficult to control because of the lack of sensitivity and specificity of available diagnostic assays (5) and the lack of an efficacious vaccine (4,33,37). Available assays, such as M. avium subsp.…”

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confidence: 99%

Analysis of the Immune Response toMycobacterium aviumsubsp.paratuberculosisin Experimentally Infected Calves

Koo

Park

Hamilton³

et al. 2004

Infect Immun

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Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp.

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“…Idealmente, también la inactivación de genes deberá generar cepas que sean compatibles con pruebas diagnósticas y que permitan una rápida y eficaz diferenciación entre individuos vacunados e infectados. Además, las cepas atenuadas deberán ser productos finales carentes de marcadores, principalmente marcadores que confieran resistencia a agentes quimioterapéuticos, para evitar la transferencia de estos genes a los aislamientos clínicos (141).…”

Section: Consideraciones Finalesunclassified

Inactivación de genes de Mycobacterium tuberculosis y su potencial utilidad en la prevención y el control de la tuberculosis.

et al. 2004

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El conocimiento derivado del genoma de Mycobacterium tuberculosis, junto con el desarrollo de sofisticados sistemas para la manipulación genética del bacilo, ofrece la mayor promesa para el desarrollo de herramientas nuevas y más eficientes para prevenir y controlar la tuberculosis. Se han desarrollado métodos más eficientes para la inactivación de genes micobacterianos que se han convertido en el pilar de la genómica funcional micobacteriana. La generación de mutantes mediante la inactivación génica, apoyada directa o indirectamente por el desciframiento del genoma micobacteriano, ha permitido la generación de un número significativo de mutantes de M. tuberculosis. En algunos casos, el análisis de estas mutantes ha establecido relaciones entre los productos génicos y sus funciones en la fisiología y la patogenicidad de la micobacteria. En esta revisión se describen los estudios más representativos basados en dichas mutantes.Palabras claves: Mycobacterium tuberculosis, inactivación de genes, genómica funcional, mutantes atenuadas. Gene inactivation in Mycobacterium tuberculosis and its use in tuberculosis control and preventionAvailability of the M. tuberculosis genome sequence and the development of sophisticated systems for genetic manipulation of bacilli offer the potential for new and effective tools to prevent and control tuberculosis. Efficient methods to inactivate mycobacterial genes have been developed. These methods have become the cornerstone for the application and development of mycobacterial functional genomics. Specific mutants are generated to establish the role of targetted genes associated with mycobacterial physiology and pathogenesis. Gene inactivation, supported directly or indirectly by the deciphering of the mycobacterial genome, has permitted the generation of large numbers of M. tuberculosis mutants. Analysis of these mutants has (in some cases) established relationships between gene products and their role in mycobacterial physiology and pathogenesis.

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Vaccines against Intracellular Pathogens

Cited by 5 publications

References 144 publications

Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions

Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions

Analysis of the Immune Response toMycobacterium aviumsubsp.paratuberculosisin Experimentally Infected Calves

Inactivación de genes de Mycobacterium tuberculosis y su potencial utilidad en la prevención y el control de la tuberculosis.

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