Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis
Mem. Inst. Oswaldo Cruz
; 112(3): 203-208, Mar. 2017. graf
Article
en En
| LILACS
| ID: biblio-841772
Biblioteca responsable:
BR1.1
ABSTRACT
BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
LILACS
Contexto en salud:
2_ODS3
/
3_ND
Problema de salud:
2_enfermedades_transmissibles
/
3_neglected_diseases
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3_tuberculosis
Asunto principal:
Macrófagos
/
Mycobacterium tuberculosis
/
N-Glicosil Hidrolasas
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Mem. Inst. Oswaldo Cruz
Asunto de la revista:
MEDICINA TROPICAL
/
PARASITOLOGIA
Año:
2017
Tipo del documento:
Article
/
Project document
País de afiliación:
Brasil