Dataset Information

Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

ABSTRACT: Nuclear factor-kappa B (NF?B) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NF?B has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NF?B are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NF?B in host defense in humans is not fully understood. We sought to examine the role of NF?B activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NF?B activation using BAY 11-7082 (BAY, an inhibitor of I?B? kinase) or an adenovirus construct with a dominant-negative I?B? significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NF?B inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NF?B inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

SUBMITTER: Bai X

PROVIDER: S-EPMC3636238 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

Bai Xiyuan X Feldman Nicole E NE Chmura Kathryn K Ovrutsky Alida R AR Su Wen-Lin WL Griffin Laura L Pyeon Dohun D McGibney Mischa T MT Strand Matthew J MJ Numata Mari M Murakami Seiji S Gaido Loretta L Honda Jennifer R JR Kinney William H WH Oberley-Deegan Rebecca E RE Voelker Dennis R DR Ordway Diane J DJ Chan Edward D ED

PloS one 20130425 4

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role ...[more]

PMID: 23634218

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Project description:Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

Dataset Information

Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

Publications

Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

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