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• W2052717571 abstract "Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species." @default.
• W2052717571 created "2016-06-24" @default.
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• W2052717571 date "2012-10-01" @default.
• W2052717571 modified "2023-10-05" @default.
• W2052717571 title "Mycobacterial Shuttle Vectors Designed for High-Level Protein Expression in Infected Macrophages" @default.
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• W2052717571 doi "https://doi.org/10.1128/aem.01674-12" @default.
• W2052717571 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3457510" @default.
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