Document Detail

Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages.
MedLine Citation:
PMID:  22820329     Owner:  NLM     Status:  MEDLINE    
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.
Jennifer L Eitson; Jennifer J Medeiros; Ashley R Hoover; Shashikant Srivastava; Kole T Roybal; José A Aínsa; Eric J Hansen; Tawanda Gumbo; Nicolai S C van Oers
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-07-20
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  78     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-09-10     Completed Date:  2013-01-23     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6829-37     Citation Subset:  IM    
The Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
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MeSH Terms
Antigens, Bacterial / biosynthesis,  genetics
Bacterial Proteins / biosynthesis,  genetics
Escherichia coli / genetics
Gene Expression*
Genes, Reporter
Genetic Vectors*
Genetics, Microbial / methods*
Green Fluorescent Proteins / biosynthesis,  genetics
Immune Evasion
Immune Tolerance
Macrophages / microbiology*
Molecular Biology / methods*
Mycobacterium / genetics*,  pathogenicity
Recombinant Proteins / biosynthesis,  genetics
Virulence Factors / biosynthesis,  genetics
Grant Support
1 DP2 OD001886/OD/NIH HHS
Reg. No./Substance:
0/Antigens, Bacterial; 0/Bacterial Proteins; 0/ESAT-6 protein, Mycobacterium tuberculosis; 0/Recombinant Proteins; 0/Virulence Factors; 147336-22-9/Green Fluorescent Proteins

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