Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages.

Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7605801 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-5336 (Electronic) Linking ISSN: 00992240 NLM ISO Abbreviation: Appl Environ Microbiol Subsets: MEDLINE

Original Publication: Washington, American Society for Microbiology.

Gene Expression* ; Genetic Vectors*; Genetics, Microbial/*methods ; Macrophages/*microbiology ; Molecular Biology/*methods ; Mycobacterium/*genetics; Antigens, Bacterial/biosynthesis ; Antigens, Bacterial/genetics ; Bacterial Proteins/biosynthesis ; Bacterial Proteins/genetics ; Escherichia coli/genetics ; Fluorescence ; Genes, Reporter ; Green Fluorescent Proteins/biosynthesis ; Green Fluorescent Proteins/genetics ; Immune Evasion ; Immune Tolerance ; Mycobacterium/pathogenicity ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Virulence Factors/biosynthesis ; Virulence Factors/genetics

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.

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DP2 OD001886 United States OD NIH HHS; 1 DP2 OD001886 United States OD NIH HHS

0 (Antigens, Bacterial)
0 (Bacterial Proteins)
0 (ESAT-6 protein, Mycobacterium tuberculosis)
0 (Recombinant Proteins)
0 (Virulence Factors)
147336-22-9 (Green Fluorescent Proteins)

Date Created: 20120724 Date Completed: 20130123 Latest Revision: 20211021

20221213

PMC3457510

10.1128/AEM.01674-12

22820329