title: Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages.
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.
authors: The Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors: Jennifer L
authors: JL
authors: Eitson
authors: Jennifer J
authors: JJ
authors: Medeiros
authors: Ashley R
authors: AR
authors: Hoover
authors: Shashikant
authors: S
authors: Srivastava
authors: Kole T
authors: KT
authors: Roybal
authors: José A
authors: JA
authors: Aínsa
authors: Eric J
authors: EJ
authors: Hansen
authors: Tawanda
authors: T
authors: Gumbo
authors: Nicolai S C
authors: NS
authors: van Oers
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057131:Immune Evasion; D007108:Immune Tolerance; D008264:Macrophages / Q000382:microbiology*; D008967:Molecular Biology / Q000379:methods*; D009161:Mycobacterium / Q000235:genetics* / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D037521:Virulence Factors / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D037521:Virulence Factors; D049452:Green Fluorescent Proteins