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

Appl Environ Microbiol. 2012 Oct;78(19):6829-37. doi: 10.1128/AEM.01674-12. Epub 2012 Jul 20.

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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • ESAT-6 protein, Mycobacterium tuberculosis
  • Recombinant Proteins
  • Virulence Factors
  • Green Fluorescent Proteins