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

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 Oersa

Research output: Contribution to journalArticle

5 Scopus citations

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 (pSUMkan-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.

Original languageEnglish (US)
Pages (from-to)6829-6837
Number of pages9
JournalApplied and environmental microbiology
Volume78
Issue number19
DOIs
StatePublished - Oct 2012

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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    Eitson, J. L., Medeiros, J. J., Hoover, A. R., Srivastava, S., Roybal, K. T., Aínsa, J. A., Hansen, E. J., Gumbo, T., & Van Oersa, N. S. C. (2012). Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages. Applied and environmental microbiology, 78(19), 6829-6837. https://doi.org/10.1128/AEM.01674-12