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

4 Citations (Scopus)

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

Fingerprint

Mycobacterium smegmatis
genetic vectors
Genetic Vectors
macrophages
interleukin-6
protein synthesis
Macrophages
Interleukin-6
protein
cell lines
Escherichia coli
Proteins
Cell Line
lipopolysaccharides
Lipopolysaccharides
inflammation
promoter regions
replication origin
Replication Origin
Mycobacterium avium

ASJC Scopus subject areas

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

Cite this

Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages. / Eitson, Jennifer L.; Medeiros, Jennifer J.; Hoover, Ashley R.; Srivastava, Shashikant; Roybal, Kole T.; Aínsa, José A.; Hansen, Eric J.; Gumbo, Tawanda; Van Oersa, Nicolai S C.

In: Applied and Environmental Microbiology, Vol. 78, No. 19, 10.2012, p. 6829-6837.

Research output: Contribution to journalArticle

Eitson, JL, Medeiros, JJ, Hoover, AR, Srivastava, S, Roybal, KT, Aínsa, JA, Hansen, EJ, Gumbo, T & Van Oersa, NSC 2012, 'Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages', Applied and Environmental Microbiology, vol. 78, no. 19, pp. 6829-6837. https://doi.org/10.1128/AEM.01674-12
Eitson, Jennifer L. ; Medeiros, Jennifer J. ; Hoover, Ashley R. ; Srivastava, Shashikant ; Roybal, Kole T. ; Aínsa, José A. ; Hansen, Eric J. ; Gumbo, Tawanda ; Van Oersa, Nicolai S C. / Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages. In: Applied and Environmental Microbiology. 2012 ; Vol. 78, No. 19. pp. 6829-6837.
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AU - Aínsa, José A.

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