Identification of strong promoter elements of<i>Mycobacterium smegmatis</i>and their utility for foreign gene expression in mycobacteria

Spratt, Joanne M.; Britton, Warwick J.; Triccas, James A.

doi:10.1016/s0378-1097(03)00442-7

Cited by 12 publications

(9 citation statements)

References 18 publications

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“…Both pJEX57 and pMV261 were transformed into M. bovis BCG Pasteur strain 1173P2 as previously described (21). BCG strains were grown, as described previously (28), and mycobacterial cell lysates were prepared by sonication of cultures harvested at mid-log phase. The expression of mMCP-3 was verified by immunoblotting using the 12CA5 monoclonal antibody specific for the hemagglutinin epitope tag present at the N terminus of the mature protein.…”

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confidence: 99%

Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection

et al. 2007

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A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCG MCP-3 ) was developed. Mice vaccinated with BCG MCP-3 displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCG MCP-3 was equivalent to that with control BCG; however, immunodeficient mice infected with BCG MCP-3 survived significantly longer than mice infected with the control BCG strain. Therefore, BCG MCP-3 may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.Tuberculosis (TB) remains a leading cause of worldwide mortality alongside human immunodeficiency virus and malaria. Recent figures suggest that each year there are approximately 8 million new cases and 2 million deaths (17). The current vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is only partially effective against lung TB, the contagious form of the disease, with vaccine efficacy ranging from 0 to 80% (median of 50%) (7). Consequently, although BCG is considered a safe vaccine and has been administered since 1921 to an estimated 3 billion people, it has proved insufficient to control the spread of TB (2). BCG is amenable to genetic manipulation, and overexpression of protective antigens in BCG can improve the efficacy of the vaccine in animal models of TB (9,20,23). An alternative approach is to express immunostimulatory molecules in BCG. Murine cytokines, including interleukin-2, interleukin-18, gamma interferon (IFN-␥), and granulocyte-macrophage colony-stimulating factor, can be secreted by BCG in a functional form; however, the ability of these vaccines to protect against TB has not been tested (13,16,18). The monocyte chemotactic proteins (MCP) are a subset of the CC or ␤-chemokines consisting of four members, MCP-1, -2, -3, and -4 (with MCP-5 in the mouse only), which are induced by viral and bacterial infections in many cell types (26, 31). MCP-3 is a pleiotropic chemokine that binds the receptors CCR1, CCR2, and CCR3 (15). These receptors are expressed on mononuclear phagocytes, T cells, B cells, natural killer cells, basophils, eosinophils, neutrophils, and particularly immature dendritic cells (1,3,11,26,32). MCP-3 is highly expressed by macrophages exposed to mycobacterial components or M. tuberculosis-infected mice (27, 30). Mice lacking CCR2 were acutely susceptible to M. tuberculosis infection and exhibited a significant reduction in early macrophage recruitment and a later defect in dendritic cell and T-cell migration (22). These results clearly demonstrate that chemokines, such as MCP-3, are vital in the response to M. tuberculosis infection, as they are required for the selective migration of the cells essential for the generation of a protective immune response.In this report, we demonstrate that murine MCP-3 (mMCP-3) can be expressed and secreted by BCG in an active form. Secretion of MCP-3 markedly improved the immunogenicity of BCG a...

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Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection

et al. 2007

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“…These symptoms may be due to toxicity of these particular proteins, imposing heavy physiological burden on to the cells. A number of methods could be used to resolve this issue, including using plasmids with lower copy numbers, use of weaker promoters with lower expression level or using a promoter that is more tightly regulated . Alternatively, other expression hosts that are described in this manuscript could be of use.…”

Section: Expression Hosts For Mycobacterial Proteinsmentioning

confidence: 99%

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Bashiri

Baker

2014

Protein Science

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Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to overexpress and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.

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“…The G13 promoter was the first mycobacterial promoter described that was highly active in an E. coli background, and others have since been described (Satchidanandam et al, 2003). Recently, Spratt et al (2003) reported that a series of strong promoters isolated from an M. smegmatis library using a similar gfp reporter system was similar to both E. coli and mycobacterial consensus promoter sequences. This same group indicated that no correlation existed between promoter sequence and strength of gfp expression.…”

Section: Analysis Of Promoter Activity In Mycobacterium Marinummentioning

confidence: 99%

Differential green fluorescent protein expression from mycobacterial promoter constructs inEscherichia coliandMycobacterium marinum

Gall

Barker

2006

FEMS Microbiology Letters

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The Mycobacterium marinum G13 promoter is a sigma 70-like promoter that is more active by green fluorescent protein (gfp) differential fluorescence induction (DFI) assays when M. marium resides in an intracellular compartment as compared with growth in media. In assays using DFI, we found that the mycobacterial G13 promoter was also more active in a background of lower nutrient availability during logarithmic growth. This promoter, contained in an insert cloned upstream of a gfp reporter gene, is also active in Escherichia coli. When gfp expression assays of different plasmid constructs were performed in parallel with E. coli and M. marinum, expression in E. coli was maintained after deletion of both upstream and/or downstream regions proximal to the core promoter sequence. In M. marinum, however, although upstream deletions had no appreciable effect on gfp expression, promoter constructs with deleted downstream regions expressed 20- to 40-fold less gfp over all growth phases. The high-level expression of gfp was restored, however, in a clone containing approximately 100 bp downstream of the transcriptional start point. We have therefore utilized this gfp reporter assay of promoter activity to distinguish possible differences in requirements for gfp expression between different genera that utilize sigma 70-like promoter elements. We found that high levels of expression of gfp from the G13 promoter in M. marinum require downstream regions not necessary for gfp expression in E. coli.

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Identification of strong promoter elements ofMycobacterium smegmatisand their utility for foreign gene expression in mycobacteria

Cited by 12 publications

References 18 publications

Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection

Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Differential green fluorescent protein expression from mycobacterial promoter constructs inEscherichia coliandMycobacterium marinum

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