Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

doi:10.1016/j.plasmid.2004.11.002

Plasmid

Volume 53, Issue 3, May 2005, Pages 269-273

Communicated by Julian Rood

https://doi.org/10.1016/j.plasmid.2004.11.002 Get rights and content

Abstract

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409–419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.

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Acknowledgments

Ag85B was obtained through the tuberculosis research material and vaccine testing contract, NIH, NIAID, No. 1 AI-75320. OT-II transgenic mice were kindly provided by Dr. William Heath, WEHI, Melbourne, Australia. We thank Dr. Stuart Tangye (Centenary Institute) for the SAP-myc vector. This work was supported by a University of Sydney Sesqui Research and Development grant and the National Health and Medical Research Council of Australia.

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There are more references available in the full text version of this article.

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Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis
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The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses, and its genome encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family. To understand its role in M. tb pathophysiology we have characterised the PPE50 (Rv3135)-PPE51 (Rv3136) gene cluster, one of nine PPE–PPE clusters in the genome. We demonstrate here that this cluster is operonic, and that PPE50 and PPE51 interact - the first demonstration of PPE–PPE interaction. THP-1 macrophages infected with recombinant Mycobacterium smegmatis strains expressing PPE50 and PPE51 showed lower intracellular viability than the control, which correlated with an increase in transcript levels of iNOS2. Infected macrophages also exhibited an upregulation in levels of IL-10, indicating an immunomodulatory role for these proteins. Using pull-downs and signalling assays, we identified TLR1 to be the cognate receptor for PPE50 - all the phenotypes observed on infection of THP-1 macrophages were reversed on pre-treatment with an anti-TLR1 antibody, validating the functional outcome of PPE50-TLR1 interaction. Our data reveals a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance.
The same well-characterized T cell epitope SIINFEKL expressed in the context of a cytoplasmic or secreted protein in BCG induces different CD8<sup>+</sup> T cell responses
2010, Immunology Letters
Mycobacterium bovis BCG is still the most widely used vaccine against tuberculosis and CD8⁺ T cells play important roles in fighting infection. We investigated how well antigen is processed and presented to CD8⁺ T cells using the same well-characterized CD8⁺ T cell epitope SIINFEKL expressed in either a cytoplasmic (GFP-OVA) or secreted (85B-OVA) context from BCG. We report that secreted SIINFEKL from 85B-OVA BCG is presented better than cytoplasmic SIINFEKL expressed by GFP-OVA BCG.
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¹: These authors contributed equally to this work.

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Plasmid

Abstract

Section snippets

Acknowledgments

References (13)

Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains

Tuberculosis

Introduction of soluble protein into the class I pathway of antigen processing and presentation

Cell

Recombinant BCG vaccines

Vaccine

An inducible expression system permitting the efficient purification of a recombinant protein from Mycobacterium smegmatis

FEMS Micro. Lett.

Host responses and antigens involved in protective immunity to Mycobacterium tuberculosis

Scand. J. Immunol.

Mycobacterium bovis BCG vaccines exhibit defects in alanine and serine catabolism

Infect. Immun.

Cited by (12)

Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis

The same well-characterized T cell epitope SIINFEKL expressed in the context of a cytoplasmic or secreted protein in BCG induces different CD8<sup>+</sup> T cell responses

Septum site placement in Mycobacteria – identification and characterisation of mycobacterial homologues of Escherichia coli MinD

An immunomodulatory role for the Mycobacterium tuberculosis region of difference 1 locus proteins PE35 (Rv3872) and PPE68 (Rv3873)

The Mycobacterium tuberculosis PE Proteins Rv0285 and Rv1386 Modulate Innate Immunity and Mediate Bacillary Survival in Macrophages

Recombinant BCG as a vaccine vehicle to protect against tuberculosis

Short communicationEpitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

Abstract

Section snippets

Acknowledgments

Tuberculosis

Cell

Vaccine

FEMS Micro. Lett.

Host responses and antigens involved in protective immunity to Mycobacterium tuberculosis

Scand. J. Immunol.

Mycobacterium bovis BCG vaccines exhibit defects in alanine and serine catabolism

Infect. Immun.

Short communication
Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria