Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum

Abstract

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH 2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH 2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.

Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85

“…For unclear reasons, transposon mutagenesis has generated only an antigen 85C-deficient mutant but not the other expected antigen 85A-and 85B-deficient mutants (Jackson et al, 1999). Thanks to the isolation and characterization of a csp1-inactivated mutant strain and the previous success of the heterologous expression of antigen 85A in C. glutamicum (Salim et al, 1997), we expressed the different antigens 85 in the csp1-inactivated mutant strain in order to identify the in vivo acceptors of the mycoloyl residues transferred by the various antigens 85. Expression of antigens 85A, 85B and 85C in the csp1-inactivated mutant strain restored the cell wall-bound corynomycolate content and the cell wall permeability to small hydrophilic substances; this mycoloyltransferase function was expected because, like the mycobacterial antigens 85, the recombinant antigen 85A has been shown to be biologically active and its signal peptide has been shown to be cleaved off at the predicted site (Salim et al, 1997).…”

“…Thanks to the existence of the CGL2022 mutant strain with an inactivated csp1 gene and the recent demonstration of the heterologous expression of the mycobacterial antigen 85A in C. glutamicum (Salim et al, 1997), the nature of the in vivo acceptors of the various mycobacterial mycoloyltransferase antigens 85 complex was investigated using appropriate C. glutamicum±E. coli shuttle plasmids containing the various fbp genes.…”

“…Interestingly, a csp1-disrupted mutant of C. glutamicum has been generated (Joliff et al, 1992), providing a clue in the investigation of the in vivo function of the putative corynomycoloyltransferase PS1. Furthermore, heterologous expression of antigen 85A in C. glutamicum demonstrated the secretion of the mycobacterial antigen in corynebacteria (Salim et al, 1997), facilitating the identification of the in vivo acceptors of the mycoloyltransferase activity of the individual antigen 85 by expressing the various antigens 85 in the csp1-disrupted mutant. Accordingly, we investigated the consequences of the disruption of the csp1 gene on the cell envelope structure and on the permeability of the mutant and the effects of the overexpression of PS1 and truncated forms of the protein, as well as that of the various antigens 85, in order to establish the in vivo functions of the corresponding gene products in the transfer of corynomycoloyl residues.…”

Development of a Potential Protein Display Platform in Corynebacterium glutamicum Using Mycolic Acid Layer Protein, NCgl1337, as an Anchoring Motif

“…[17][18][19] Particularly, C. glutamicum is a generally recognized as safe (GRAS) strain; hence, it is suitable for the display of viral antigens, which can be used as oral vaccines. [20,21] For the display of proteins on the surface of C. glutamicum, several anchoring motifs have been used, including NCgl1221, NCgl0933 (Porin B, PorB), PorC from C. glutamicum, and PgsA of Bacillus subtilis. [18,19,[22][23][24] However, similar to most other bacterial hosts, the choice of anchoring motifs is crucial for the display efficiency in C. glutamicum.…”

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476

“…Also, expression of heterologous proteins such as B. subtilis protease subtilisin has been achieved in corynebacteria using vector pEP2 [10]. The gene encoding ¢bronectin binding protein 85A of Mycobacterium tuberculosis has also been expressed in C. glutamicum using pBL1 replicon based vectors containing tac promoter of E. coli or endogenous cspB gene promoter [11]. It was reported earlier by Morinaga et al [12] that common E. coli promoters such as lacUV5, tac and trp function very well in Brevibacterium lactofermentum.…”