Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Abstract

Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae, Mycobacterium bovis BCG and Mycobacterium smegmatis mc 2 155 was evaluated using a range of characterized mycobacterial promoter sequences (hsp60, hsp70, PAN, 18kDa and 16S rRNA) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis. Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis. Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria.

Plasmid instability when the <i>hsp</i>60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

“…In our study all rBCGpUS977dtb PW8 clones stably expressed significant amounts of DTB while rBCGpUS973dtb PW8 clones were unstable and, consequently, unsuitable for DTB expression. Both hsp60 and P AN are constitutive mycobacterial promoters and have been widely used in recombinant BCG studies [18,35,36]. The reason for the instability of rBCGs transformed with the pUS973 construct designed with the stronger hsp60 promoter is not yet clear and was not the objective of this study.…”

“…Our best results came out with the BCG transformed with the pUS977dtb PW8 construct in which the expression of DTB is driven by the P AN promoter, a regulatory sequence derived from M. paratuberculosis. The P AN sequence, a weak mycobacterial promoter [18], has been successfully used by others for the expression of the α-galactosidade of E. coli [30], the LacZ of E. coli [31], the Gp63 surface antigen of Leishmania [32], the Nef antigen of Simian Immunodeficiency Virus [33], the Nef, Gag, Env antigens of Simian Immunodeficiency Virus [34], the S1 subunit of the Bordetella pertussis toxin [24], the hepatitis B surface antigens [35] and the LipL32 antigen of Leptospira interrogans [28]. In our study all rBCGpUS977dtb PW8 clones stably expressed significant amounts of DTB while rBCGpUS973dtb PW8 clones were unstable and, consequently, unsuitable for DTB expression.…”

“…The PCR reaction was performed in the Gene Amp PCR System 2400 thermo cycler (Perkin Elmer) with 30 cycles of 5 minutes denaturation at 94˚C, 1.5 minutes annealing at 55˚C and 2 minutes extension at 50˚C. The PCR product was analyzed in agarose gel and the 1051 bp amplicon band corresponding to the size of the dtb PW8 DNA was purified, digested with XbaI and then cloned into the same site of the expression vectors pUS973 and pUS977 [17,18], giving rise to pUS973dtb PW8 and pUS977dtb PW8 constructs, respectively. The identity of the DNA inserts was confirmed by DNA sequencing.…”

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

“…Despite the success of heterologous antigen expression, and in some cases protection achieved when using rBCG, in vitro and in vivo instability of the recombinant vaccines has been reported (Medeiros et al, 2002;Michelon et al, 2006). This instability is observed mainly when replicative vectors are used, which are lost during BCG replication in vivo (Edelman et al, 1999).…”

Vaccine Against Entheropathogenic E. colii: A Systematic Review

“…With this expression of BfpA, Intimin and EspA recombinants using specific primers, the normal development and maintenance of memory cells during vaccination will be preserved [68]. This strategy is based on the BCG ∆leuD ability to multiply inside macrophages only in the presence of complementation [69].…”