First conjugation directed traverse of gene cassettes harboring a1,3GT from fast-growing Mycobacterium smegmatis mc2 155 to slow-growing pathogen Mycobacterium tuberculosis H37Rv, presumably opening up new scopes in tuberculosis treatment

Mycobacterium smegmatis strain mc(2) 155 is a fast-growing and non-pathogenic mycobacteria and widely used in genetic studies of mycobacteria. It has been shown that this species of mycobacterium can transfer its genomic DNA fragments to other species of mycobacteria during the conjugation process. Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal) glycan epitope is a highly immunogenic epitope exerted by the enzyme alpha 1-3-galactosyltransferase (alpha 1,3GT) in mammalian cells on the glycan skeleton. However, the enzyme is inactive in humans, primates and Old World monkeys as a result of evolutionary mutations. The robust immunogenicity induced by the epitope in human, has attracted much attention to apply the epitope in vaccine research. In this study we proved successful transfer of desired expression cassettes from fast-growing Mycobacterium smegmatis mc(2 )155, to the slow-growing pathogen Mycobacterium tuberculosis H37Rv. We designed gene cassettes encoding the alpha 1,3GT enzyme under control the potent G13 promoter and the cassette containing hygromycin resistance gene under a Mtb specific promoter, Ptpa in the vector pMV306DIG13 +FflucRT (harboring attP site). The resulting construct was electroporated into mc(2) 155 strain in combination with pBS-int containing the gene encoding Mycobacteriophage L5 integrase to integrate pMV306DIG13 +FflucRT-cassettes into mc(2) 155 genome. Following the integration, the recombinant clones were placed in vicinity to the Mycobacterium tuberculosis H37Rv strain to establish conjugation. Conjugated recombinant clones were selected on the medium containing the hygromycin B and transfer of the desired cassettes to Mycobacterium tuberculosis was confirmed. The enzyme alpha 1,3GT in transconjugants were also investigated.