Allelic exchange mutagenesis in Map

Abstract

Particular aspects provide efficient allelic exchange methods to generate directed mutations within genes of slow-growing stains of mycobacteria (e.g., Mycobacterium avium subsp. paratuberculosis (Map), Map 10 or GFP-expressing Map K-10) using a phage-delivery system, and demonstrate high efficiency allelic exchange. Additional exemplary aspects provide non-naturally occurring slow-growing strains of mycobacteria (e.g., Map, M. bovis, M. tuberculosis) having at least one gene deletion (e.g., pknG, relA, lsr2, panC, panD, proC, trpD, sapM (MAP3432), lysA_1, leuD, and leuC, and deletion mutants at the orthologous loci of two known virulence genes in pathogenic mycobacteria (relA and pknG) and one gene related to colony morphology and biofilm formation in fast growing mycobacteria (lsr2) were made. Further aspects provide novel vaccines comprising such deletion mutants, or portions thereof, and methods for making said vaccines. Yet further aspects provide methods for protecting a mammal from virulent Map, M. bovis, or M. tuberculosis, comprising treating the mammal with the inventive vaccines.

Dependencies are inferred from the claim text.