SITE-DIRECTED MUTAGENESIS AND VIRULENCE ASSESSMENT OF THE KATG GENE OF MYCOBACTERIUM-INTRACELLULARE

Mycobacterial catalases have been suggested as acting as virulence fac tors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase act ivity: a heat-stable catalase (KatE) and an inducible, heat-labile cat alase peroxidase (KatG). The M. intracellulare katG gene was cloned, a nd a plasmid derivative with a 4 bp insertion in the katG coding seque nce was constructed and used for site-directed mutagenesis of M. intra cellulare 1403 (ATCC 35761). The resulting katG mutant was highly resi stant to isoniazid (INH), showed an increased sensitivity to H2O2 and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshif t allele was also used for mutagenesis of the mouse virulent M. intrac ellulare isolate D673. After intravenous injection into BALB/c mice, D 673 and the isogenic katG mutant showed the same growth kinetics in th e spleen, liver and lungs of the infected mice. Our results demonstrat e that the KatG catalase peroxidase mediates resistance to H2O2 and su sceptibility to INH but is not an essential virulence factor for the s urvival and growth of M. intracellulare in the mouse.