Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare.
Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant 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 frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H2O2 and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.
Marklund
B I
BI
Swedish Institute for Infectious Disease Control, Stockholm. Britt-Inger.Marklund@micro.umu.se
Mahenthiralingam
E
E
Stokes
R W
RW
D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004795:Enzyme Stability; D005260:Female; D005798:Genes, Bacterial; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007538:Isoniazid / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D015269:Mycobacterium avium Complex / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D015270:Mycobacterium avium-intracellulare Infection / Q000209:etiology / Q000382:microbiology; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D014774:Virulence
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D015342:DNA Probes; D006861:Hydrogen Peroxide; D010544:Peroxidases; C076136:catalase HPI; D007538:Isoniazid