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Infect Immun, August 1998, p. 3626-3634, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare

Eshwar Mahenthiralingam,1 2 3 Britt-Inger Marklund,4 Lucy A. Brooks,1 2 3 Debbie A. Smith,5 Gregory J. Bancroft,5 and Richard W. Stokes1 2 3 6 *

Division of Infectious and Immunological Diseases, B.C. Children's Hospital,1 Canadian Bacterial Diseases Network,2 and Departments of Paediatrics3 and Pathology,6 University of British Columbia, Vancouver, Canada; Department of Microbiology, Umeå University, Umeå, Sweden4; and Immunology Unit, Department of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, London, United Kingdom5

Received 7 November 1997/Returned for modification 31 March 1998/Accepted 11 May 1998

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

* Corresponding author. Mailing address: Div. of Infectious and Immunological Diseases, Dept. of Paediatrics, Univ. of British Columbia, Rm. 304, The Research Institute, 950 West 28th Ave., Vancouver, B.C., Canada V5Z 4H4. Phone: (604) 875-2466. Fax: (604) 875-2226. E-mail: rstokes{at}cbdn.ca.

Infect Immun, August 1998, p. 3626-3634, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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