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Infection and Immunity, July 2003, p. 3927-3936, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3927-3936.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

Michael V. Tullius, Günter Harth, and Marcus A. Horwitz*

Division of Infectious Diseases, Department of Medicine, School of Medicine, University of California—Los Angeles, Los Angeles, California 90095-1688

Received 20 December 2002/ Returned for modification 26 February 2003/ Accepted 8 April 2003

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was ~10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, School of Medicine, UCLA, CHS 37-121, 10833 Le Conte Ave., Los Angeles, CA 90095-1688. Phone: (310) 206-0074. Fax: (310) 794-7156. E-mail: mhorwitz{at}mednet.ucla.edu.

Editor: S. H. E. Kaufmann

Infection and Immunity, July 2003, p. 3927-3936, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3927-3936.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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