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

Abstract

ABSTRACT 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.