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

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 ϳ10% 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.Glutamine and glutamate are central molecules in nitrogen metabolism. Glutamine is used as the nitrogen donor for many nitrogen-containing molecules in the cell and is synthesized from L-glutamate, ammonia, and ATP by the enzyme glutamine synthetase (GS) (33). The internal L-glutamine pool has been shown to be a sensor of external nitrogen limitation for Salmonella enterica serovar Typhimurium (21). GS is the only known biosynthetic pathway for the synthesis of glutamine and along with glutamate synthetase is responsible for ammonia assimilation under nitrogen-limiting growth conditions. In enteric bacteria, glutamate dehydrogenase can assimilate ammonia directly into glutamate at high concentrations of ammonia. However, for bacteria such as Mycobacterium tuberculosis which lack glutamate dehydrogenase, GS and glutamate synthetase are the sole means of ammonia assimilation. Due to its central role in nitrogen metabolism, GS is subject to varied and complex forms of transcriptional and posttranslational regulation as well as feedback inhibition by several products of glutamine metabolism (7,33).There are at least four major forms of GS (25). In enteric bacteria, a single glnA gene encodes a GS type I (GSI) enzyme, and glnA null mutants are glutamine auxotrophs. Other bacteria have been shown to possess two or three different types of GS. In the case of Sinorhizobium meliloti (formerly Rhizobium meliloti), all three GS genes...

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

“…Only glnA-1 has been shown to be essential for M. tuberculosis growth (Harth et al, 2005). Disruption of glnA-1 in M. tuberculosis results in glutamine auxotrophy, indicating its role in the synthesis of glutamine for the bacillus (Tullius et al, 2003). The genome of the saprophyte Mycobacterium smegmatis mc 2 155 also contains homologues of glnA-1-4, in addition to several sequences that encode GSs similar to those present in soil-dwelling bacteria but absent from the mycobacterial genome (Amon et al, 2009).…”

Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

“…tuberculosis auxotrophs are attenuated [30][31][32][33][34][35][36] . It seems likely that a threonine auxotroph would also be attenuated in vivo.…”

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

“…6 Finally, glnA1 has been found to be essential for M. tuberculosis virulence. 7 The two most active known GS inhibitors described in the literature, L-methionine-(S)-sulfoximine (MSO, 1) and phosphinothricin (PPT, 2) have a profound effect on the growth of M. tuberculosis. 8 These are also the only known inhibitors of MtGS.…”