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 approximately 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.

FIG. 1.

Allelic exchange vector, pEX1, used in the construction of the glnA1 mutant. Unique restriction sites are shown.

FIG. 2.

Construction of the M. tuberculosis glnA1 mutant. (A) Maps of the wild-type glnA1 locus and the disrupted allele, which contains a Km^r cassette (aphA-2) inserted into the unique EcoNI site in the middle of the glnA1 coding region. Only a small portion of the 3′ end of glnE is present on the SmaI fragment. (B) Genomic DNA from the M. tuberculosis wild-type strain and the glnA1 mutant was digested to completion with SmaI and probed with a 1.8-kb fragment containing glnA1 (hatched bar in panel A). M, molecular mass markers in kilobases; wt, wild-type.

FIG.3.

Glutamine requirement of the M. tuberculosis glnA1 mutant in broth culture. Cultures of the glnA1 strain (A and B) and the wild-type strain (C) were inoculated into 7H9-OADC-TW containing various concentrations of l-glutamine (indicated next to the corresponding lines on the graphs) to an initial calculated A₅₅₀ of 0.0001. Growth was monitored by assaying absorbance (A and C) and CFU (B). Data are the means ± standard errors for duplicate or triplicate cultures. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <14% of the mean. The limits of detection were 0.02 absorbance units (A and C) and 1.22 log CFU/ml (16 CFU/ml) (B), as indicated by the dashed lines (measurements below the detection limit were scored as equal to the detection limit). The experiment was repeated once with similar results (only the 0 and 0.2 mM l-glutamine cultures were plated for CFU in the second experiment). Growth of the wild-type strain was unaffected by the l-glutamine concentration. Mtb, M. tuberculosis.

FIG. 4.

GS expression in M. tuberculosis wild-type, glnA1, and complemented strains. (A) SDS-PAGE analysis of cell lysates (∼18 μg of total protein per lane). (B) Immunoblot analysis of the cell lysates (∼18 μg of total protein per lane). Blots were probed with polyclonal rabbit anti-M. tuberculosis GS and, as a control, rabbit anti-M. tuberculosis superoxide dismutase antibody. The GS band (arrow) present in the wild-type lysates is absent in the M. tuberculosis glnA1 pNBV1 and M. tuberculosis glnA1 pNBV1-StGS lysates. GS is overexpressed in the M. tuberculosis glnA1 pNBV1-MtbGS strain due to its expression from a multicopy plasmid. The M. tuberculosis GS polyclonal antibodies used for immunodetection of GS were not capable of detecting <1 μg of purified S. enterica serovar Typhimurium GS (data not shown); therefore, the absence of a band for S. enterica serovar Typhimurium GS shows only that expression is <1 μg. +l-Gln, cultures grown in the presence of 20 mM l-glutamine; M, molecular mass markers in kilodaltons; Mtb, M. tuberculosis.

FIG. 5.

Intracellular growth of M. tuberculosis wild-type, glnA1, and complemented strains in human THP-1 macrophages. The tissue culture medium included the standard amount of l-glutamine (2 mM) (A) or 0.2 mM l-glutamine (B). When THP-1 cells were cultured in the presence of 2 mM l-glutamine, the glnA1 mutant multiplied intracellularly but at a reduced rate compared with the wild-type strain. When THP-1 cells were cultured in the presence of only 0.2 mM l-glutamine, the strain did not multiply and slowly died (∼0.5 log reduction in 6 days). The wild-type strain and both complemented strains grew normally in the presence of 0.2 mM l-glutamine. Data are the means ± standard errors for three wells per time point. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <2% of the mean. The experiment was repeated once with similar results. Mtb, M. tuberculosis.

FIG. 6.

Glutamine requirement of the M. tuberculosis glnA1 mutant during intracellular growth in human THP-1 macrophages. The concentration of l-glutamine in the tissue culture medium was varied from 0.2 to 10 mM. At the highest concentration, the mutant grew at a rate similar to that of the wild-type strain. Data are the means ± standard errors for three wells per time point. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <2% of the mean. The experiment was repeated once with similar results. Mtb, M. tuberculosis.

FIG. 7.

The M. tuberculosis glnA1 mutant is avirulent in guinea pigs. Guinea pigs were infected by aerosol with M. tuberculosis strains as indicated, and 10 weeks later, bacterial load in the right lung (A) and spleen (B) was quantified. Guinea pigs were infected with the M. tuberculosis glnA1 mutant at the standard dose (1×) used for the other strains and at 10× and 100× the standard dose. Data are the means ± standard errors for all animals in a group (n = 5). No CFU were detected in plated aliquots of the lung and spleen for any of the M. tuberculosis glnA1-infected animals, and all of these organs were scored as 2.1 log CFU for statistical purposes (2.1 log CFU/organ, the limit of detection, is indicated by the dashed lines). Mtb, M. tuberculosis.