Fig 1.
Schematic illustration depicting central nitrogen metabolism in the mycobacterium tuberculosis complex.
Fig 2.
The effect of glutamate as a major carbon source on survival of M. bovis BCG wild type, mutant (Δgdh and ΔgltBD) and complemented strains.
Exponential phase cultures were diluted to an OD600 of 0.0005 into (A & B) 7H9 supplemented with glycerol, dextrose and Tween 80, (C & D) 7H9 without glycerol, dextrose, or Tween 80 (replaced with tyloxapol) or (E & F) 7H9 without glycerol, dextrose, or Tween80 and supplemented with cholesterol (0.25 mM). Symbols and error bars are means and standard errors calculated from triplicate plating obtained from two independent experiments. Data was analysed by a regular two-way ANOVA test with Bonferroni post-testing to compare mean CFU/ml of mutant (Δgdh and ΔgltBD) and wild type strains. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3.
The effect of pH on survival of M. bovis BCG wild type, mutant (Δgdh and ΔgltBD) and complemented strains.
Exponential phase cultures were diluted to an OD600 of 0.0005 into 7H9 of which the pH was adjusted to 4.5 (A & B) or 5.5 (C & D). Symbols and error bars are means and standard errors calculated from triplicate plating obtained from two independent experiments. Data was analysed by a regular two-way ANOVA test with Bonferroni post-testing to compare mean CFU/ml of mutant (Δgdh and ΔgltBD) and wild type strains. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4.
The effect of diethylenetriamine/nitric oxide adduct (DETE/NO) exposure on survival of M. bovis BCG wild type, mutant (Δgdh and ΔgltBD) and complemented strains.
Exponential phase cultures of wild type, Δgdh and Δgdh complement (A) or ΔgltBD and ΔgltBD complement (B) were diluted to an OD600 of 0.0005 into 7H9 (without catalase), which was supplemented with 500 μM of diethylenetriamine/nitric oxide adduct (DETE-NO) (Sigma-Aldrich, USA) and cultured for 48 hours at 37°C without agitation. Symbols and error bars are means and standard errors calculated from three independent experiments. Data was analysed by a regular two-way ANOVA test with Bonferroni post-testing to compare mean CFU/ml of mutant (Δgdh and ΔgltBD) and wild type strains. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5.
Survival of M. bovis BCG wild type, Δgdh mutant and Δgdh complemented strains in macrophages.
RAW 264.7 macrophages (A) or BMDM (B) were infected with M. bovis BCG wild type, Δgdh mutant and Δgdh complemented strains or (C) the ΔgltBD mutant and complemented strains and the survival of the bacteria was determined by measuring CFU obtained from macrophage lysates. Means and standard errors were calculated from three independent experiments. For the BMDM infections a different mouse was used in each experiment. Data was analysed by repeated measures two-way ANOVA with Bonferroni post-testing to compare mean CFU/ml of Δgdh mutant and wild type strains. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 6.
The role(s) of enzymes related to central mycobacterial glutamate metabolism and metabolites in the resistance against acid and nitrosative stress.
Glutamates is an important nitrogen source (i), carbon source (ii) protectant against acidic stress (iii) as well as against nitrosative stress (iv). Metabolites shown in red are responsible for resistance against either nitrosative stress or acidic stress. Enzymes marked with circles have been demonstrated to be required for resistance against cellular stress.