Figure 1.
Schematic representation of the glycolytic pathway.
Key committing step of glycolysis is catalyzed by pfkA/pfkB.
Table 1.
Primers used in this study.
Figure 2.
Deletion of pfkA and pfkB genes in M. tuberculosis.
Schematic representation of the genomic regions of (A) pfkA in WT and ΔpfkA mutant, (C) pfkB in WT and ΔpfkB mutant, and location of restriction sites and probes. (B) and (D) are Southern blots confirming the knockout of pfkA and pfkB genes respectively. res: sites of resolvase; hyg: hygromycin resistance cassette.
Table 2.
pfkA encodes a functional phosphofructokinase.
Figure 3.
Western blot analysis of PFKB expression in wild-type M. tuberculosis and mutants.
(A) Detection of PFKB with rabbit-anti-PFKB antibodies. (B) Ponseus-S stained of the membrane showing equal loading of cell-free extracts. Lane 1: WT; 2: ΔpfkB ; 3: pfkB-complemented ΔpfkA; 4: ΔpfkA ; 5: purified His-PFKB as control.
Figure 4.
Phenotypic complementation of ΔpfkAΔpfkB E. coli mutant with mycobacterial pfkA and pfkB.
(i) ΔpfkAΔpfkB E. coli RL257, (ii) RL257 complemented with Mtb pfkA, (iii) RL257 complemented with Mtb pfkB and (iv) RL257 transformed with empty vector were grown on M9 minimal agar supplemented with (A) 0.2% glucose, (B) 0.2% glucose and IPTG and (C) glycerol.
Figure 5.
In vitro growth kinetic of ΔpfkA and ΔpfkB mutants on various carbon sources.
Growth in liquid medium with glucose, glycerol or acetate as sole carbon source (as indicated) was monitored for Wild-type, ΔpfkA, ΔpfkA complemented with pfkA, ΔpfkA complemented with pfkB, and ΔpfkB M. tuberculosis strains (as indicated). Bacterial growth was monitored by OD absorbance at 600 nm over time. Results are representative of at least two independent experiments.
Figure 6.
Infection profile of ΔpfkA mutant in mouse.
8-weeks old female BALB/c mice were nasally infected with the wild-type (black circle) or ΔpfkA (open circle) strains. Four animals per time point per group were used. Bacterial loads in lung (A) and spleen (B) were determined by CFU counts. Data are expressed in Log10 CFU per organ as the mean ± SD of four mice per group.
Figure 7.
Growth kinetic of ΔpfkA mutant under aerobic or hypoxic conditions.
Growth in aerobic (A, B) or hypoxic (Wayne model) (C, D) conditions was monitored over time for wild-type (open circle), ΔpfkA (open square) and complemented ΔpfkA (black triangle) strains as determined by OD600 nm (A, B) or CFU counts (mean ± SD of triplicates) (C, D) in Dubos medium with (A, C) or without (B, D) glucose. Results are representative of at least two independent experiments.
Table 3.
Concentration of intracellular glucose-6-phosphate and fructose-6-phosphate in aerobic M. tuberculosis strains.
Figure 8.
Growth kinetic of M. tuberculosis H37Rv under hypoxia in the presence or absence of glucose.
Growth under hypoxia (Wayne model) of wild-type M. tuberculosis was monitored by determining the number of CFU at various time-point up to Day 60 in the presence (black square) or absence (open square) of glucose. Data are expressed as mean ± SD of triplicates. Results are representative of two independent experiments. Arrow heads mark the start of decolourization of methylene blue and full arrows mark the complete decolourization of methylene blue in culture medium with (black) or without (red) glucose.
Table 4.
Concentration of intracellular glucose-6-phosphate and fructose-6-phosphate in hypoxic non-replicating M. tuberculosis strains.