Inactivation of fructose-1,6-bisphosphate aldolase prevents optimal co-catabolism of glycolytic and gluconeogenic carbon substrates in Mycobacterium tuberculosis

Abstract

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.

Figure 1. FBA depletion inhibits growth of Mtb in single carbon sources, but not in the presence of both a glycolytic and a gluconeogenic carbon source.

Growth of WT (black squares) and FBA-DUC in the absence (grey circles) and presence (open circles) of 500 ng/ml atc in carbon defined media containing (A) 0.4% glucose (B) 0.4% glycerol (C) 0.1% butyrate (D) 0.2% glucose and 0.2% glycerol (E) 0.2% glucose and 0.1% butyrate and (F) no carbon. Bacteria were cultured in 25 cm² flasks. Data are representative of at least two independent experiments.

Figure 2. FBA is required for replication and persistence of Mtb in mice.

(A) Growth and survival of WT (squares) and FBA-DUC (circles) in mouse lungs (left panel) and spleens (right panel). Mice infected with FBA-DUC received doxy-containing food from the day of infection (day 0), day 10, day 35 or not at all as indicated. CFU were not detected in spleens from mice infected with FBA-DUC and treated with doxy starting day 0. The limit of detection was 4 CFU in lungs and spleens. Data are means ± SD of four mice, except for three data points which derive from 3 or 2 mice due to the appearance of atc/doxy resistant revertants in the lungs (day 57 FBA-DUC+doxy day 10, day 85 FBA-DUC+doxy day 10 and day 112 FBA-DUC+doxy day 35). (B) Gross pathology and H&E staining of lung sections from mice infected with FBA-DUC. Lungs were isolated at day 35 (upper panel) and day 112 (middle and lower panel) from mice not treated and administered doxy starting day 35 post infection. A second short course infection experiment reproduced the phenotype of FBA-DUC in mice not treated or treated with doxy starting on the day of infection.

Figure 3. FBA essentiality is carbon source dependent.

(A) Growth of replacement transformants of Mtb P_smyc-fba-fba::hyg with a plasmid not containing fba and thus resulting in Δfba on agar plates containing the indicated carbon sources. (B) Growth of Δfba in 7H9 base liquid media with identical carbon sources as in the above plate conditions.

Figure 4. FBA is required for growth and survival in single carbon sources.

(A) Growth measured by absorbance of WT (black squares), Δfba (open triangles) and Δfba-comp (grey triangles) in carbon-defined media containing glucose, glycerol and butyrate at the indicated concentrations. Bacteria were cultured in 25 cm² flasks. (B) Survival measured by culturing for CFU on growth-permissive agar plates of the indicated Mtb strains in media with single carbon sources and no carbon addition at different time points post-inoculation. Stippled lines indicate that next data point was below the limit of detection.

Figure 5. FBA is required for replication in macrophages and growth and survival in mouse lungs.

(A) Bacterial loads in resting and IFNγ activated mouse bone marrow derived macrophages (BMDM) infected with carbon-starved WT, Δfba and Δfba-comp. Data are means +/− SD of triplicate wells. *P≤0.05; **P≤0.005 by Student's t-test (B) Growth and survival of WT and Δfba in mouse lungs. Data are means +/− SD from four mice. Limit of detection was 4 CFU and Δfba was not detectable on days 10 and 21.

Figure 6. Metabolic consequences of fba deletion.

Intrabacterial pool sizes of selected metabolites (nmol/mg protein) in the indicated Mtb strains after 24 hours culture on media containing either glucose (Gluc), glycerol (Gly) or a combination of both. nd = not detected. All values are means of measurements from two independent experiments, each performed with triplicate cultures ± SEM. *P≤0.05; **P≤0.005; ***P≤0.0005 by Student's t-test. ALA, alanine; α-KG, α-ketoglutarate; ASP, aspartate; CIT/ISOCIT, citrate/isocitrate; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; FBA, fructose-1,6-bisphosphate aldolase; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; FUM, fumarate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; MAL, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; R5P, ribose 5-phosphate; RU5P, ribulose 5-phosphate, SH7P, sedoheptulose 7-phosphate; SUC, succinate; X5P, xylulose 5-phosphate.

Figure 7. Mtb lacking FBA requires a balanced carbon diet for growth.

Growth of WT and Δfba was in media containing (A) 0.2% glucose with varying concentrations of butyrate and (B) 0.1% butyrate with varying concentrations of glucose. Bacteria were cultured in 96-well plates and absorbance was measured at the indicated time points. Data are means of triplicate cultures +/− SEM and representative of two independent experiments.

Figure 8. Vulnerability of Mtb to FBA depletion depends on the carbon source.

Growth of WT and FBA-DUC without and with indicated amounts of atc in (A) 0.4% glucose or (C) 0.1% butyrate. (B) FBA immunoblot in protein extracts from 0.4% glucose cultures on day 15 after atc addition. (D) FBA immunoblot in protein extracts from 0.1% butyrate cultures on day 11 after atc addition. PrcB served as loading control.