Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.

Keywords: CoA; CoaBC; drug discovery; pantetheine; tuberculosis.

Figure 1

Pan and CoA biosynthesis pathways of Mtb. CoaBC bypass occurs via PanK-mediated phosphorylation of PantSH to produce P-PantSH.

Figure 2

ATc dose dependence of growth of cKD mutants of Mtb. Strains were grown to early log phase and equivalent numbers of cells were inoculated onto 7H10 agar containing the indicated concentrations of ATc (ng/mL) and/or Pan (25 μg/mL) and PantS (2.5 mg/mL) and incubated for 9 days. Tet-ON_M, TetR expressed from intermediate-strength promoter; Tet-OFF, reverse TetR expressed from intermediate-strength promoter; Tet-ON_S, TetR expressed from strong promoter.

Figure 3

Effect of transcriptional silencing of panB, panC, coaBC, and coaE on the viability of Mtb in vitro. Tet-ON_M and Tet-OFF cultures of each conditional mutant were grown in the presence and absence of ATc prior to plating serial dilutions of each on 7H10 agar in the presence (200 ng/mL) and absence of ATc. CFUs were scored at the indicated time points, and the results are representative of three independent experiments. The limit of detection was 1 CFU. Error bars represent standard deviation. SCO, promoter replacement mutant generated by single crossover (SCO) homologous recombination; (ATc–/−), incubated in the absence of ATc and plated in the absence of ATc; (AT−/+), incubated in the absence of ATc and plated in the presence of ATc; (ATc+/−), incubated in the presence of ATc and plated in the absence of ATc; (ATc+/+), incubated in the presence of ATc and plated in the presence of ATc.

Figure 4

Mass spectrometric quantitation of CoaBC-specific peptides. Label-free quantitative analysis of six CoaBC-specific peptides in the coaBC Tet-OFF mutant with and without silencing for 3 and 6 days. Strains were grown in the presence of PantS to prevent the emergence of unresponsive repressor mutants and to ensure equivalent growth rates of all strains. All data are representative of three biological replicates and were normalized to SigA peptide abundance. ATc200, anhydrotetracycline (200 ng/mL); PantS, pantethine (2.5 mg/mL).

Figure 5

PantS supplementation temporarily rescues the bactericidal effect of coaBC silencing in Mtb in vitro in a dose-dependent manner. The coaBC Tet-OFF cKD mutant was grown in the presence and absence of ATc prior to plating serial dilutions on 7H10 agar in the presence (200 ng/mL) and absence of ATc, as well as on 7H10 agar containing various concentrations of PantS (mg/mL). CFUs were scored at the indicated time points, and the results are representative of three independent experiments. The limit of detection was 1 CFU. Error bars represent standard deviation. (ATc–/−), incubated in the absence of ATc and plated in the absence of ATc; (ATc−/+), incubated in the absence of ATc and plated in the presence of ATc; (ATc+/−), incubated in the presence of ATc and plated in the absence of ATc; (ATc+/+), incubated in the presence of ATc and plated in the presence of ATc.

Figure 6

Metabolic impact of genetic silencing of pantothenate and CoA biosynthesis enzymes in ATc-regulated cKD mutant strains. Steps in the biosynthetic pathway that were subject to transcriptional silencing are indicated in orange diamonds with black lettering; other steps are shown as pink diamonds with gray lettering. Relative metabolite abundances (based on ion intensities) are indicated in heatmap format below each pathway intermediate, with columns indicating the duration of silencing (1.5 or 3 days) and rows denoting the specific gene silenced. Asterisks indicate metabolites for which levels varied in direction between two independent experiments after 3 days of silencing. Primary data are included in Figures S6 and S7.

Figure 7

CoaBC is required for growth and persistence of Mtb in mouse lungs (a) and spleen (b). Mice were infected with the coaBC Tet-OFF mutant and received food containing doxycycline starting from the day of infection (day 0), at day 8 (during acute infection), at day 35 (during chronic infection), or not at all, as indicated. The limit of detection was 4 CFU in lungs and spleens. The data are representative of four mice per time point; error bars represent standard deviation. Doxy day 8 + PantS, lung homogenate from mice fed with doxy from day 8 post-infection inoculated onto agar containing PantS (2.5 mg/mL); Doxy day 35 + PantS, lung homogenate from mice fed with doxy from day 35 post-infection inoculated onto agar containing PantS (2.5 mg/mL).