Combatting melioidosis with chemical synthetic lethality

Abstract

Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei, the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis. In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and β-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei. Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens.

Keywords: Burkholderia thailandensis; FolE2; antibiotics; folate biosynthesis; natural products.

Fig. 1.

FolE2 is essential and required for robust growth in the presence of low-dose TMP. (A) Abbreviated one-carbon metabolic pathway focusing on steps catalyzed by FolE, FolE2, FolP, and FolA. The latter two are inhibited by SMX and TMP, respectively. (B) Transcriptional and translational changes of one-carbon metabolite genes in response to low-dose TMP. FolE2 is transcriptionally the most induced gene and translationally the second-most induced protein in the presence of low-dose TMP. (C) Growth curves of wt B. thailandensis vehicle control (blue), wt B. thailandensis with 20 μM TMP (orange), ΔfolE2 vehicle control (green), and ΔfolE2 with 20 μM TMP (red). Bars represent SE from three biological replicates in panels B and C.

Fig. 2.

Chemical synthetic lethality screen with low-dose TMP. (A) High-throughput screen with 1,320 compounds monitoring wt B. thailandensis growth in the absence (orange squares) or presence (blue circles) of low-dose TMP. Hits are those that reduce growth by 2 sigma or more in the presence of TMP but do not do so in its absence. (B) Structures of 10 inhibitors that satisfy hit criteria and validated independently in two assay formats (96-well plates and flask culture) and are commercially available.

Fig. 3.

DHL forms a chemical-synthetic lethal combination with TMP. (A) Growth curves of wt B. thailandensis in the presence of vehicle control (dark blue), low-dose TMP (light blue), DHL (dark green), and low-dose TMP and DHL (light green). Also shown are traces for ΔfolE2 vehicle control (red) and ΔfolE2 with low-dose TMP (light red). (B) Quantification of endpoint OD600 (at 48 h) from panel A. The combination of DHL and low-dose TMP results in a 20-fold lower OD600. (C) Half-maximal inhibitory concentration of DHL against wt B. thailandensis in the presence of 0 (red triangle), 5 (yellow diamond), 10 (green square), and 20 (blue circles) μM TMP. (D) Quantification of IC₅₀ from the data in panel C. Error bars represent SE from three biological replicates in all panels.

Fig. 4.

Validation of FolE2 inhibitors. (A) Reaction carried out by FolE2 (and FolE). (B) Michaelis–Menten curve for FolE2 yields k_cat and K_m of 74 min⁻¹ and 22 μM. (C–E) Inhibition of FolE2 by DHL (C), parthenolide (D), and β-lapachone (E). Shown are inhibition curves at substrate K_m yielding apparent K_i values of 87 ± 21, 21.9 ± 4.0, and 2.9 ± 0.5.

Fig. 5.

X-ray crystal structure of B. pseudomallei FolE2 bound to the covalent inhibitor DHL. (A) Overview of the apo FolE2 homotetramer solved to 2.02 Å. (B) Metal-binding site of the as-purified enzyme. (C) Structure of the DHL-inhibited enzyme in which the metal-coordinating Cys154 is covalently linked to DHL. Locations of possible additional crosslinks are denoted with asterisks. (D) Cys154 reacts with the exo-enone of DHL to yield an inhibited complex, which is observed crystallographically (see also SI Appendix, Figs. S8 and S11).

Fig. 6.

Specificity of chemical-synthetic lethal combinations. TMP/DHL, TMP/BL, and TMP/parthenolide form synthetic lethal combinations that are highly selective against B. thailandensis. Among several commensal strains, only E. coli growth is inhibited. Note that other common antibiotics are not only toxic toward B. thailandensis but also cause significant collateral damage.