A multi-strategy antimicrobial discovery approach reveals new ways to treat Chlamydia
Fig 7
Identification of Chlamydia FabH as a molecular target of c1e.
(A) Volcano plot displaying results from the thermal proteome profiling analysis of HeLa cells infected with CTL2 and treated with various concentrations of c1e for 60 min at 24 hpi. Marked in color are the top 100 hits based on log2 of F-statistic. (B) Heatmap displaying results from the thermal proteome profiling for FabH, the top stabilized bacterial protein. At each temperature, displayed are fold changes of remaining soluble protein in treated vs. untreated samples. (C) Illustration of the branched-chain fatty acid synthesis pathway in CTL2, based on recent literature [52,53,54]. (D, E) Inhibition of chlamydial branched-chain fatty acid biosynthesis by c1e. HeLa cells infected for 22 h with CTL2 were incubated for 1 or 2 h with l-[U-14C]-isoleucine in presence of the indicated compounds (c1e, 1 µM; CER, 50 µM; DOX, 1 µM; DMSO). In (E) cells were incubated for 2 h in presence of the indicated concentrations of c1e. 14C incorporation into lipids is displayed as background-subtracted counts per minute (mean ± SD, n = 3, two-way ANOVA with Dunnett’s multiple comparisons test vs. uninfected control (D) or DMSO control (E)). (F) Enzyme kinetics progress curves of CTL2 FabH (ctFabH, 20 µM) and Escherichia coli FabH (ecFabH, 5 µM) with acetyl-CoA (100 µM) and malonyl-CoA substrates (100 µM). Where indicated, reactions were conducted in the presence of c1e (30 µM) and/or CoA (30 µM) with or without preincubation (pre-inc) for 10 min prior to starting the reaction. (G) Intact protein mass spectrometry of purified acyl-modification-free ctFabH (10 µM) after incubation with the indicated acyl-CoAs or c1e (50 µM), revealing covalent modification of ctFabH by c1e. (H) Proposed mechanism of ctFabH acylation by substrate and mode of inhibition by c1e through covalent bond formation with the active site cysteine. CER, cerulenin; DOX, doxycycline. The data underlying this figure can be found in S9 Data.