Identification of Chlamydia FabH as molecular target of c1e.

The data underlying Figs 7 and S14–S15. (A) Results from two-dimensional thermal proteome profiling. logfc columns display the log2 fold-changes in intensity compared to the untreated control at each temperature. Intensity columns display the log2 signal intensity from the mass spectrometer. Data are displayed in Fig 7A and 7B. (B) Statistical analysis of two-dimensional thermal proteome profiling data using the TPP2D package. Abundance/stability score was calculated as the square root of the difference of residual sum squares of the null model (RSS0) and the alternative model (RSS1) and reflects how much more variance is explained by a dose–response model compared to a linear model. log2_Fstat is the log2(F-statistic+1) calculated from the TPP2D package. Data are displayed in Fig 7A. (C) Increase in the concentrations of c1e used during parallel passaging of three CTL2 strains evolving resistance. Data are displayed in S14A Fig. (D) Growth of the three evolved CTL2 strains in HeLa cells treated with the indicated concentrations of c1e, as measured by inclusion area. Data are displayed in S14B Fig. (E) Expression levels of fabH in the three evolved CTL2 strains during infection of HeLa cells, as measured at 46 hpi by qRT-PCR. Data are displayed in S14D Fig. (F) Results from the mass spectrometric comparison of the proteomes of wild-type CTL2 and the c1e-resistant mutant 3. The analysis included four samples: WT_DMSO (infected with wild-type CTL2, treated with solvent-only during last hour), WT_c1e (infected with wild-type CTL2, treated with 0.1 µM c1e during last hour), R3_DMSO (infected with resistant CTL2 mutant 3, treated with solvent-only during last hour), R3_c1e (infected with resistant CTL2 mutant 3, treated with 0.1 µM c1e during last hour), each in triplicate. Statistical significance was determined using limma as described in the methods. Data are displayed in S14E Fig. (G) Inhibition of chlamydial branched chain fatty acid biosynthesis by c1e and CER but not DOX. Data are displayed in Fig 7D. (H) Inhibition of chlamydial branched chain fatty acid biosynthesis by different concentrations of c1e. Data are displayed in Fig 7E. (I) Enzyme kinetics progress curves of CTL2 FabH (ctFabH) and Escherichia coli FabH (ecFabH) with acetyl-CoA and malonyl-CoA substrates. Data are displayed in Fig 7F. (J) Intact protein mass spectrometry of recombinant ctFabH after purification from E. coli, demonstrating a large proportion of the protein to be modified by acyl groups, primarily pentanoyl groups. Data are displayed in S15A Fig. (K) Intact protein mass spectrometry of purified acyl-modification-free ctFabH after incubation with the indicated acyl-CoAs or c1e, revealing covalent modification of ctFabH by c1e. Data are displayed in Fig 7G. (L) Intact protein mass spectrometry of purified acyl-modification-free ctFabH after sequential 10-min incubations with isovaleryl-CoA (IV-CoA) and c1e in the indicated (>) order. Data are displayed in S15B Fig. (M) Single nucleotide polymorphisms in the genomes of the three resistant CTL2 mutants. (N) Insertions and deletions in the genomes of the three resistant CTL2 mutants. (XLSX)