Proteomic Signature of Fatty Acid Biosynthesis Inhibition Available for In Vivo Mechanism-of-Action Studies

Abstract

Fatty acid biosynthesis is a promising novel antibiotic target. Two inhibitors of fatty acid biosynthesis, platencin and platensimycin, were recently discovered and their molecular targets identified. Numerous structure-activity relationship studies for both platencin and platensimycin are currently being undertaken. We established a proteomic signature for fatty acid biosynthesis inhibition in Bacillus subtilis using platencin, platensimycin, cerulenin, and triclosan. The induced proteins, FabHA, FabHB, FabF, FabI, PlsX, and PanB, are enzymes involved in fatty acid biosynthesis and thus linked directly to the target pathway. The proteomic signature can now be used to assess the in vivo mechanisms of action of compounds derived from structureactivity relationship programs, as demonstrated for the platensimycin-inspired chromium bioorganometallic PM47. It will further serve as a reference signature for structurally novel natural and synthetic antimicrobial compounds with unknown mechanisms of action. In summary, we described a proteomic signature in B. subtilis consisting of six upregulated proteins that is diagnostic of fatty acid biosynthesis inhibition and thus can be applied to advance antibacterial drug discovery programs.

Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

“…Protein extracts for 2D-PAGE were prepared as described previously (48). In short, 5 ml of a bacterial culture in early exponential growth phase were exposed to 6 g/ml gallidermin, 30 g/ml mersacidin, 10 g/ml valinomycin, or 1 g/ml gramicidin S or left untreated as the control.…”

“…Bacillus subtilis 168 (trpC2) (1) was grown at 37°C under steady agitation in Belitzky minimal medium (BMM) (42). MICs were determined in a modified MIC test described previously to match the growth conditions of the proteome experiment, specifically using chemically defined medium and supplying sufficient oxygen (48). Briefly, in a test tube, 2 ml of defined medium was inoculated with 5 ϫ 10 5 bacteria per ml and incubated with different lantibiotic concentrations at 37°C under steady agitation for 18 h. The MIC was defined as the lowest concentration inhibiting visible growth.…”

“…The physiological response of B. subtilis manifest in the proteome after antibiotic stress was previously shown to correlate with the antibacterial mechanism (2, 4, 47) and contributed to elucidating the antibacterial mechanisms of novel antibiotic compounds (12,48). Proteomic profiles of B. subtilis treated with several cell wall biosynthesis inhibitors have been reported (4,39,46), but proteomic signatures indicative of specific aspects of cell wall biosynthesis inhibition have not yet been described.…”

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

“…Fifty-five g of protein for analytical and 300 g for preparative gels were loaded onto 24-cm immobilized pH 4 -7 gradient strips (GE Healthcare) by passive rehydration for 18 h. Proteins were separated in a first dimension by isoelectric focusing and in a second dimension by SDS-PAGE using 12.5% acrylamide gels. Analytical gel images were analyzed as described previously (39,40) using Decodon Delta 2D 4.1 image analysis software (Decodon, Greifswald, Germany). Proteins more than 2-fold up-regulated in three independent biological replicates were defined as marker proteins.…”

Perturbation of Staphylococcus aureus Gene Expression by the Enoyl-Acyl Carrier Protein Reductase Inhibitor AFN-1252

“…With antibiotics targeting bacterial fatty acid synthesis, the upregulation of fab genes is a transcriptional signature that is not shared by other antibiotic classes (33,34). In S. aureus, the expression of the fab gene set is controlled by the transcriptional regulator FapR (35), and the upregulation of fab gene expression following the inhibi-tion of fatty acid synthesis is reflected by increased levels of mRNA encoding the Fab enzymes (36). Despite these correlations between patterns of virulence gene expression and specific pathway inhibitors in the laboratory, there is little understanding of whether these experiments translate in vivo.…”