Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties

Wang et al. 10.1073/pnas.0700746104.

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Fig. 4. FabF was expressed in S. aureus strain RN4220 from the plasmid pTet15 under control of a tetracycline-inducible promoter. Under inducing conditions with 45 ng/ml anhydrotetracycline, the pTet15-FabF strain seeded in chloramphenicol-LB/agar showed resistance to platencin compared to the control strain as measured by zone of inhibition sizes. Under noninducing conditions pTet15-FabF showed similar sensitivity to platencin as the control strain.

SI Text

Overexpression of Target Proteins in S. aureus. The details of FabF overexpression assay was described in ref. 1. Briefly, S. aureus fabF was cloned from strain RN4220 and placed in the vector pTet15 under the control of a tetracycline-inducible promoter. S. aureus cells carrying either pTet15-FabF or pTet15 alone were seeded in chloramphenicol-LB/agar plates with or without 45 ng/ml of the inducer anhydrotetracycline. Varying concentrations of platencin in DMSO were applied to wells in each plate. Plates were then incubated for 18 h at 37°C and images were captured. In the absence of the inducer, the pTet15-FabF strain was as sensitive to platencin as the control strain as measured by zone of inhibition sizes. In the presence of the inducer, however, the pTet15-FabF strain showed resistance to platencin as measured by smaller or nonexistent zones of inhibition (SI Fig. 4). We have constructed a number of S. aureus strains over-expressing FabH protein using pTet10 or pTet15 with or without a Flag-tag and tested under various induction conditions. All strains over-expressing FabH protein showed a slight increase in sensitivity to platencin, platensimycin, thiolactomycin, cerulenin, and kanamycin but no increased sensitivity to cycloserine or triclosan. To our knowledge, this is the first report describing FabH over-expression in S. aureus and its phenotype. Tsay et al. reported that overproduction of FabH in E. coli did not impart thiolactomycin resistance (2), which is similar to our observation. However, the reason that FabH over-expression enhances sensitivity to selective antibiotics and does not show resistance to the FabH inhibitor is not understood and remains under further investigation.

1. Wang J, Soisson SM, Young K, Shoop W, Kodali S, Galgoci A, Painter R, Parthasarathy G, Tang YS, Cummings R, et al. (2006) Nature 441:358-361.

2. Tsay JT, Rock CO, Jackowski S (1992) J Bacteriol 174:508-513.