Genome Wide Expression Profiling for Modeof Action Characterization

Transcriptome- and proteome-based expression profiling techniques are applied for the identification of modes of action of novel antibiotics, since expression profiles reflect the bacterial defense mechanisms against antibiotic stress. In order to deduce characteristic expression signatures for antibiotics, comparative global expression analyses need to be performed on the basis of a large collection of diverse expression profiles that represent different cellular stress states. Such so-called reference compendia of expression profiles are suitable for comparison with profiles of novel antibacterial agents with unknown function [75]. Bandow et al. investigated the effects of 30 antibacterial compounds on the B. subtilis pro-teome [17], while Freiberg et al. and Hutter et al. studied the genome-wide transcriptional response of B. subtilis to 14 and 37 antibiotics, respectively [73, 76]. As a proof of principle, the mechanism of action of the novel natural-product-derived pyrimidinone antibiotic BAY 50-2369 was correctly predicted as peptidyltransfer-ase inhibition by proteome analysis. An additional example for successful mode-of-action identification is provided by the novel class of phenylthiazolylurea sulfo-namides, which originate from a lead optimization program on a screening hit from a biochemical target assay. Compendia of proteome and transcriptome profiles revealed that these compounds triggered the increased expression of the direct target phenylalanyl-tRNA synthetase as well as the stringent response [33, 76]. An essential prerequisite for these findings is that antibiotics with closely related modes of action are members of the reference data set. The identification of completely novel mechanisms may remain difficult, but can be facilitated by inclusion of profiles derived from mutants conditionally expressing antibacterial targets. The equivalence of conditional mutant profiles and antibiotics-triggered profiles was demonstrated with a B. subtilis mutant downregulating the peptide deformylase. The proteome profile of this mutant correlated well with the profile of the wild type treated with the deformylase inhibitor actinonin [17].

An example of prediction of a new antibiotic mode of action using conditional mutant profiles is provided by the recently published mechanistic characterization of the natural product antibiotic moiramide B. The study was based on a reference compendium of transcriptome profiles of antibiotic-stressed B. subtilis cells as well as of diverse strains with downregulated essential genes. Moiramide B was predicted to be the first antibiotic targeting the bacterial acetyl-CoA carboxylase (ACC), since it triggered a transcriptional response strongly resembling the one of a mutant downregulating subunits of the ACC [76]. To be sure, such predictions need to be verified by additional biochemical and genetic tests such as performed for moiramide B [72].

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