The influence of established and novel antibiotics on the activity of the WalRK and the LiaRS two-component systems of Bacillus subtilis

Due to the increasing number of antibiotic-resistant bacteria, the demand for new antibiotics and new antibiotic targets is growing. Two-component systems (TCS) are attractive targets for antimicrobials. They consist of a histidine kinase (HK) that detects a signal and a response regulator (RR) that conveys adaption of gene expression in response to environmental changes. Mainly, HK and RR genes are encoded on the bacterial genome, a few are found in lower eukaryotes. TCS genes were not found in mammalian genomes. Therefore, a TCS is an optimal new target for old and new antibiotics without affecting human cells. WalRK represents the only essential TCS in Gram-positive bacteria like the pathogen Staphylococcus aureus. It controls cell wall metabolism and the expression of autolysins, which are involved in cell wall modeling during cell growth and division. WalRK activates the expression of ssaA and ihnhibits iseA. The upstream sequences of liaI, iseA and ssaA were fused to the luminescence gene cluster of Photorhabdus luminescens and integrated into the B. subtilis genome. With these reporter clones, luminescence assays were performed using a TECAN plate reader in the presence of different antibiotics. The activity of the reporter strains was evaluated and showed typical expression patterns for all three clones in the presence of different antibiotic classes. The expression of ssaA was always downregulated. Lipid II binders enhanced the expression of liaI and iseA. A low activation of liaI and a strong upregulation of iseA were observed with β-lactams. The expression of all three promoters was downregulated in the presence of protein, RNA and DNA biosynthesis inhibitors. Published WalRK inhibitors resulted in a downregulation of iseA and ssaA at concentrations below the minimal inhibitory concentration and an upregulation of liaI at higher concentrations. WalRK is a target for new antibiotics in bacteria. In this study, reporter systems were created in B. subtilis, to test compounds for an inhibitory effect against WalRK. The system showed not the expected upregulation of iseA in the presence of published WalRK inhibitors. The expression pattern was not comparable with the observed profiles in presence of established antibiotics. Therefore, an inhibition of the phosphorylation and dephosphorylation activity of WalK is discussed.