Resistant Bacteria – RUNOr better – WalKThe influence of established and novel antibiotics on the activity of the WalRK and the LiaRS two-component systems of Bacillus subtilis

Introduction: Bacterial two-component systems (TCS) are not present in mammalian cells and, therefore, represent attractive targets for new antimicrobials (Gotoh et al., 2010,J Antibiot (Tokyo) 63 (3): 127–134). WalRK (YycG) is the only essential TCS in Gram-positive bacteria with a low G+C content, e.g. Bacillus subtilis and Staphylococcus aureus (Dubrac et al., 2008). WalRK influences the maturation and turnover of the peptidoglycan as well as the separation of daughter cells. Therefore, the WalRK regulon comprises mainly genes that are involved in cell wall lysis (Dubrac et al., 2008,Mol Microbiol 70 (6): 1307–1322). Phosphorylated WalR activates transcription of several autolysins, e.g. SsaA in S. aureus (Dubrac et al., 2008,Mol Microbiol 70 (6): 1307–1322), and inhibits transcription of the autolysin inhibitor IseA in B. subtilis (Dobihal et al., 2022,J Bacteriol 204 (2): e0053321). However, the activity of WalRK is also influenced by the Ser/Thr kinase PrkC, which phosphorylates the amino acid Thr101 of WalR (Libby et al., 2015,PLoS Genet 11 (6): e1005275).Method: Promoter sequences of iseA and ssaA were fused to the luminescence gene cluster of Photorhabdus luminescens and integrated into the B. subtilis genome. A liaI reporter (Kobras et al., 2017,Methods Mol Biol 1520: 121–131) was included in the experiments to detect inhibition of cell wall biosynthesis. These reporter strains were validated with known antibiotics and natural products. Additionally, ΔprkC knockout mutants of the reporter strains were constructed. In vitro assays were performed with the purified WalRK of S. aureus.Results: The activity of the reporter strains showed typical expression patterns for all three strains. Lipid II binders enhanced the expression of liaI and iseA and β-lactam antibiotics led to an upregulation of iseA, indicating that the activity of WalRK is inhibited in the presence of cell wall biosynthesis inhibitors. The expression of all three promoters was downregulated in the presence of protein, RNA and DNA biosynthesis inhibitors, indicating that the signal is specific for cell wall biosynthesis or WalK inhibitors. An additional knockout of prkC in the reporter strains resulted in differences in the expression of the promoters in presence of glycopeptide antibiotics.Summary: In this study, reporter strains for screening of WalRK inhibitors were created. Validation with established antibiotics showed expression patterns dependent on the antibiotic group and the presence of PrkC.