RESUMO
The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Histidina Quinase/genética , Quinonas/farmacologia , Antibacterianos/farmacologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Parede Celular/metabolismo , Histidina Quinase/efeitos dos fármacos , Concentração Inibidora 50 , Mutação , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.
Assuntos
Antibacterianos/farmacologia , Histidina Quinase/antagonistas & inibidores , Histidina Quinase/química , Quinonas/farmacologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Sequência Conservada , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Histidina Quinase/genética , Modelos Estruturais , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , FosforilaçãoRESUMO
Two-component signal transduction systems (TCSs) represent one of the primary means by which bacteria sense and respond to changes in their environment, both intra- and extracellular. The highly conserved WalK (histidine kinase)/WalR (response regulator) TCS is essential for cell wall metabolism of low G+C Gram-positive bacteria and acts as a master regulatory system in controlling and coordinating cell wall metabolism with cell division. Waldiomycin, a WalK inhibitor, has been discovered by screening metabolites from actinomycetes and belongs to the family of angucycline antibiotics. In the present study, we have shown that waldiomycin inhibited autophosphorylation of WalK histidine kinases in vitro from Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans at half-maximal inhibitory concentrations of 10.2, 8.8, 9.2, and 25.8 µM, respectively. Quantitative RT-PCR studies of WalR regulon genes have suggested that waldiomycin repressed the WalK/WalR system in B. subtilis and S. aureus cells. Morphology of waldiomycin-treated S. aureus cells displayed increased aggregation instead of proper cellular dissemination. Furthermore, autolysis profiles of S. aureus cells revealed that waldiomycin-treated cells were highly resistant to Triton X-100- and lysostaphin-induced lysis. These phenotypes are consistent with those of cells starved for the WalK/WalR system, indicating that waldiomycin inhibited the autophosphorylation activity of WalK in cells. We have also confirmed that waldiomycin inhibits WalK autophosphorylation in vivo by actually observing the phosphorylated WalK ratio in cells using Phos-tag SDS-PAGE. The results of our current study strongly suggest that waldiomycin targets WalK histidine kinases and inhibits the WalR regulon genes expression, thereby affecting both cell wall metabolism and cell division.
