Characterization of H-box region mutants of WalK inert to the action of waldiomycin in <i>Bacillus subtilis</i>

Abstract

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 (ICs) 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.

Synthesis and biochemical characterization of naphthoquinone derivatives targeting bacterial histidine kinases

“…In contrast, the addition of 2b, 2h, or 2l increased the MS signal of the collected waldiomycin by about twofold compared to the signal in the presence of only waldiomycin (Fig. 6, columns 5,6,9,10,11,12). These results suggest that the binding of 2b, 2h, or 2l to WalK is not competitive with that of waldiomycin, but rather enhances the binding of waldiomycin.…”

“…The naphthoquinone moiety of waldiomycin is believed to bind to the H-box region of the intracellular dimerization domain of HK (Fig. 2b) [8,9]. This region is present in bacteria but not in mammalian cells, including humans [10].…”

Strainy: phasing and assembly of strain haplotypes from long-read metagenome sequencing

“…For example, mutations in MAG 71 ( Firmicutes ) genes involved in cell wall synthesis are consistent with developing resistance via limiting drug uptake. The mutations in WalK contribute to the development of vancomycin resistance in Staphylococcus aureus (Zhu et al 2021), as well as waldiomycin resistance in Bacillus subtilis (Kato et al 2017). MurG encodes an enzyme involved in the biosynthesis of peptidoglycan, a crucial component of bacterial cell walls.…”

Recent Advances in Histidine Kinase-Targeted Antimicrobial Agents

“…A series of experiments showed that waldiomycin inhibited HK in an ATP-non-competitive manner, suggesting that the binding site of waldiomycin may not be located in the ATP active site in the CA domain. Additionally, more detailed experiments, including nuclear magnetic resonance (NMR) spectroscopy and protein amino acid mutation, indicated that waldiomycin may bind to the H-box region of the DHp domain, which contains the conserved His residue, to inhibit the class-I HKs and suppress the autophosphorylations ( Eguchi et al, 2017 ; Kato et al, 2017 ). The broad-spectrum HK inhibition activities of waldiomycin showed that the H-box could be a promising target for the design of novel broad-spectrum HK inhibitors especially those HKs with the WalK-type H-box region.…”

“…Another important direction for the discovery of new HK inhibitors is to design inhibitors by targeting the DHp domain, which is formed by two long helices (α1 and α2), where α1 contains a phosphorylatable conserved His residue (His391, as shown in Figure 3 ) ( Casino et al, 2014 ). This domain contains His391 is called H-box (shown in Figure 1 , the position of the histidine phosphorylation site is marked with an “H” indicating the H-box region), which is the site of autophosphorylation ( Wang et al, 2013 ; Kato et al, 2017 ). This structural homology of HKs indicates the possibility that multiple TCSs can be inhibited simultaneously by one inhibitor, and decrease the appearance of drug-resistant strains.…”