Development of an Antivirulence Drug against Streptococcus mutans : Repression of Biofilm Formation, Acid Tolerance, and Competence by a Histidine Kinase Inhibitor, Walkmycin C

Abstract

Two-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential of Streptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors of S. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK in Bacillus subtilis, inhibited the in vitro autophosphorylation activity of three purified S. mutans HKs, i.e., VicK, CiaH, and LiaS. Although S. mutans does not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 g/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes of S. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs.

Two-Component System VicRK Regulates Functions Associated with Establishment of Streptococcus sanguinis in Biofilms

“…During colonization of tooth surfaces, S. sanguinis interactions with dental pellicle, biofilm extracellular matrix, and/or coadherent microorganisms likely require dynamic cell surface fitting in response to host and microbial stimuli, a process that involves two-component regulatory systems. The TCS VicRK Sm of S. mutans, a competitor of S. sanguinis which promotes cariogenic biofilms, regulates biofilm formation and virulence (12), which raises interest in this TCS (also called WalRK) as a therapeutic target (34). In this study, we showed that VicRK Ss regulates critical functions necessary for S. sanguinis to initiate biofilms, including chain formation, tolerance to oxidative stress, production of hydrogen peroxide, and amounts of eDNA.…”

Inhibition of Streptococcus mutans biofilm formation, extracellular polysaccharide production, and virulence by an oxazole derivative

“…Natural products and derivatives, such as green tea catechins (Hirasawa et al 2006), cranberry constituents , Galla chinensis (Xie et al 2008), citrus lemon oil (Liu et al 2013), mushroom extracts (Yano et al 2010), and several bioactive macromolecules (Shang et al 2014;Lee and Park 2015), have been reported to be effective against the growth and adherence of S. mutans. In addition, small molecules, such as tt-farnesol (Koo et al 2003), walkmycin C (Eguchi et al 2011), and anthraquinones (Coenye et al 2007), have been shown to inhibit the growth of cariogenic bacteria, and even the development of caries, but few of these compounds target S. mutans biofilms. Therefore, there is an urgent need to identify new approaches capable of targeting cariogenic bacteria in the biofilm state.…”

Study on <i>in vivo</i> effects of bacterial histidine kinase inhibitor, Waldiomycin, in <i>Bacillus subtilis</i> and <i>Staphylococcus aureus</i>

“…The cytoplasmic WalK domains (with C-terminal His tag) of Gram-positive B. subtilis, S. aureus, E. faecalis, and S. mutans were expressed and purified as described previously using E. coli BL21 (DE3) containing pETYycG-tru (Okada et al, 2010), pYycGSa (Watanabe et al, 2003), pETEfWalK (Watanabe et al, 2012), and pETSMVicK31-450 (Eguchi et al, 2011), respectively. The WalK cytoplasmic domains were preincubated in a kinase buffer {50 mM Tris-HCl (pH 8.5), 100 mM KCl, 100 mM NH 4 Cl, 5 mM MgCl 2 } at 30°C, as previously reported (Eguchi et al, 2011;Okada et al, 2007Okada et al, , 2010Watanabe et al, 2003). Waldiomycin was then added.…”