Inhibition of Gene Expression of S. aureus Virulence Factors by CCG-203592
The chemical series of compounds represented by CCG-203592 was shown to inhibit gene expression in GAS [20]. As a result, we hypothesized that CCG-203592 might also inhibit gene expression in S. aureus. A number of S. aureus RN6390 genes were selected that were reported to play roles in S. aureus virulence and biofilm formation (Table 1). Among the 15 genes tested, AgrA and ebps demonstrated no significant changes of gene expression when treated with 50 mM CCG-203592 (Figure 6).Figure 3. The effect of 50 mM CCG-203592 on S. aureus RN1, NRS234 and NRS235 biofilm formation. Biofilm formation was determined by OD595 nm reading of crystal violet stain solubilized by ethanol with DMSO treatment as controls. The data is presented as mean6standard error of means for a total of 9 samples (pooled from 3 independent experiments in triplicate). ** p,0.01.
Figure 4. The effect of CCG-203592 on S. aureus biofilm formation on silicone wafer. A) RN6390 biofilm formation on medical grade silicone wafer at different concentrations as determined by OD595 nm reading of crystal violet stain solubilized by ethanol. The data is presented as mean6standard error of means for a total of 9 samples (pooled from 3 independent experiments in triplicate). * p,0.05, ** p,0.01. B) Scan electron microscopy representative images of RN6390 biofilm formation on silicone wafer treated with different concentrations of CCG-203592 from triplicate. Figure 5. The effect of CCG-203592 on S. aureus and mammalian cell viability. A) Growth curves for RN6390 in the presence of CCG-203592 (50 mM) (grey curve) or DMSO alone (dark curve) as determined by OD600 nm. The data is presented as mean6standard error of means for a total of 9 samples (pooled from 3 independent experiments in triplicate). B) HeLa cell viability (as determined by mitochondrial reduction of MTT substrate) in the presence of CCG-203592 at different concentrations normalized to the value for DMSO treated samples which was defined as 100%. The data is presented as mean6standard error of means for a total of 12 samples (pooled from 3 independent experiments in quadruplicate).
Table 1. Virulence factor genes tested by Real time RT-PCR.
Gene 16S rRNA icaA dltD atlA Psma operon SPA lrgA sdrD sspB SigB AgrA RNAIII CodY ebpS cidA HlaFunction Internal standard gene Polysaccharide intercellular adhesion/ polymeric N-acetyglucosamine production Figure 6. The effect of CCG-203592 on expression of selected S. aureus genes. Real time RT-PCRs were performed at mid-logarithmic growth phase (ML), late logarithmic growth phase (LL) and stationary (S) phase. The values are presented as the fold of change of gene transcriptional level of samples treated with 50 mM CCG-203592 versus that of samples treated with DMSO as calculated by 2(2DD Ct) method. The data is presented as mean6standard error of means for a total of 9 samples (pooled from 3 independent experiments in triplicate). * p,0.05, ** p,0.01. Other genes demonstrated significant changes of gene expression by CCG-203592 (Figure 6). Expression of icaA gene of the icaADBC operon that is responsible for polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) production [30] was increased by 88.3630.2% (p,0.02) at midlogarithmic (ML) growth phase and decreased by 40.2613% (p,0.05) at stationary (S) phase compared to control by treatment with 50 mM CCG-203592. Expression of dltD gene of the dlt operon that is involved in D-alanine incorporation into teichoic acids [31] was decreased by 20.664.5% (p,0.01) at S phase. Bifunctional autolysin atl gene expression [32] was reduced by 51.765.6% (p,0.01) at S phase. Expression of psma operon which produces phenol soluble modulins a (PSMa1?) peptides [33] was decreased by 71.663.8% (p,0.01) at S phase. Immunoglobulin G-binding protein A (SPA) gene expression was decreased by 68.166.1% (p,0.01) at ML phase, 56.267.2% (p,0.01) at late logarithmic (LL) growth phase and decreased by 50.666.2% (p,0.01) at S phase. Murein hydrolase regulator lrgA gene expression was decreased by 66.765.1% (p,0.01) at LL phase and decreased by 85.662.3% (p,0.01) at S phase. SD-repeatcontaining protein D (sdrD) gene expression was reduced by 62.667.2% (p,0.01) at S phase. Cysteine protease (sspB) gene expression was decreased by 37.469.1% (p,0.01) at S phase. Sigma B (SigB) gene expression was decreased by 73.565.1% (p,0.01) at S phase. RNAIII gene expression was reduced by 20.164.5% (p,0.01) at S phase. Gene repressor CodY expression was reduced by 32.7610.9% (p,0.05) at S phase. The murein hydrolysin regulator cidA gene expression was increased by 65.1627.2% (p,0.05) at ML phase and 294.0658.7% (p,0.01) at LL phase. The alpha-toxin (Hla) gene expression was decreased by 37.163.8% (p,0.01) at LL phase.
Discussion
In recent years, antibiotic resistance has become one of the biggest threats to public health. Conventional antibiotics aim to kill or inhibit the growth of bacteria, leading to a strong selective advantage for resistant pathogens. As a result, a new approach to developing antimicrobial agents has been proposed that entails targeting virulence of the pathogens without inhibiting their growth, thereby reducing or slowing the selection for resistance [17?0,34]. In our previous studies, we identified a novel chemical series of low molecular weight compounds that can inhibit expression of group A streptococcus virulence gene expression, leading to in vivo efficacy at protecting mice against GAS infection [20]. These compounds demonstrated little interference with GAS growth following the new approach above to develop novel antimicrobial agents [17?9,34]. In order to further improve the potency and pharmacokinetic properties of this class of anti-virulence compounds, we have been carrying out Structure Activity Relationship (SAR) studies by synthesizing and characterizing more compounds in this chemical series (manuscript in preparation). In an effort to test whether these anti-virulence compounds have broad spectrum efficacy against other gram positive pathogens, we tested their effects on S. aureus biofilm formation. A total of 68 compounds (those that were active against GAS SK expression) from the SAR program were tested for effects on biofilm formation of S. aureus Newman strain. Two of the compounds, CCG-203592 and CCG-205363, demonstrated consistent inhibition of biofilm formation. These two compounds were further tested for their potency at inhibiting biofilm formation using the widely studied biofilm strain, RN6390.
