Abstract
Antimicrobial drug discovery against drug-resistant bacteria is an urgent need. Beyond agents with direct antibacterial activity, anti-virulent molecules may also be viable compounds to defend against bacterial pathogenesis. Using a high throughput screen (HTS) that utilized Caenorhabditis elegans infected with methicillin-resistant Staphylococcus aureus (MRSA) strain of MW2, we identified 4-(1,3-dimethyl-2,3-dihydro-1H-benzimidazol-2-yl)phenol (BIP). Interestingly, BIP had no in vitro inhibition activity against MW2, at least up to 64 μg/ml. The lack of direct antimicrobial activity suggests that BIP could inhibit bacterial virulence factors. To explore the possible anti-virulence effect of the identified molecule, we first performed real-time PCR to examine changes in virulence expression. BIP was highly active against MRSA virulence factors at sub-lethal concentrations and down-regulated virulence regulator genes, such as agrA and codY. However, the benzimidazole derivatives omeprazole and pantoprazole did not down-regulate virulence genes significantly, compared to BIP. Moreover, the BIP-pretreated MW2 cells were more vulnerable to macrophage-mediated killing, as confirmed by intracellular killing and live/dead staining assays, and less efficient in establishing a lethal infection in the invertebrate host Galleria mellonella (p = 0.0131). We tested the cytotoxicity of BIP against human red blood cells (RBCs), and it did not cause hemolysis at the highest concentration tested (64 μg/ml). Taken together, our findings outline the potential anti-virulence activity of BIP that was identified through a C. elegans-based, whole animal based, screen.
Highlights
Fatal infections from antibiotic-resistant bacteria are predicted to rise and expected to exceed deaths caused by cancer by the year 2050 (de Kraker et al, 2016), putting forth a critical need for novel antibiotics (Zaman et al, 2017)
We report a molecule 4-(1,3-dimethyl-2,3-dihydro-1H-benzimidazol-2-yl)phenol (BIP) (Figure 1) that produced an average Z-score of 9.61 and rescued the nematode C. elegans from MW2 infection
Growth curves were generated using S. aureus cells. (A) After BIP treatment, MW2 cells continued to grow, at a slower rate than cells exposed to DMSO. (B) After BIP was removed by washing, the MW2 cells exhibited normal growth comparable to DMSO pre-treated cells
Summary
Fatal infections from antibiotic-resistant bacteria are predicted to rise and expected to exceed deaths caused by cancer by the year 2050 (de Kraker et al, 2016), putting forth a critical need for novel antibiotics (Zaman et al, 2017). The multiplicity of infection (MOI) of 25 (i.e., 25 bacterial cells per macrophage) was used for the assay in which BIP-pretreated MW2, untreated MW2 alone, or agr mutant S. aureus RN4220 cells were added to macrophage cultures for 2 h to interrogate phagocytosis.
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