Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) represents one of the most serious infectious disease concerns worldwide, with the CDC labeling it a “serious threat” in 2019. The current arsenal of antibiotics works by targeting bacterial growth and survival, which exerts great selective pressure for the development of resistance. The development of novel anti-infectives that inhibit quorum sensing and thus virulence in MRSA has been recurrently proposed as a promising therapeutic approach. In a follow-up of a study examining the MRSA quorum sensing inhibitory activity of extracts of Italian plants used in local traditional medicine, 224C-F2 was reported as a bioactive fraction of a Castanea sativa (European chestnut) leaf extract. The fraction demonstrated high activity in vitro and effective attenuation of MRSA pathogenicity in a mouse model of skin infection. Through further bioassay-guided fractionation using reverse-phase high performance liquid chromatography, a novel hydroperoxy cycloartane triterpenoid, castaneroxy A (1), was isolated. Its structure was established by nuclear magnetic resonance, mass spectrometry and X-ray diffraction analyses. Isomers of 1 were also detected in an adjacent fraction. In a series of assays assessing inhibition of markers of MRSA virulence, 1 exerted activities in the low micromolar range. It inhibited agr::P3 activation (IC50 = 31.72 µM), δ-toxin production (IC50 = 31.72 µM in NRS385), supernatant cytotoxicity to HaCaT human keratinocytes (IC50 = 7.93 µM in NRS385), and rabbit erythrocyte hemolytic activity (IC50 = 7.93 µM in LAC). Compound 1 did not inhibit biofilm production, and at high concentrations it exerted cytotoxicity against human keratinocytes greater than that of 224C-F2. Finally, 1 reduced dermonecrosis in a murine model of MRSA infection. The results establish 1 as a promising antivirulence candidate for development against MRSA.
Highlights
Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), remains a pathogen of great concern in the United States and across the world
In order to determine which fraction was most likely to contain quorum sensing inhibitory compounds against S. aureus, a preliminary screen of the fractions was performed using a panel of four yellow fluorescent protein (YFP) agr::P3 reporter strains representing the four agr subtypes (Kirchdoerfer et al, 2011)
Fraction 224C-F2c was fractionated by C-18 reverse phase high-performance liquid chromatography (HPLC) to produce 43 “preparative fractions,” all given the prefix “preparative fractions” (PFs).” The 43 PFs were screened in the YFP agr::P3 reporter strain panel, which revealed that PF22-28 and 39-42 were the most bioactive (Supplementary Figure S2)
Summary
Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), remains a pathogen of great concern in the United States and across the world. The World Health Organization (WHO) has designated S. aureus, including MRSA, as a leading international pathogen of concern both in the community and in hospitals, and in 2017 it cited S. aureus a high priority pathogen for which new antibiotics are urgently needed (WHO, 2017; WHO, 2020). Both organizations cite antibiotic resistance in S. aureus as one of its most threatening characteristics. The inhibition of virulence has been repeatedly cited and demonstrated in the literature as a promising antiinfective strategy against S. aureus, including MRSA (Salam and Quave, 2018; Tan et al, 2018; Wu et al, 2019)
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