Attenuation of Staphylococcus aureus virulence by protocatechuic acid ethyl ester through inhibition of alpha-hemolysin secretion

Staphylococcus aureus is a major human pathogen whose secreted toxins are key factors in the pathogenesis of life-threatening conditions such as sepsis and pneumonia. In this study, we evaluated the regulatory effect of protocatechuic acid ethyl ester (EDHB), a naturally occurring small-molecule compound, on the inhibition of hemolytic activity in S. aureus at sub-inhibitory concentrations. The minimum inhibitory concentrations (MICs) of EDHB for S. aureus isolates were determined to be 1024 µg/mL. Hemolysis assays showed that EDHB, at concentrations ranging from 8 to 256 μg/mL (1/128 to 1/4 MIC for Newman and USA300, and 1/64 to 1/4 MIC for JP21), dose-dependently inhibited the hemolytic activity of the commonly used laboratory standard strain Newman, the methicillin-resistant S. aureus (MRSA) strain USA300, and the clinical isolate JP21. Real-time quantitative PCR analyses indicated that EDHB reduced the expression of saeR , a key component of the SaeRS virulence regulatory system, which was associated with the downregulation of hla gene transcription and a consequent decrease in Hla protein levels. The saeR -dependent anti-hemolytic potential of EDHB was validated by constructing knockout and overexpression strains of saeR in S. aureus Newman. Using promoter-reporter assays, we found that EDHB inhibited the activity of both the saeR P1 promoter and the hla promoter. Consistent with the in vitro findings, in vivo studies using a murine skin abscess model showed that EDHB reduced abscess size, decreased local bacterial load, and alleviated tissue inflammation. In summary, this study suggests that EDHB attenuates the virulence of S. aureus by targeting saeR to regulate virulence gene expression. These findings provide experimental evidence supporting EDHB as a potential compound for the development of anti-virulence strategies against S. aureus infections.