Novel anti-virulence compounds disrupt exotoxin expression in MRSA

Hemolysins are lytic exotoxins expressed in most strains of S. aureus, but hemolytic activity varies between strains. We have previously reported several novel anti-virulence compounds that disrupt the S. aureus transcriptome, including hemolysin gene expression. This report delves further into our two lead compounds, loratadine and a structurally related brominated carbazole, and their effects on hemolysin production in methicillin-resistant S. aureus (MRSA). To gain understanding into how these compounds affect hemolysis, we analyzed these exotoxins at the DNA, RNA, and protein level after in vitro treatment. While lysis of red blood cells varied between strains, DNA sequence variation did not account for it. We hypothesized that our compounds would modulate gene expression of multiple hemolysins in two hospital-acquired strains of MRSA, both with staphylococcal cassette chromosome mec (SCCmec) type II. RNA-seq analysis of differential gene expression in untreated and compound-treated cultures revealed hundreds of differentially expressed genes, with a significant enrichment in genes involved in hemolysis. The brominated carbazole and loratadine both displayed the ability to reduce hemolysis in strain 43300 but displayed differential activity in strain USA100. These results corroborate gene expression studies as well as western blots of alpha hemolysin. Together, this work suggests that small molecules may alter exotoxin production in MRSA but that the directionality and/or magnitude of the difference are likely strain dependent. Overall design: Differential gene expression was examined between MRSA cultures either untreated, treated with oxacillin only, treated with compound 8 or loratadine alone, or cotreated with oxacillin and loratadine or compound 8. Each of the six treatments was performed with biological triplicates.