Miller, H. B. | bioRxiv Science

bioRxiv2025

Loratadine decreases virulence of methicillin-resistant Staphylococcus aureus by widespread disruption of hemolysis in both Stk1-dependent and -independent fashions

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that relies on expression of a wide range of virulence factors. Hemolysins are a diverse collection of cytolytic toxins that promote virulence by being excreted and lysing host blood cells. We previously reported on novel anti-virulence compounds that modulate hemolysis in vitro, including a brominated carbazole (compound 8) and the active ingredient in Claritin (loratadine). We analyzed expression and activity of MRSA hemolysins in two hospital-acquired strains. Even with the same staphylococcal cassette chromosome mec (SCCmec) type, the strains displayed unique responses to loratadine where both repression and elevation of hemolysis occurred. These compounds are potentially binding and regulating the master regulatory protein, Stk1, which has known ties to hemolysis in S. aureus. We hypothesized that modulation of hemolysis by these putative Stk1 inhibitors would extend to community-acquired strains of MRSA. Furthermore, we wanted to determine if Stk1 was the only hemolysis regulatory protein being engaged by these compounds. To test this, we examined compound 8 and loratadines effects on two different USA300 strains, in addition to transposon mutants for a range of hemolysis regulatory genes. We observed strain-specific regulation of hemolysis by Stk1. Loratadine continued to downregulate both alpha and beta hemolytic activity in USA300 JE2 in both Stk1-dependent and independent pathways. Additionally, we report that this anti-virulence compound effectively disrupts hemolysis by modulating both agr and vraSR global regulators, contributing to widespread cytotoxin repression. Both alpha hemolysin (hla) and RNAIII/delta hemolysin (hld) mRNA reductions occurred in vitro and in a human cell line model of MRSA infection. Together, these results expand our knowledge of loratadines mode of action as a virulence modulator.

microbiology↗

bioRxiv2024

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 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 a laboratory strain and a clinically relevant hospital-acquired strain of MRSA, both with 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 the laboratory strain, but displayed differential activity in a hospital-acquired strain. 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 is likely strain-dependent.

microbiology↗