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.溶血素（hemolysin）是多数金黄色葡萄球菌（Staphylococcus aureus，简称S. aureus）菌株表达的溶细胞外毒素，但不同菌株的溶血活性存在差异。我们此前已报道多种可干扰金黄色葡萄球菌转录组（包括溶血素基因表达）的新型抗毒力化合物。本研究进一步聚焦于两款先导化合物——氯雷他定（loratadine）与一种结构相关的溴代咔唑（brominated carbazole），并探讨二者对耐甲氧西林金黄色葡萄球菌（methicillin-resistant S. aureus，简称MRSA）溶血素产生的影响。为阐明此类化合物调控溶血的分子机制，我们在体外处理菌株后，从DNA、RNA及蛋白三个层面对这些外毒素开展了分析。尽管不同菌株的红细胞裂解活性存在差异，但DNA序列变异并非该差异的成因。我们提出假说：此类化合物可调控两株医院获得性耐甲氧西林金黄色葡萄球菌（均携带II型葡萄球菌盒式染色体mec（staphylococcal cassette chromosome mec，简称SCCmec））中多种溶血素的基因表达。对未处理组与化合物处理组的培养物进行RNA测序（RNA-seq）差异基因表达分析后发现，共有数百个差异表达基因，其中与溶血相关的基因显著富集。溴代咔唑与氯雷他定均可降低菌株43300的溶血活性，但二者在菌株USA100中表现出不同的活性模式。上述结果与基因表达分析及α溶血素（alpha hemolysin）的蛋白质免疫印迹实验结果相符。综上，本研究表明小分子化合物可改变耐甲氧西林金黄色葡萄球菌的外毒素产生，但这种差异的方向和/或程度大概率因菌株而异。 实验设计：本研究对六组耐甲氧西林金黄色葡萄球菌培养物进行差异基因表达分析，分别为未处理组、仅用苯唑西林（oxacillin）处理组、单独使用化合物8或氯雷他定处理组，以及苯唑西林与氯雷他定或化合物8联合处理组。每组处理均设置3次生物学重复。