SUMMARY

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

 概括

使用 DNA 亲和层析，我们证明金黄色葡萄球菌调节蛋白 MgrA、Rot、SarA 和 SarS 结合源自与编码金黄色素溶血素、ScpAB、SspABC 和 SplA-F 的基因相关的启动子区域的 DNA 诱饵。四种诱饵中的三种也结合了 SarR 和 SarZ，两种情况下的例外是与 ScpAB 相关的诱饵。使用 USA300（甲氧西林抗性菌株 LAC）和 USA200（甲氧西林敏感菌株 UAMS-1），我们在单独编码这些蛋白质以及与sarA组合的每一种蛋白质的基因中产生突变，并检查了对蛋白酶生产、高水平积累的影响。分子量蛋白质和生物膜形成。这些研究证实，多个调控位点参与限制蛋白酶的产生，并在一定程度上影响所有这些表型，但也证明sarA在这方面发挥着主导作用。使用无法单独产生单个蛋白酶和彼此组合的sarA突变体，我们还证明金黄色素溶血素和 ScpA 产量的增加对于定义 LAC 和 UAMS-1 sarA突变体的生物膜缺陷表型特别重要，而金黄色素溶血素单独发挥作用使用成骨细胞和破骨细胞评估，在确定减少α毒素积累和总体细胞毒性方面发挥着关键作用。