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 DNA 诱饵，这些诱饵源自与编码 aureolysin、ScpAB、SspABC 和 SplA-F 的基因相关的启动子区域。四个诱饵中的三个也绑定了 SarR 和 SarZ，两种情况下的例外是 ScpAB 相关诱饵。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和与 sarA 组合的基因中产生了突变并检查了对蛋白酶生产、高分子量蛋白质积累和生物膜形成的影响。这些研究证实，多个调节位点参与将蛋白酶生产限制到影响所有这些表型的程度，但也证明sarA在这方面起着主导作用。使用sarA突变体无法单独产生单独的蛋白酶以及相互组合，我们还证明增加的 aureolysin 和 ScpA 的产生对于定义 LAC 和 UAMS-1 sarA 的生物膜缺陷表型尤为重要突变体，而单独的 aureolysin 在确定使用成骨细胞和破骨细胞评估的 α 毒素积累减少和总体细胞毒性方面起着关键作用。