Loughran Allister J, Gaddy Dana, Beenken Karen E, Meeker Daniel G, Morello Roy, Zhao Haibo, Byrum Stephanie D, Tackett Alan J, Cassat James E, Smeltzer Mark S
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Infect Immun. 2016 Aug 19;84(9):2586-94. doi: 10.1128/IAI.00152-16. Print 2016 Sep.
We used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureus clinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarA mutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarA mutants. These results confirm that sarA is required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureus osteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarA mutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psmα mutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.
我们使用急性创伤后骨髓炎的小鼠模型来评估两种不同的金黄色葡萄球菌临床分离株（USA300菌株LAC和USA200菌株UAMS-1）及其同基因sarA突变体的毒力。结果证实，在骨髓炎急性期，这两种菌株引起的骨溶解程度和反应性新骨形成程度相当。来自两种菌株稳定期培养物的条件培养基（CM）对已建立的细胞系（MC3TC-E1和RAW 264.7细胞）、原代小鼠颅骨成骨细胞和骨髓来源的破骨细胞具有细胞毒性。在同基因sarA突变体中，CM的细胞毒性和骨中的反应性变化均显著降低。这些结果证实，sarA对于限制成骨细胞和破骨细胞活力、从而在金黄色葡萄球菌骨髓炎急性期促进骨破坏和反应性骨形成的细胞外毒力因子的产生和/或积累是必需的。蛋白质组学分析证实了LAC和UAMS-1 sarA突变体中多种细胞外蛋白的积累减少。其中包括酚溶性调节素（PSM）的α类，其先前被认为是骨髓炎中成骨细胞细胞毒性以及骨破坏和修复过程的重要决定因素。相应操纵子的突变降低了来自UAMS-1和LAC培养物的CM对成骨细胞和破骨细胞的细胞毒性。它还显著降低了来自LAC培养物的CM引起的反应性骨形成和皮质骨破坏。然而，对于来自UAMS-1 psmα突变体培养物的CM并非如此，从而表明在这类菌株中仍有待鉴定的其他毒力因子的参与。
