Supplementary Material for: Rapid Neutrophil Destruction following Phagocytosis of Staphylococcus aureus

Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence.社区获得性耐甲氧西林金黄色葡萄球菌（community-associated methicillin-resistant Staphylococcus aureus, CA-MRSA）增强毒力表型的潜在机制尚未完全阐明，目前推测其包含逃避人类多形核白细胞（polymorphonuclear leukocytes, PMNs或中性粒细胞）杀伤的过程。为深入理解这一现象，我们针对临床主流CA-MRSA菌株USA300被吞噬后引发中性粒细胞快速裂解的分子基础展开了研究。USA300临床分离株经吞噬作用存活后，最终可导致中性粒细胞裂解。摄入USA300的中性粒细胞会出现与细胞凋亡（apoptosis）相符的形态学改变，但随后会在6小时内快速裂解；而经Fas介导的细胞凋亡（FAS-mediated apoptosis）或吞噬诱导的细胞死亡（phagocytosis-induced cell death）过程的中性粒细胞则仍保持完整结构。在中性粒细胞被破坏前，吞噬体膜（phagosome membranes）始终保持完整，这提示裂解并非由金黄色葡萄球菌从吞噬体逃逸或孔道形成毒素（pore-forming toxins）的溶细胞作用所介导。对代表性社区获得性金黄色葡萄球菌及医疗相关性MRSA菌株进行吞噬处理后，对中性粒细胞转录组（transcriptome）的基因芯片分析（Microarray analysis）显示，社区获得性金黄色葡萄球菌菌株存在特异性转录变化，例如参与钙稳态（calcium homeostasis）调控的转录本表达上调。综上，本研究数据表明，USA300被吞噬后引发的中性粒细胞破坏，在一定程度上属于程序性坏死（programmed necrosis），而非金黄色葡萄球菌孔道形成毒素直接介导的细胞裂解。我们提出，CA-MRSA菌株诱导中性粒细胞程序性坏死的能力，是其毒力增强的重要组成部分。