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

Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant <i>Staphylococcus aureus</i> (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 <i>S. aureus</i> from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated <i>S. aureus</i> and healthcare-associated MRSA strains revealed changes unique to community-associated <i>S. aureus</i> 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 <i>S. aureus</i> 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，推测其中包括逃避免疫系统中人多形核白细胞（polymorphonuclear leukocytes, PMNs 或中性粒细胞neutrophils）的杀伤作用。为更好地理解这一现象，我们针对临床常见CA-MRSA菌株美国300（USA300）被吞噬后引发中性粒细胞快速裂解的机制展开了研究。USA300临床分离株被吞噬后的存活最终会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）一致的形态学变化，但随后会在6小时内快速裂解；而经FAS介导的细胞凋亡或吞噬诱导的细胞死亡的细胞则保持完整。在中性粒细胞被破坏前，吞噬体膜始终保持完整，这表明裂解并非由金黄色葡萄球菌从吞噬体逃逸或成孔毒素（pore-forming toxins）的溶细胞作用所导致。对代表性社区获得性金黄色葡萄球菌及医疗相关性MRSA菌株进行吞噬后的中性粒细胞转录组（transcriptome）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的基因表达变化，例如参与钙稳态（calcium homeostasis）调控的转录本出现上调。综合来看，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成孔毒素直接介导的裂解。我们认为，CA-MRSA菌株诱导中性粒细胞程序性坏死的能力，是其增强毒力的关键组成部分之一。