Supplementary Material for: agr-Dependent Interactions of Staphylococcus aureus USA300 with Human Polymorphonuclear Neutrophils

The emergence of serious infections due to community-associated methicillin-resistant <i>Staphylococcus aureus </i>(CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of <i>agr</i>-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC <i>agr </i>KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC <i>agr </i>KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs &gt;1, required α-hemolysin <i>(hla)</i> as USA300 (LAC <i>agr</i> KO) and USA300 (LAC <i>hla</i> KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the <i>agr</i> operon in human PMN with the subsequent production of α-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.

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）引发重症感染的现象，引发了学界对特定葡萄球菌毒力因子在临床疾病中致病贡献的广泛关注。为评估agr依赖型因子对多形核中性粒细胞（polymorphonuclear neutrophils, PMN）内病原菌存亡状态的影响，我们以不同感染复数（multiplicity of infection, MOI），分别用野生型CA-MRSA（LAC菌株）与agr敲除型CA-MRSA（LAC agr KO）感染PMN，探究其对病原菌与宿主细胞的影响。被吞噬的病原菌会以时间与感染复数依赖的方式快速上调RNAIII的转录水平；尽管胞外USA300（LAC菌株）具备响应自体诱导肽富集培养基的能力，却并未上调RNAIII的表达。次氯酸（HOCl）介导的细胞损伤与病原菌胞内存活率，在野生型菌株与agr敲除型USA300（LAC agr KO）之间无显著差异。被吞噬的USA300（LAC菌株）引发PMN裂解的效应呈时间与感染复数依赖性；当感染复数大于1时，该过程需要α-溶血素（α-hemolysin, hla）介导：仅在高感染复数条件下，USA300（LAC agr KO）与USA300（LAC hla KO）才可诱导PMN裂解。综合以上实验结果，本研究证实人类PMN吞噬体中agr操纵子（agr operon）的激活，以及后续α-溶血素的产生与PMN裂解过程。人类PMN吞噬体中的这些事件，对USA300（LAC菌株）感染患者发病率与死亡率升高的贡献程度，仍有待进一步研究阐明。