Human neutrophils and Staphylcoccus aureus: microbial targets and responses

描述(由申请人提供)： 金黄色葡萄球菌(SA)感染仍然是一个重要的临床挑战，尽管有强大的抗生素。新的治疗进展等待阐明持久性、慢性化和转移性传播的分子基础--即SA感染的特征。金黄色葡萄球菌产生严重和慢性人类疾病的显著倾向部分反映了它表达的一系列毒力因子，包括在其对数生长后期分泌的磷酸肌醇特异性磷脂酶C(PI-PLC)。PI-PLC是在感染过程中分泌的，具有免疫原性，使其成为一个候选的毒力因子。人多形核白细胞(PMN)处于细胞天然免疫介导的宿主抵抗SA感染的第一线，PMN发挥其对吞噬体内SA的所有抗菌作用。在PMN吞噬过程中，随着吞噬细胞NADPH氧化酶的激活，颗粒与新生的吞噬小体融合，并将其内容物释放到吞噬小体中，从而使摄入的微生物暴露在一系列毒剂和氧化剂中，这些毒剂和氧化剂协同杀死吞噬小体中的微生物。然而，尽管存在这种有效的PMN抗菌系统，10%-20%的摄入SA仍然在PMN中存活，但不能复制。为了应对吞噬小体中的PMN毒素，持续存活的SA刺激PMN启动转录程序，决定PMN的命运。携带SA的PMN有两种潜在的命运：它们可能从非典型的凋亡进展到坏死，释放活性的SA，或者可以被巨噬细胞(MF)摄取，从而触发表型反应，促进进一步的炎症。虽然典型的凋亡性PMN摄取MF不会触发促炎级联反应(即非炎性反应)，但我们最近已经证明，摄取MF摄取SA-PMN促进了异常的细胞因子反应。金黄色葡萄球菌在吞噬细胞内的持续存在具有深远的临床后果，因为金黄色葡萄球菌感染的特征是复发、转移和对金黄色葡萄球菌敏感的抗生素失效。在小鼠感染模型中，从脓肿中回收的PMN相关SA可以将感染传播给NA小鼠，证明PMN可以作为一个“特洛伊木马”，偷偷进入巨噬细胞(MF)，然后扩散导致转移性感染。鉴于我们的初步数据，我们建议进行研究，以检验SA PI-PLC作为SA毒力因子的总体假设，因为它具有破坏吞噬细胞固有宿主防御的能力。我们假设PI-PLC可能在两个不同的阶段促进金黄色葡萄球菌感染：(1)在感染早期，在进行有效的细胞宿主防御和生物摄取之前，细胞外PI-PLC将攻击脆弱的底物，包括那些重新招募的吞噬细胞，并破坏它们的正常反应能力(目标1)；和(2)后来，吞噬细胞吞噬后，在吞噬体内表达SA的PI-PLC将改变PMN-MF的相互作用，干扰炎症的正常消退，并促进感染和传播(目标2)。本研究的具体目的包括：1.确定细胞外SA PI-PLC对吞噬细胞功能和固有宿主防御的影响--着重于PMN NADPH氧化酶的启动和直接激动剂对吞噬细胞的作用；2.确定在PMN内表达的PI-PLC在SA感染的启动和促进中的作用--重点关注PMN和MF的体外去向和体内感染的后果。我们已经开发了必要的分析工具和实验模型来研究PI-PLC对吞噬细胞反应的影响，我们正在进行的含有活性SA的PMN与MF之间相互作用的研究为体外模拟携带SA的PMN在感染中可能发生的命运提供了一个框架。

DESCRIPTION (provided by applicant): Infection with Staphylococcus aureus (SA) remains an important clinical challenge despite potent antibiotics. Novel therapeutic advances await elucidation of the molecular bases for persistence, chronicity, and metastatic spread - i.e. the hallmarks of SA infection. The remarkable proclivity of SA to produce severe and chronic human disease reflects, in part, the array of virulence factors it expresses, including a phosphoinositol-specific phospholipase C (PI-PLC) that is secreted during its late logarithmic growth phase. PI-PLC is secreted during infection and is immunogenic, making it a candidate as a virulence factor. Human polymorphonuclear leukocytes (PMN) are at the front-line of cellular innate immune-mediated host defense against SA infection, and PMN exert ~ all of their antimicrobial effort against SA internalized within phagosomes. During PMN phagocytosis and coincident with activation of the phagocyte NADPH oxidase, granules fuse with nascent phagosomes and release their contents into the phagosome, thereby exposing the ingested microbe to an array of toxic agents and a flux of oxidants that collaborate to kill microbes in phagosomes. However, despite the presence of this potent PMN antimicrobial system, 10-20% of ingested SA remain viable, but not replicating, in PMN. In response to PMN toxins in phagosomes, persisting viable SA stimulate PMN to initiate transcriptional programs that dictate the fate of PMN. SA-laden PMN have two potential fates: they may progress from atypical apoptosis to necrosis, with release of viable SA, or can be ingested by macrophages (Mf), thereby triggering phenotypic responses that promote further inflammation. Whereas Mf uptake of typical apoptotic PMN occurs without triggering proinflammatory cascades (i.e. a non phlogistic response), we have recently demonstrated that Mf ingestion of SA-PMN promoted aberrant cytokine responses. Persistence of SA within phagocytes has profound clinical consequences, as signature features of SA infection are relapse, metastases, and failure of antibiotics to which SA are susceptible. In murine infection models, PMN-associated SA recovered from an abscess can transmit infection to na¿ve mice, demonstrating that PMN can serve as a "Trojan horse", covertly entering macrophages (Mf) and then disseminating to cause metastatic infection. Given our preliminary data, we propose studies to test the overall hypothesis that SA PI-PLC serves as a virulence factor for SA by virtue of its ability to undermine phagocyte innate host defense. We hypothesize that PI-PLC may contribute to SA infection at two distinct stages: (1) early in infection, before effective cellular host defenses have been engaged and organisms ingested, extracellular PI-PLC will attack vulnerable substrates, including those on recruited phagocytes, and undermine their capacity to respond normally (Aim 1); and (2) later, after ingestion by phagocytes PI-PLC-expressing SA within phagosomes will alter PMN-Mf interactions, interfere with normal resolution of inflammation, and promote infection and dissemination (Aim 2). Our Specific Aims include: Aim 1. To determine the impact of extracellular SA PI-PLC on phagocyte function and innate host defense - focusing on priming of PMN NADPH oxidase and direct agonist effects phagocytes, and Aim 2. To determine the contribution of PI-PLC expressed within PMN on initiation and promotion of SA infection - focusing on PMN and Mf fate in vitro and consequences in infection in vivo. We have generated the analytical tools and developed experimental models necessary to study the effects of PI-PLC on phagocyte responses, and our ongoing studies of interactions between PMN containing viable SA and Mf provide a framework to model in vitro the fate of SA-laden PMN as might occur in infection.