STAPHEX II: Dissecting Staphylococcus aureus macrophage exit

鼻腔携带金黄色葡萄球菌是严重和侵袭性感染的主要危险因素。即使在最先进的抗生素治疗之后，临床感染的复发也并不罕见，这表明细菌可能隐藏在细胞内。最近对小鼠实验模型的研究表明，金黄色葡萄球菌持续存在于肝脏或腹膜巨噬细胞的细胞内储存库中，在脓毒症的病理发展中起关键作用。在骨髓炎等慢性感染期间，金黄色葡萄球菌也可能在细胞内存活较长时间。在分子和细胞水平上，金黄色葡萄球菌的传播和宿主细胞的退出或持续还不是很清楚。进入血液后，金黄色葡萄球菌很容易被多种吞噬细胞吞噬，例如巨噬细胞。然而，这种病原体能够在人类巨噬细胞内存活并从内部逃逸。我们以前可以证明，苯酚可溶性调制素(PSM)和人类特有的两组分杀白素LukAB和/或PVL在这一过程中起着至关重要的作用。PSMS可以非依赖于细胞受体的方式裂解细胞，介导金黄色葡萄球菌从吞噬小体逃逸到胞浆中，并在小鼠和人身上发挥作用。相反，LukAB和PVL是高度依赖受体的，仅限于人类，阻碍了在传统小鼠模型中对这些毒素的分析。我们的数据表明，LukAB以一种非常规的方式促进人巨噬细胞逃逸和细胞死亡：尽管NLRP3炎症小体被激活，导致IL-1β的释放，但不涉及下垂或凋亡；相反，MLKL/坏死下垂特征被激活，但不足以导致毒素依赖的细胞死亡和退出。使用新的基因标记的细菌和细胞系，可以监测出口的特征，并可能涉及通过泡吞噬作用直接传播到未受感染的巨噬细胞。与SPP的主要目标非常一致，这里将解决由此产生的三个问题：1)ExitStaph中LukAB诱导的哪些机制与MLKL协同或独立发挥作用？宿主细胞是如何重新编程的？2)LukAB的活性是严格依赖于经典的CD11b结合和/或信号，还是涉及其他细菌和/或宿主因素？3)细胞到细胞的传播是否包括真正的细菌从细胞中退出并释放，以便随后再次感染，或者它更需要结合细胞碎片(泡沫化)摄取细菌？两种不同的细菌转移途径有什么不同？通过结合我们在分子和细胞免疫细胞信号(Weber)和金黄色葡萄球菌生理学和基因工程(Wolz)方面的专业知识，我们将确定导致细胞死亡或金黄色葡萄球菌持续存在的关键决定因素。了解金黄色葡萄球菌逃避细胞内杀伤、存活和逃逸的机制可以为更好地对临床金黄色葡萄球菌表型进行分类和使用新的抗感染治疗方法铺平道路。

Nasal carriage of Staphylococcus aureus is a major risk factor for severe and invasive infections. Clinical infection relapse even after state-of-the-art antibiotic therapy is not uncommon, suggesting that bacteria may hide inside cells. Recent studies in murine experimental models indicate that S. aureus persistence in intracellular reservoirs in macrophages in the liver or the peritoneum is critical for the pathology of sepsis development. During chronic infections such as osteomyelitis, S. aureus may also persist within cells for prolonged time. On a molecular and cellular level S. aureus dissemination and host cell exit or persistence are not well understood. After entering the bloodstream, S. aureus is readily phagocytosed by multiple types of phagocytes, e.g. macrophages. However, the pathogen is able to survive within human macrophages and escape from within. We could previously show that phenol soluble modulins (PSMs) and human-specific two component leukocidins LukAB and/or PVL are crucial for this process. PSMs can lyse cells receptor-independently, mediate the escape of S. aureus from phagosomes into the cytosol and function in mouse and humans. Conversely, LukAB and PVL are highly receptor dependent and restricted to humans, hampering analysis of these toxins in conventional mouse models. Our data indicate that LukAB promotes human macrophage escape and cell death in a non-conventional manner: although the NLRP3 inflammasome is activated resulting in IL-1beta release, pyroptosis or apoptosis are not involved; rather MLKL/necroptosis features are activated but are not sufficient for toxin-dependent cell death and exit. Using novel genetically labeled bacteria and cell lines features of exit can be monitored and may involve direct spread to uninfected macrophages by efferocytosis. In good agreement with the SPP’s key aims three resulting questions will be addressed here: 1) Which mechanisms induced by LukAB in ExitStaph synergize with MLKL or act independently? How is the host cell reprogrammed? 2) Is the activity of LukAB from within strictly dependent on classical CD11b binding and/or signaling or involves other bacterial and/or host factors? 3) Does cell-to-cell spread involve bona fide exit and release of free bacteria from cells for subsequent re-infection or does it rather entail uptake of bacteria in combination with cellular debris (efferocytosis)? What are the differences of the two alternative routes of bacterial transfer? By combining our expertise in the molecular and cellular immune cell signaling (Weber) and S. aureus physiology and genetic engineering (Wolz), we will define the key determinants driving cell death or S. aureus persistence. Understanding the mechanisms employed by S. aureus to evade intracellular killing, survive and escape could pave the way for better classification and treatment of clinical S. aureus phenotypes using novel anti-infective treatment approaches.