C3-Dependent Intracellular Killing in Innate Immunity and Bacterial Pathogenesis

总结 细菌感染仍然是世界范围内发病率和死亡率的主要原因，也是一个关键的公共卫生问题 由于抗生素耐药性增加和疫苗有限。许多最严重的细菌感染 起源于粘膜表面，如肠道、呼吸道或皮肤，然后通过粘膜传播到其他组织。 血流引起粘膜和侵袭性疾病的两种主要人类病原体是革兰氏- 阴性肠道沙门氏菌（例如，血清型鼠伤寒沙门氏菌，STm）和革兰氏阳性A组链球菌 （GAS）。补体系统的功能是先天性宿主抵抗血流感染的重要手段 蛋白质及其激活级联反应，特别是C3的调理作用，再加上蛋白质的杀菌活性 吞噬细胞包括巨噬细胞（MΦ）和嗜中性粒细胞。STm和GAS的致病菌株可以破坏 吞噬溶酶体的功能是在离体和体内的MΦ细胞内存活，因此自噬系统 成为病原体生存/杀灭的关键战场。该项目汇集了两个高度 具有互补专业知识的经验丰富且富有成效的医生-科学家研究者：革兰氏阴性 细菌发病机理、粘膜免疫和无菌小鼠模型（MPI，M. Raffatellu）偶联至革兰氏- 阳性细菌发病机制、先天免疫和细菌-吞噬细胞相互作用（MPI，V. Nizet）。一起 我们最近发现了C3的一种新的、重要的细胞内功能：将细菌靶向于自噬， MΦ中的杀伤系统--一个可能挑战无数MΦ细菌研究结论的发现 在不存在活性血清的情况下进行的相互作用。此外，我们已经发现STm丝氨酸蛋白酶 PgtE、GAS丝氨酸蛋白酶SpyCEP和GAS半胱氨酸蛋白酶SpeB允许相应的病原体 并在MΦ细胞内复制。我们的中心假设是细胞内C3依赖性 自噬对宿主的先天防御至关重要，并且侵袭性病原体如STm和GAS的能力， 抵消这一过程大大增加了他们的致病潜力。最近的数据也表明， 微生物组在皮肤和粘膜补体产生中起着重要作用，这可能代表另一个 肠道微生物影响侵袭性细菌感染风险的关键因素。在此，我们建议继续 研究细胞内C3在感染过程中的作用，了解这一新原理的分支， 先天免疫对宿主-病原体相互作用的影响以及两种最重要的人类感染性疾病的结果 疾病我们的方法很可能揭示新的毒力基因和宿主免疫途径， 机制，解决长期存在的知识差距，并导致具有广泛相关性的新的调查途径 涉及细菌发病机制，包括潜在的新治疗靶点和先导物。

SUMMARY Bacterial infections remain a leading cause of morbidity and mortality worldwide and a critical public health issue due to increasing antibiotic resistance and limited vaccines. Many of the most consequential bacterial infections originate at mucosal surfaces, such as the gut, respiratory tract or skin, then disseminate to other tissues via the bloodstream. Two preeminent human pathogens causing both mucosal and invasive diseases are Gram- negative Salmonella enterica (e.g., serovar Typhimurium, STm) and Gram-positive group A Streptococcus (GAS). Pivotal to innate host defense against bloodstream infection is the function of complement system proteins and their activation cascades, especially opsonization by C3, coupled with the bactericidal activity of phagocytic cells including macrophages (MΦ) and neutrophils. Pathogenic strains of STm and GAS can subvert phagolysosome function to survive intracellularly in MΦ ex vivo and in vivo, whereupon the autophagy system emerges as a critical battleground for pathogen survival/killing. This project brings together two highly experienced and productive physician-scientist investigators with complementary expertise: Gram-negative bacterial pathogenesis, mucosal immunity and gnotobiotic mouse models (MPI, M. Raffatellu) coupled to Gram- positive bacterial pathogenesis, innate immunity, and bacterial-phagocyte interactions (MPI, V. Nizet). Together we have recently discovered a novel, essential intracellular function of C3: targeting of bacteria to the autophagy system for killing in MΦ – a discovery that may challenge conclusions of countless studies of MΦ-bacterial interactions performed in the absence of active serum. Further, we have discovered that the STm serine protease PgtE, the GAS serine protease SpyCEP, and the GAS cysteine protease SpeB allow the respective pathogens to inactivate C3 and to replicate intracellularly in MΦ. Our central hypothesis is that intracellular C3-dependent autophagy is critical to host innate defense, and that the ability of invasive pathogens such as STm and GAS to counteract this process substantially increases their disease-causing potential. Recent data also indicate the microbiome plays an essential role in skin and mucosal complement production, which may represent another crucial factor by which commensal microbes affect invasive bacterial infection risk. Here we propose to continue to investigate the role of intracellular C3 during infection, to understand the ramifications of this new principle of innate immunity on host-pathogen interactions and the outcome of two of the most important human infectious diseases. Our approaches are likely to reveal new virulence genes and host immune pathways that will connect mechanisms, resolve longstanding knowledge gaps, and lead to new avenues of investigation of broad relevance to bacterial pathogenesis including potential novel therapeutic targets and leads.