A novel family of type VI-secreted toxins affecting Rho GTPases, actin dynamics and inflammation

要想成功，病原体必须克服体液和细胞天生的免疫障碍。宿主先天免疫细胞(如巨噬细胞)吞噬并将细菌捕获在被称为吞噬小体的膜结合的小泡中。然而，吞噬病原体会解除巨噬细胞的武装，并通过部署颠覆关键细胞通路的蛋白质(效应器)来对抗免疫。宿主细胞骨架在检测细菌病原体和动员抗菌反应方面的关键作用是感染生物学中一个新兴的主题。事实上，病原体诱导的肌动蛋白网络解体是一种显著的抗宿主策略，巨噬细胞也将其视为驱动炎症和细胞死亡的危险信号。然而，在损害巨噬细胞功能的慢性疾病(例如囊性纤维化、糖尿病、慢性阻塞性肺疾病)的背景下，肌动蛋白网络还没有被探索。我们和其他人发现，囊性纤维化(CF)基因缺陷的巨噬细胞无法清除被吞噬的细菌，并在维持与疾病相关的慢性炎症状态方面发挥核心作用。基于CF模型，我们假设细胞内病原体被巨噬细胞吞噬，结合潜在的慢性缺陷，导致肌动蛋白网络的破坏，从而导致高度促炎状态。我们将使用细胞内机会性病原体Burkholderia cenocepacia来检验这一假设，这种病原体感染全球2%-10%的CF患者，迅速加速他们的临床衰退。与其他CF病原体不同，盲肠伯克霍尔德氏菌不存在于患者肺部的生物膜中，而主要存在于巨噬细胞中。我们建立了伯克霍尔德氏菌作为细胞微生物学的模式生物。具体地说，一种伯克霍尔德氏菌VI型分泌系统(T6SS-BC)扰乱了肌动蛋白网络，并引起巨噬细胞下垂(细胞死亡，伴有夸大的促炎反应)。这些表型依赖于TecA(“影响细胞骨架结构的T6SS效应蛋白”)，这是我们实验室最近发现的一种新的毒素，可以使Rho GTP酶失活。这一里程碑式的发现支持了拟议的研究，该研究结合细胞和分子微生物学方法，研究细胞内的伯克霍尔德氏菌如何操纵巨噬细胞的肌动蛋白网络。我们将解决三个问题：(I)T6SS-BC是如何分泌TecA的？(Ii)吡咯炎症体是如何感知到有缺陷的肌动蛋白聚合的？(Iii)在感染cenocepacia的Burkholderia cenocepacia感染的人外周单核细胞中，派林炎症体的状态是什么？了解机会性病原体如何调节巨噬细胞的肌动蛋白网络来驱动炎症，将有助于发现新的检查点来控制继发于细菌感染的失调炎症，这可能成为开发治疗慢性潜在免疫疾病患者的新疗法的靶点。

To be successful, pathogens must overcome humoral and cellular innate immune barriers. Host innate immune cells (e.g. macrophages) engulf and trap bacteria in membrane-bound vesicles known as phagosomes. Engulfed pathogens, however, disarm macrophages and counteract immunity by deploying proteins (effectors) that subvert key cellular pathways. The host cell cytoskeleton's critical role in both detection of bacterial pathogens and mobilization of antibacterial responses is an emerging theme in infection biology. Indeed, pathogen-induced disorganization of the actin network is a remarkable anti-host strategy, also perceived by macrophages as a danger signal driving inflammation and cell death. However, the actin network in the context of chronic diseases that impair macrophage functions (e.g. cystic fibrosis, diabetes, chronic obstructive pulmonary disease) has not been explored. We and others discovered that cystic fibrosis (CF) gene-defective macrophages fail to clear engulfed bacteria and play a central role in maintaining the chronic inflammation state associated with disease. Based on the CF model, we hypothesize that intracellular pathogens engulfed by macrophages, combined with an underlying chronic defect, induce disorganization of the actin network that leads to a highly proinflammatory state.We will examine this hypothesis using the intracellular opportunistic pathogen Burkholderia cenocepacia, which infects 2-10% of CF patients worldwide, rapidly accelerating their clinical decay. Unlike other CF pathogens, Burkholderia cenocepacia does not reside in biofilms in the lungs of patients but primarily within macrophages. We established Burkholderia cenocepacia as a model organism for cellular microbiology. Specifically, a Burkholderia cenocepacia type VI-secretion system (T6SS-Bc) disrupts the actin network and elicits pyroptosis in macrophages (cell death with exaggerated proinflammatory responses). These phenotypes depend on TecA ("T6SS effector protein affecting cytoskeletal Architecture"), a novel toxin recently discovered in our laboratory that inactivates Rho GTPases. This landmark discovery underpins the proposed study combining cellular and molecular microbiology approaches to investigate how intracellular Burkholderia cenocepacia manipulates the macrophages' actin network. We will address 3 questions:(i)How TecA is secreted by the T6SS-Bc?(ii)How defective actin polymerization is sensed by the Pyrin inflammasome?(iii) What is the status of the Pyrin inflammasome in Burkholderia cenocepacia infected CF gene-defective human peripheral monocytes?Understanding how opportunistic pathogens modulate the macrophages' actin network to drive inflammation will enable the discovery of new checkpoints for controlling dysregulated inflammation secondary to bacterial infection, which could be targeted to develop novel therapies to treat patients with chronic, underlying immune disorders.