Deciphering microbial virulence mechanisms during Legionella pneumophila infection

嗜肺军团菌是一种可能危及生命的肺炎的病原体，称为军团病。一旦被人类吸入，军团菌进入肺部，在那里它可以感染肺泡巨噬细胞，专门的免疫细胞内复制。军团菌不被巨噬细胞降解，而是利用感染的细胞进行细胞内复制循环。如果不及时治疗，这种呼吸道感染的死亡率高达30%。在过去的15年里，美国的军团病病例数量增加了四倍，使军团菌成为一个重大的健康威胁和相当大的经济负担。 我们致力于研究军团菌如何绕过我们的免疫系统并引起疾病，以便我们能够开发更好的方法来对抗其毒力策略。 人类经常暴露于军团菌，因为军团菌普遍存在于淡水栖息地，如冷却塔，水箱，淋浴喷头或喷泉。军团病的大规模爆发发生在污染水源的水被雾化并随后被人类吸入时。免疫功能低下的个体、婴儿或老年人感染的风险较高。 像许多其他微生物病原体一样，军团菌已经开发出各种策略来利用它们的人类宿主并引起疾病。他们使用一种称为IV型分泌系统（T4SS）的专门蛋白质易位机器将大量蛋白质（所谓的效应物）注入受感染的宿主细胞。效应子调节宿主内的信号事件，以创造有利于军团菌增殖的条件。详细了解军团菌的效应子及其毒力策略对于开发能够预防和治疗这种危险的肺炎的新型疗法至关重要，并将深刻改善人们的生活和福祉。 在过去的资助期间，我们继续在破译嗜肺军团菌的毒力策略方面取得重大进展。 以前对军团菌的研究一直被这种细菌产生近300种效应子的事实所混淆，这些效应子通常具有重叠的功能。这些效应子之间的功能冗余对研究人员提出了挑战，以确定这些效应子中最关键的，最有希望的药物靶点。我们现在已经在军团菌中开发了一种新的基因沉默工具，利用CRISPR干扰（CRISPRi）的力量不仅抑制单个基因，而且抑制整个细菌基因组。使用这种CRISPRi工具，我们询问了来自嗜肺军团菌的200多种毒力因子，现在正在观察细胞内病原体的表型，其中以前很少有报道，从而为解密嗜肺军团菌毒力机制奠定了基础。 最近，我们开发了一种改进的CRISPRi工具，该工具允许多重基因沉默来寻找同时沉默时使军团菌毒性降低的基因。 在一项概念验证研究中，我们使用这种方法来探测一组高度保守的跨膜效应子，以了解它们在人类巨噬细胞中军团菌复制过程中的重要性。几个基因组合被确定为至关重要的，这些命中已成为我们未来研究的重点，目标是开发抑制性化合物。 在感染人类免疫细胞的过程中，军团菌驻留在一个膜封闭的隔室或空泡中，以远离宿主细胞。然而，当细菌开始复制时，这种“拯救避风港”是一个挑战，因为周围的空泡也必须扩大，以便为越来越多的军团菌后代提供空间。我们的研究发现军团菌通过毒力因子VpdC控制空泡扩张。VpdC催化改变液泡膜的脂质组成，以促进其扩张。太多或太少的VpdC干扰适当的空泡扩张，使军团菌毒性降低，这表明阻断其空泡的协调扩张是治疗军团菌和相关病原体感染的一种新的治疗方法。

The bacterium Legionella pneumophila is the causative agent of a potentially life-threatening pneumonia called Legionnaires' disease. Upon inhalation by humans, Legionella enters the lung where it can infect and replicate within alveolar macrophages, specialized immune cells. Instead of being degraded by macrophages, Legionella uses the infected cell for its intracellular replication cycle. If not treated promptly, this respiratory infection ends fatal in up to 30 percent of all cases. The number of Legionnaires' disease cases in the U.S. has increased four-fold over the past 15 years, making Legionella a significant health threat and a considerable economic burden. We are committed to studying how Legionella can bypass our immune system and cause disease so that we can develop better ways to counteract its virulence strategies. Humans are frequently exposed to Legionella since Legionella is ubiquitously found in freshwater habitats such as cooling towers, faucets, shower heads, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. Like many other microbial pathogens, Legionella bacteria have developed a variety of strategies to exploit their human host and to cause disease. They use a specialized protein translocation machine called Type IV Secretion System (T4SS) to inject an abundance of proteins, so-called effectors, into the infected host cell. The effectors modulate signaling events within the host to create conditions favorable for Legionella proliferation. Obtaining a detailed understanding of Legionella's effectors and its virulence strategy is essential for the development of novel therapeutics capable of preventing and treating this dangerous pneumonia and will profoundly improve people's lives and wellbeing. Over the past funding period, we have continued to make significant progress in deciphering the virulence strategies of Legionella pneumophila. Previous investigations of Legionella have been confounded by the fact that this bacterium produces nearly 300 effectors, which often have overlapping functions. Functional redundancy among these effectors represents a challenge to investigators to identify the most critical of these effectors the most promising drug targets. We have now developed a novel gene silencing tool in Legionella that harnesses the power of CRISPR-interference (CRISPRi) to suppress not only individual genes but entire groups of bacterial genes. Using this CRISPRi tool, we interrogated more than 200 virulence factors from Legionella pneumophila and are now observing phenotypes in an intracellular pathogen in which few had previously been reported, thus laying the foundation for decrypting the mechanisms of Legionella pneumophila virulence. More recently, we generated an improved CRISPRi tool that allows multiplexed gene silencing to look for genes that, when silenced simultaneously, render Legionella less virulent. In a proof-of-concept study, we used this approach to probe a group of highly conserved transmembrane effectors for their importance during replication of Legionella in human macrophages. Several gene combinations were identified as vital, and those hits have become the focus of our future research with the goal of developing inhibitory compounds. During infection of human immune cells, Legionella resides within a membrane-enclosed compartment, or vacuole, to his from the host cell. Yet, this 'save haven' represents a challenge when the bacteria start to replicate, as the surrounding vacuole has to be expanded as well to give space to the growing number of Legionella progeny. Our studies discovered that Legionella controls vacuole expansion using the virulence factor VpdC. VpdC catalytically modifies the lipid composition of the vacuolar membrane to promote its expansion. Too much or too little VpdC interfered with proper vacuole expansion and rendered Legionella less virulent, suggesting that blocking the coordinated expansion of their vacuole is a novel therapeutic approach to treat infections with Legionella and related pathogens.