Deciphering microbial virulence mechanisms during Legionella pneumophila infection

嗜肺军团菌是一种潜在威胁生命的肺炎的病原体，这种肺炎被称为军团病。军团菌一旦被人类吸入，就会进入肺部，在那里它可以感染并在肺泡巨噬细胞内复制，肺泡巨噬细胞是一种特殊的免疫细胞。军团菌不是被巨噬细胞降解，而是利用感染的细胞进行细胞内复制周期。如果不及时治疗，这种呼吸道感染最终会导致高达30%的病例死亡。在过去的15年里，美国退伍军人症的病例数量增加了四倍，这使得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而导致疾病，以便我们能够开发更好的方法来对抗其毒力策略。 人类经常接触军团菌，因为军团菌普遍存在于淡水栖息地，如冷却塔、水龙头、淋浴喷头或饮水机。当来自受污染来源的水被雾化，然后被人类吸入时，军团病的主要暴发就会发生。免疫功能受损的个人、婴儿或老年人感染的风险更高。 像许多其他微生物病原体一样，军团菌已经开发了各种策略来利用它们的人类宿主并导致疾病。他们使用一种名为IV型分泌系统(T4SS)的特殊蛋白质转运机，向受感染的宿主细胞注入大量蛋白质，即所谓的效应器。效应器调节宿主内的信号事件，为军团菌的增殖创造有利条件。对军团菌的效应物及其毒力策略的详细了解对于开发能够预防和治疗这种危险的肺炎的新疗法至关重要，并将深刻改善人们的生活和福祉。 在过去的资助期间，我们在破译嗜肺军团菌毒力策略方面继续取得重大进展。 以前对军团菌的研究因这种细菌产生近300个效应器的事实而感到困惑，这些效应器往往具有重叠的功能。这些效应器之间的功能冗余对研究人员来说是一个挑战，他们要确定这些效应器中最关键的、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，它利用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。利用这个CRISPRi工具，我们从嗜肺军团菌中询问了200多个毒力因子，现在正在观察一种以前鲜有报道的细胞内病原体的表型，从而为破解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，它允许多路基因沉默来寻找当同时沉默时使军团菌毒力降低的基因。在一项概念验证研究中，我们使用这种方法来探索一组高度保守的跨膜效应器在军团菌在人巨噬细胞中复制过程中的重要性。有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，目标是开发抑制化合物。 在感染人类免疫细胞的过程中，军团菌居住在宿主细胞对他的膜包裹的隔间或液泡中。然而，当细菌开始复制时，这种“拯救天堂”代表着一个挑战，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控制液泡扩张。VPDC催化修饰液泡膜的脂质成分以促进其扩张。VPDC过多或过少都会干扰适当的液泡扩张，并使军团菌的毒力降低，这表明阻断其液泡的协调扩张是治疗军团菌感染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.