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。 在过去的筹资期间，我们在开发旨在破译嗜肺军团菌毒力策略的新型遗传工具方面继续取得重大进展。 军团菌产生近300个效应物，这些效应物往往具有重叠的功能，这一事实扰乱了以前对军团菌毒力的研究。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。 在另一个项目中，我们已经朝着开发更智能的抗生素迈出了第一步，这种抗生素可以选择性地针对病原体。多重耐药病原体是对人类健康的一种新威胁。由于传统抗生素不仅针对病原体，而且还能根除有益的人体微生物区系，因此它们往往会造成额外的临床并发症。因此，迫切需要开发一种治疗方法，在不影响有益共生体的情况下选择性地针对病原体。细菌IV型分泌系统(T4SS)对各种病原体的毒力是必不可少的，但大多数共生细菌都不存在，因此可以被认为是病原体的阿喀琉斯之踵。通过识别干扰T4SS功能的小分子，我们能够有力地抑制人巨噬细胞内军团菌的生长。我们的抑制化合物也抑制了另一种细胞内病原体--Q热的病原体--伯氏柯克斯体的生长，但不影响共生细菌大肠杆菌的生长，与军团菌和柯克斯体不同的是，它的生长不需要T4SS。我们的研究代表了追求精确医学的第一步，通过开发能够治疗感染而不对共生细菌造成伤害的病原体选择性疗法。

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 developing novel genetic tools aimed at deciphering the virulence strategies of Legionella pneumophila. Previous investigations of Legionella virulence 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. In another project, we have taken the first step towards the development of smarter antibiotics that selectively target pathogens. Multi-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial human microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but mostly absent from commensal bacteria and can, thus, be considered a pathogens Achilles heel. By identifying small molecules that interfere with the function of the T4SS, we were able to robustly suppress growth of Legionella within human macrophages. Our inhibitory compounds also suppressed growth of another intracellular pathogen, Coxiella burnetii, the causative agent of Q fever, but did not affect growth of the commensal bacterium Escherichia coli which, unlike Legionella and Coxiella, does not require a T4SS for growth. Our study represents the first step in the pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.