Deciphering microbial virulence mechanisms during Legionella pneumophila infection

破译嗜肺军团菌感染期间微生物的毒力机制

• 批准号: 10691795
• 负责人: Matthias Machner
• 金额: $ 155.49万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10691795
• 关键词: Affect; Alveolar Macrophages; Antibiotics; Bacteria; Bacterial Genes; Bypass; CRISPR interference; Cells; Clinical; Contracts; Coxiella; Coxiella burnetii; Dangerousness; Development; Disease; Disease Outbreaks; Drug Targeting; Economic Burden; Elderly; Escherichia coli; Event; Exposure to; Foundations; Fresh Water; Funding; Gene Silencing; Genes; Genetic; Goals; Growth; Habitats; Head; Health; Human; Immune; Immune system; Individual; Infant; Infection; Infection prevention; Inhalation; Investigation; Knowledge; Legionella; Legionella pneumophila; Legionnaires' Disease; Life; Lung; Molecular; Personal Satisfaction; Persons; Phenotype; Pneumonia; Process; Protein translocation; Proteins; Q Fever; Reporting; Research; Research Personnel; Respiratory Tract Infections; Risk; Signal Transduction; Source; Therapeutic; Type IV Secretion System Pathway; Virulence; Virulence Factors; Water; aerosolized; commensal bacteria; contaminated water; emerging pathogen; human microbiota; improved; macrophage; microbial; multi-drug resistant pathogen; novel; novel therapeutics; pathogen; pathogenic bacteria; pathogenic microbe; precision medicine; prevent; small molecule; therapeutic development; tool

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.

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。人类吸入后，军团菌进入肺部，可以感染肺泡巨噬细胞（专门的免疫细胞）并在其中复制。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，我们继续在开发新的遗传工具方面取得重大进展，旨在破译嗜肺军团菌的毒力策略。 以前的军团菌毒力研究一直被这样一个事实所混淆，即这种细菌产生近300种效应子，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。 在另一个项目中，我们已经朝着开发选择性靶向病原体的更智能抗生素迈出了第一步。多重耐药病原体是对人类健康的一个新威胁。由于传统抗生素不仅针对病原体，而且还根除有益的人类微生物群，因此它们通常会导致额外的临床并发症。因此，迫切需要开发选择性靶向病原体而不影响有益生物的治疗剂。细菌IV型分泌系统（T4SS）对于多种病原体的毒力是必不可少的，但大多数不存在于肠道细菌中，因此可以被认为是病原体的阿喀琉斯之踵。通过鉴定干扰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。我们的抑制性化合物还抑制了另一种细胞内病原体贝氏柯克斯体（Q热的病原体）的生长，但不影响细菌大肠杆菌的生长，与军团菌和柯克斯体不同，大肠杆菌的生长不需要T4SS。我们的研究代表了通过开发能够治疗感染而不对肠道细菌造成伤害的病原体选择性疗法来追求精准医学的第一步。