Deciphering microbial virulence mechanisms during Legionella pneumophila infection

破译嗜肺军团菌感染期间的微生物毒力机制

• 批准号: 9550425
• 负责人: Matthias Machner
• 金额: $ 114.66万
• 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/9550425
• 关键词: Agrobacterium; Air Conditioning; Alveolar Macrophages; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Bacteria; Bacterial Proteins; Binding; Biological; Biology; Breathing; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Chlamydia; Contracts; Coxiella; Dangerousness; Detection; Diagnosis; Disease; Disease Outbreaks; Economic Burden; Elderly; Enzymes; Event; Evolution; Fresh Water; Funding; Genus Mycobacterium; Goals; Gram-Negative Bacteria; Habitats; Health; Helicobacter; Human; Immune; Individual; Infant; Infection; Laboratories; Legionella; Legionella pneumophila; Legionnaires' Disease; Life; Lung; Membrane; Molecular; Monitor; New York City; Phosphorylation; Plants; Pneumonia; Polyubiquitination; Post-Translational Protein Processing; Process; Protein Array; Proteins; Public Health; Research; Respiratory Tract Infections; Risk; Role; Salmonella; Signal Transduction; Source; System; Technology; Type IV Secretion System Pathway; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Virulence; Water; aerosolized; combat; contaminated water; disorder prevention; flexibility; improved; insight; macrophage; microbial; microorganism; novel; novel therapeutics; pathogen; prevent; programs; protein protein interaction; tool; ubiquitin-protein ligase

Microbial pathogens have developed a variety of strategies to infect their human host and cause disease. Many Gram-negative bacteria use type IV secretion systems (T4SSs) to deliver bacterial proteins, called effectors, into host cells. The effectors help to modulate signaling events within the host in order to create conditions favorable for bacterial survival. We are committed to the in-depth analysis of microbial virulence strategies. We use as a model organism the bacterium Legionella pneumophila, the causative agent of a potentially fatal respiratory infection known as Legionnaires' disease. Each year more individuals in the U.S. contract Legionnaires' disease (8,000 to 18,000) than there are cases of ALS (Amyotrophic Lateral Sclerosis or Lou Gehrig's Disease), thus making L. pneumophila a significant health threat and a considerable economic burden. Moreover, the infection cycle of L. pneumophila shows numerous parallels to the virulence programs of Salmonella, Chlamydia, Mycobacterium, Coxiella, and many other human pathogens that manipulate host cells from within a membrane-enclosed compartment. In addition, given that a type IV secretion system (T4SS), the major virulence apparatus of L. pneumophila, is present in numerous animal and plant pathogens including Helicobacter or Agrobacterium, the in-depth analysis of this translocation system and its cargo proteins, called effectors, is of great importance for our general understanding of microbial virulence. Last but not least, the effector proteins that are used by L. pneumophila to manipulate host cell processes display remarkable parallels to eukaryotic proteins, and deciphering their function will yield valuable insight into mechanistic and regulatory concepts about processes that occur within our own cells. Thus, obtaining a detailed understanding of Legionella's biology and its virulence strategies is essential to more effectively prevent, diagnose, and treat this dangerous pneumonia, and will profoundly improve people's lives and wellbeing. L. pneumophila is ubiquitously found in freshwater habitats such as cooling towers, air conditioning systems, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. That was the case during an outbreak of Legionnaires disease in New York City in 2015, where more than 120 individuals got infected and 12 died of the disease. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires' disease cases within the U.S. has doubled over the past decade, making this microorganism an emerging public health threat. Upon inhalation, L. pneumophila infects and replicates within alveolar macrophages, specialized immune cells within our lung. L. pneumophila delivers close to 300 proteins, called effectors, through a T4SS into the host cell. Most L. pneumophila effector proteins have not been characterized in detail, and their activities and host targets remain unknown. Interference with T4SS activity renders L. pneumophila avirulent, underscoring the important role of the translocated effectors for infection. Over the past funding period, we have made important progress in developing and applying new research tools to decipher the biological role of effectors. We revealed that during infection L. pneumophila translocates several effectors that mimic host cell proteins with E3 ubiquitin ligase activity. E3 ubiquitin ligases catalyze the final step in an enzymatic cascade that results in the transfer of the small protein ubiquitin from E2 ubiquitin-conjugating enzymes to a particular target protein. Poly-ubiquitination of target proteins alters their cellular fate, often resulting in their proteasomal degradation. By encoding its own E3 ligases, L. pneumophila can hijack the host cell ubiquitination machinery and use it for its own benefit. We found that one of the L. pneumophila effectors is an E3 ligase relic that that has been extensively modified during evolution to no longer resemble the ancestral enzyme. Despite this diversification, the mode of E2 recognition and binding has been preserved, suggesting that virulence-critical protein features are less prone to evolutionary diversification. In addition to the contributions described above, we also developed an experimental platform for the identification of human targets for L. pneumophila effectors. The platform is comprised of a protein array composed of almost 10,000 human proteins. Upon incubation with a Legionella effector, protein-protein interactions are allowed to occur that can then be directly monitored using a microarray chip scanner. We also adapted this platform for the detection of post-translational modifications, including ubiquitination and phosphorylation, and discovered several novel targets for previously uncharacterized L. pneumophila effectors. These novel host-pathogen interactions are currently being investigated in the laboratory. The flexibility of our protein platform technology allows it to be easily adapted to the study of effectors from other microbial pathogens, thus holding the key to obtaining in-depth insight into the virulence program not only of L. pneumophila but related pathogens as well.

微生物病原体已经发展出多种策略来感染人类宿主并引起疾病。许多革兰氏阴性细菌使用 IV 型分泌系统 (T4SS) 将细菌蛋白（称为效应子）输送到宿主细胞中。效应器有助于调节宿主内的信号事件，以创造有利于细菌生存的条件。我们致力于微生物毒力策略的深入分析。我们使用嗜肺军团菌作为模型生物，它是一种潜在致命的呼吸道感染（称为军团病）的病原体。在美国，每年感染退伍军人病的人数（8,000 至 18,000 人）比 ALS（肌萎缩侧索硬化症或卢伽雷氏病）病例还要多，从而使嗜肺军团菌成为严重的健康威胁和相当大的经济负担。此外，嗜肺军团菌的感染周期与沙门氏菌、衣原体、分枝杆菌、柯克斯体和许多其他人类病原体的毒力程序有许多相似之处，这些病原体在膜封闭的隔室内操纵宿主细胞。此外，鉴于嗜肺军团菌的主要毒力装置IV型分泌系统（T4SS）存在于包括螺杆菌或农杆菌在内的许多动植物病原体中，因此对该易位系统及其货物蛋白的深入分析，称为效应器，对于我们对微生物毒力的一般理解非常重要。最后但并非最不重要的一点是，嗜肺军团菌用来操纵宿主细胞过程的效应蛋白与真核蛋白表现出显着的相似之处，破译它们的功能将为我们自己细胞内发生的过程的机制和调控概念提供有价值的见解。因此，详细了解军团菌的生物学及其毒力策略对于更有效地预防、诊断和治疗这种危险的肺炎至关重要，并将深刻改善人们的生活和福祉。 嗜肺军团菌普遍存在于冷却塔、空调系统或喷泉等淡水栖息地中。当来自受污染水源的水被雾化并随后被人类吸入时，就会发生军团病的大规模爆发。 2015 年纽约市爆发退伍军人病时就是这种情况，当时有 120 多人被感染，12 人死于该病。 免疫功能低下的个体、婴儿或老年人感染感染的风险较高。据美国疾病控制与预防中心 (CDC) 称，过去十年，美国确诊的退伍军人病病例数量增加了一倍，使这种微生物成为新的公共卫生威胁。 吸入后，嗜肺军团菌会感染肺泡巨噬细胞（我们肺部的特殊免疫细胞）并在其中复制。嗜肺军团菌通过 T4SS 向宿主细胞输送近 300 种蛋白质（称为效应子）。大多数嗜肺军团菌效应蛋白尚未得到详细表征，其活性和宿主靶点仍然未知。干扰 T4SS 活性使嗜肺军团菌无毒，强调了易位效应子对感染的重要作用。 在过去的资助期间，我们在开发和应用新的研究工具来破译效应器的生物学作用方面取得了重要进展。我们发现，在感染过程中，嗜肺军团菌易位了几个模拟具有 E3 泛素连接酶活性的宿主细胞蛋白的效应子。 E3 泛素连接酶催化酶级联的最后一步，导致小蛋白泛素从 E2 泛素结合酶转移到特定的靶蛋白。靶蛋白的多聚泛素化改变了它们的细胞命运，通常导致它们的蛋白酶体降解。通过编码自己的 E3 连接酶，嗜肺军团菌可以劫持宿主细胞泛素化机制并将其用于自身利益。我们发现嗜肺军团菌效应子之一是 E3 连接酶遗迹，它在进化过程中经过广泛修饰，不再类似于祖先酶。尽管存在这种多样化，E2 识别和结合的模式仍然被保留，这表明毒力关键的蛋白质特征不太容易发生进化多样化。 除了上述贡献之外，我们还开发了一个实验平台，用于识别嗜肺军团菌效应子的人类靶标。该平台由近 10,000 种人类蛋白质组成的蛋白质阵列组成。与军团菌效应器一起孵育后，允许发生蛋白质-蛋白质相互作用，然后可以使用微阵列芯片扫描仪直接监测。我们还对该平台进行了改造，用于检测翻译后修饰，包括泛素化和磷酸化，并发现了先前未表征的嗜肺军团菌效应子的几个新靶标。这些新颖的宿主-病原体相互作用目前正在实验室中进行研究。我们的蛋白质平台技术的灵活性使其能够轻松适应其他微生物病原体效应子的研究，从而掌握深入了解嗜肺军团菌和相关病原体毒力程序的关键。