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了多种策略来感染它们的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（T4 SS）将细菌蛋白质（称为效应子）递送到宿主细胞中。效应子有助于调节宿主内的信号事件，以创造有利于细菌存活的条件。我们致力于深入分析微生物毒力策略。我们使用嗜肺军团菌作为模式生物，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，每年感染军团病的人数（8,000到18,000人）比ALS（肌萎缩侧索硬化症或Lou Gehrig病）病例还多，因此使L。嗜肺菌是严重的健康威胁和相当大的经济负担。此外，L.嗜肺菌的毒力程序与沙门氏菌、衣原体、分枝杆菌、柯克斯氏菌和许多其他人类病原体的毒力程序有许多相似之处，这些病原体从膜封闭的隔室内操纵宿主细胞。此外，由于L. IV型分泌系统（T4 SS）是L. pneumophila存在于包括螺杆菌（Helicobacter）或农杆菌（Agrobacterium）在内的许多动植物病原体中，深入分析这种转运系统及其被称为效应子的货物蛋白对于我们全面了解微生物毒力具有重要意义。最后但并非最不重要的是，L. pneumophila操纵宿主细胞过程显示出与真核蛋白质的显著相似之处，并且破译它们的功能将产生关于我们自己细胞内发生的过程的机制和调节概念的有价值的见解。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。 L.嗜肺菌普遍存在于淡水栖息地，如冷却塔、空调系统或喷泉。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。2015年纽约市爆发军团病时就是这种情况，当时有120多人感染，12人死于这种疾病。 免疫功能低下的个体、婴儿或老年人感染的风险较高。根据美国疾病控制和预防中心（CDC）的数据，在过去十年中，美国确诊的军团病病例数量翻了一番，使这种微生物成为一种新的公共卫生威胁。 吸入后，L.嗜肺菌在肺泡巨噬细胞内感染和复制，肺泡巨噬细胞是我们肺内的特化免疫细胞。L. pneumophila通过T4 SS向宿主细胞递送近300种称为效应子的蛋白质。莫斯特湖尚未详细表征嗜肺菌效应蛋白，并且它们的活性和宿主靶点仍然未知。干扰T4 SS活性使L. pneumophila无毒力，强调了重要的作用，易位的效应感染。 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L。pneumophila易位几种效应子，其模拟具有E3泛素连接酶活性的宿主细胞蛋白。E3泛素连接酶催化酶级联反应的最后一步，导致小蛋白泛素从E2泛素缀合酶转移到特定的靶蛋白。靶蛋白的多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。通过编码自身的E3连接酶，L.嗜肺菌可以劫持宿主细胞的泛素化机制，并利用它为自己的利益。我们发现L.嗜肺菌效应子是E3连接酶遗物，其在进化过程中已被广泛修饰，不再类似于祖先酶。尽管这种多样性，E2的识别和结合的模式已被保存，这表明，毒性关键蛋白质的功能是不太容易进化的多样化。 除了上述贡献，我们还开发了一个实验平台，用于识别L。嗜肺效应器。该平台由近10，000种人类蛋白质组成的蛋白质阵列组成。在与军团菌效应物孵育后，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。我们还采用了这个平台，用于检测翻译后修饰，包括泛素化和磷酸化，并发现了一些新的目标，以前未知的L。嗜肺效应器。这些新的宿主-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很容易地适应于其他微生物病原体的效应子的研究，从而掌握了深入了解毒力计划的关键，不仅是L.嗜肺菌，但也有相关病原体。