Deciphering microbial virulence mechanisms during Legionella pneumophila infection

微生物病原体已经开发出各种策略来感染它们的人类宿主并导致疾病。许多革兰氏阴性细菌使用IV型分泌系统(T4SS)将被称为效应器的细菌蛋白质输送到宿主细胞中。效应器有助于调节宿主内的信号事件，以便为细菌生存创造有利条件。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，它是一种被称为军团病的潜在致命呼吸道感染的病原体。每年，美国患退伍军人症的人数(8,000至18,000人)超过肌萎缩侧索硬化症(肌萎缩侧索硬化症或卢·格里克病)的人数，从而使嗜肺乳杆菌成为一个重大的健康威胁和相当大的经济负担。此外，嗜肺性乳杆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间内操纵宿主细胞。此外，鉴于嗜肺性乳杆菌的主要毒力器官IV型分泌系统(T4SS)存在于包括幽门螺杆菌和农杆菌在内的许多动植物病原体中，深入分析这一易位系统及其被称为效应物的货物蛋白对于我们对微生物毒力的一般理解具有重要意义。最后但并非最不重要的一点是，嗜肺乳杆菌用来操纵宿主细胞过程的效应蛋白与真核蛋白显示出惊人的相似之处，破译它们的功能将使我们对发生在我们自己细胞内的过程的机械和调控概念有价值的洞察。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。 嗜肺性乳杆菌广泛存在于淡水栖息地，如冷却塔、空调系统或饮水机。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。2015年纽约市爆发退伍军人症期间就是这种情况，当时有120多人感染，12人死于这种疾病。 免疫功能受损的个人、婴儿或老年人感染的风险更高。根据疾病控制和预防中心(CDC)的数据，在过去十年中，美国境内被诊断为退伍军人病的病例数量翻了一番，使这种微生物成为一个新的公共卫生威胁。 嗜肺乳杆菌吸入后，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殊免疫细胞。嗜肺性乳杆菌通过T4SS将近300种称为效应器的蛋白质输送到宿主细胞。大多数嗜肺乳杆菌效应蛋白还没有得到详细的鉴定，它们的活性和宿主靶标也是未知的。对T4SS活性的干扰使嗜肺性乳杆菌变得无毒，突显了易位效应因子在感染中的重要作用。 在过去的资助期间，我们在开发和应用新的研究工具以破译效应器的生物学作用方面取得了重要进展。我们发现，在感染期间，嗜肺乳杆菌移位了几个效应器，这些效应器模仿具有E3泛素连接酶活性的宿主细胞蛋白。E3泛素连接酶催化了酶促反应的最后一步，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。嗜肺乳杆菌通过编码自己的E3连接酶，可以劫持宿主细胞的泛素化机制，并将其用于自身的利益。我们发现，嗜肺乳杆菌的其中一个效应物是E3连接酶残留物，它在进化过程中被广泛修改，不再与祖先的酶相似。尽管有这种多样性，但E2识别和结合的模式仍然保持不变，这表明毒力关键蛋白特征不太容易进化多样化。 除了上述贡献外，我们还开发了一个用于识别嗜肺乳杆菌效应器的人体靶标的实验平台。该平台由近10,000种人类蛋白质组成的蛋白质阵列组成。在与军团菌效应器孵育后，蛋白质-蛋白质相互作用被允许发生，然后可以使用微阵列芯片扫描仪直接监测。我们还将该平台用于检测翻译后修饰，包括泛素化和磷酸化，并发现了几个以前未确定的嗜肺乳杆菌效应器的新靶点。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其他微生物病原体的效应物的研究，从而不仅是深入了解嗜肺乳杆菌毒力计划的关键，也是深入了解相关病原体的关键。

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.