Deciphering microbial virulence mechanisms during Legionella pneumophila infection

微生物病原体已经发展出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（t4ss）将细菌蛋白（称为效应物）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，即军团病的病原体。在美国，每年有更多的人感染军团病（8000到18000），而不是ALS（肌萎缩性侧索硬化症或Lou Gehrig's病），因此嗜肺乳杆菌是一个重大的健康威胁和相当大的经济负担。此外，嗜肺乳杆菌的感染周期与沙门氏菌、衣原体、分枝杆菌、科希氏菌和许多其他人类病原体的毒力程序有许多相似之处，这些病原体在膜封闭的隔间内操纵宿主细胞。此外，考虑到IV型分泌系统（T4SS）是嗜肺乳杆菌的主要毒力装置，存在于包括幽门螺杆菌或农杆菌在内的许多动植物病原体中，对该易位系统及其货物蛋白（称为效应物）的深入分析对于我们对微生物毒力的总体理解具有重要意义。最后但并非最不重要的是，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有显著的相似之处，破译它们的功能将对发生在我们自己细胞内的过程的机制和调节概念产生有价值的见解。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。

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.