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: 8941540
• 负责人: Matthias Machner
• 金额: $ 85.7万
• 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/8941540
• 关键词: 1-Phosphatidylinositol 3-Kinase; Active Sites; Air Conditioning; Allosteric Regulation; Alveolar Macrophages; Amino Acids; Antigens; Bacteria; Bacterial Proteins; Binding; Biological Models; Breathing; Bypass; C-terminal; Cells; Centers for Disease Control and Prevention (U.S.); Complex; Contracts; Cytosol; Development; Diagnosis; Disease; Elderly; Endoplasmic Reticulum; Endosomes; Enzymes; Event; Excision; Exhibits; Failure; Fresh Water; Funding; Goals; Gram-Negative Bacteria; Growth; Guanosine Triphosphate Phosphohydrolases; Habitats; Head; Human; Immune; Individual; Infection; Integration Host Factors; Invaded; Left; Legionella; Legionella pneumophila; Legionnaires' Disease; Life; Lung; Lysosomes; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Microbe; Molecular; Monomeric GTP-Binding Proteins; N-terminal; Organelles; Organism; Phagocytosis; Phagosomes; Phosphatidylinositols; Phospholipase; Phospholipase A1; Phospholipids; Pneumonia; Process; Proteins; Public Health; Research; Respiratory Tract Infections; Risk; Signal Transduction; Structure; Surface; System; Transport Vesicles; Type IV Secretion System Pathway; Vacuole; Virulence; Water; base; combat; disorder prevention; guanine nucleotide binding protein; interest; killings; macrophage; microbial; microorganism; mutant; novel therapeutic intervention; novel therapeutics; pathogen; prevent; protein complex

Microbial pathogens have developed a variety of strategies to infect the 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 supportive of bacterial growth. We are particularly interested in understanding the virulence mechanism of Legionella pneumophila, the causative agent of a life-threatening pneumonia called Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater habitats such as water fountains, air conditioning systems, or shower heads. Consequently, humans are frequently exposed to this organism, with immune-compromised individuals or the elderly being 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 is an emerging public health threat. The primary host cell of L. pneumophila are alveolar macrophages, specialized immune cells within our lung. A crucial step in the elimination of invading microbes by macrophages is phagosomal maturation, a process where the bacteria-containing phagosome gradually fuses with endosomes and lysosomes, resulting in the acidification of its lumen and the degradation of its content. L. pneumophila bypasses the endosomal compartment by a yet unknown mechanism and proficiently replicates within the infected macrophage. Endolysosomal evasion relies on the delivery of almost 300 L. pneumophila effector proteins through the Dot/Icm T4SS into the host cytosol. We and others recently demonstrated that several of the effectors are involved in redirecting proteins and membrane material of the infected cell to the surface of the Legionella-containing vacuole, thereby establishing a protective compartment resembling host-cell endoplasmic reticulum (ER) (for review see Neunuebel & Machner (2012) Small GTPases). Our studies revealed a remarkable level of sophistication where L. pneumophila proteins with antagonistic activities are deployed in a precisely coordinated fashion in order to efficiently hijack host transport vesicles. Over the past funding period, we also investigated the molecular mechanisms underlying endolysosomal avoidance by intracellular L. pneumophila. Endosomal fusion is controlled by the host guanine nucleotide binding protein Rab5, which assembles protein complexes that include the tethering protein early endosomal antigen (EEA) 1 and the phosphatidylinositol (PI) 3-kinase hVps34. hVps34 generates PI(3)P, a phospholipid required for the assembly of EEA1 and other fusion factors on the endosome. Our studies revealed that the effector protein VipD from L. pneumophila exhibits phospholipase A1 (PLA1) activity that is activated only upon binding to endosomal Rab5 or Rab22. When produced within mammalian cells, VipD localizes to endosomes and catalyzes the removal of PI(3)P from endosomal membranes. EEA1 and other transport and fusion factors are consequently depleted from endosomes, rendering them fusion-incompetent. Consequently, we showed that during host cell infection, VipD reduces the contact of Legionella-containing vacuoles with the endosomal compartment and protects their surrounding vacuoles from acquiring Rab5. Thus, by catalyzing PI(3)P depletion in a Rab5-dependent manner, VipD specifically alters the protein composition of endosomes while leaving other host organelles unaffected. We also determined the crystal structure of VipD in complex with constitutively active Rab5. This collaborative effort uncovered how the phospholipase A1 (PLA1) activity of VipD is triggered upon binding to the host cell GTPase Rab5. A comparison of the complexed and uncomplexed form of VipD revealed that an active site-obstructing loop which originates from the C-terminal domain of VipD is repositioned upon Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of individual amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation, and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. In summary, these findings disclosed an unexpected mode of long-range allosteric regulation of the PLA1 activity of VipD and provide the basis for the development of novel therapeutic approaches that, rather than directly targeting the enzymes active site, specifically disturb the host factor-mediated activation process of VipD and related microbial phospholipases.

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，以创造支持细菌生长的条件。我们对了解嗜肺军团菌的毒力机制特别感兴趣，它是一种被称为军团病的危及生命的肺炎的病原体。 嗜肺军团菌普遍存在于淡水栖息地，如饮水机、空调系统或淋浴喷头。因此，人类经常接触这种生物体，免疫力低下的人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，使得这种微生物成为一种新兴的公共卫生威胁。 嗜肺军团菌的主要宿主细胞是肺泡巨噬细胞，是我们肺部的特殊免疫细胞。巨噬细胞消除入侵微生物的关键步骤是吞噬体成熟，在这一过程中，含有细菌的吞噬体逐渐与内体和溶酶体融合，导致其管腔酸化及其内容物降解。嗜肺军团菌通过一种未知的机制绕过内体隔室，并在受感染的巨噬细胞内高效地复制。内溶酶体逃逸依赖于将近 300 个嗜肺军团菌效应蛋白通过 Dot/Icm T4SS 递送到宿主细胞质中。我们和其他人最近证明，一些效应器参与将受感染细胞的蛋白质和膜材料重定向到含有军团菌的液泡表面，从而建立一个类似于宿主细胞内质网（ER）的保护性隔室（综述参见Neunuebel & Machner (2012) 小 GTPases)。我们的研究揭示了具有拮抗活性的嗜肺军团菌蛋白以精确协调的方式部署以有效劫持宿主转运囊泡的复杂程度。 在过去的资助期间，我们还研究了细胞内嗜肺军团菌避免内溶酶体的分子机制。内体融合由宿主鸟嘌呤核苷酸结合蛋白 Rab5 控制，该蛋白组装蛋白质复合物，其中包括束缚蛋白早期内体抗原 (EEA) 1 和磷脂酰肌醇 (PI) 3-激酶 hVps34。 hVps34 产生 PI(3)P，这是一种在内体上组装 EEA1 和其他融合因子所需的磷脂。我们的研究表明，来自嗜肺军团菌的效应蛋白 VipD 具有磷脂酶 A1 (PLA1) 活性，该活性仅在与内体 Rab5 或 Rab22 结合时被激活。当在哺乳动物细胞内产生时，VipD 定位于核内体并催化 PI(3)P 从核内体膜上去除。 EEA1 和其他转运和融合因子因此从内体中耗尽，导致它们无法融合。因此，我们发现在宿主细胞感染期间，VipD 减少了含有军团菌的液泡与内体隔室的接触，并保护其周围的液泡免于获得 Rab5。因此，通过以 Rab5 依赖性方式催化 PI(3)P 消耗，VipD 特异性改变内体的蛋白质组成，同时使其他宿主细胞器不受影响。 我们还确定了 VipD 与组成型活性 Rab5 复合物的晶体结构。这项合作揭示了 VipD 的磷脂酶 A1 (PLA1) 活性如何在与宿主细胞 GTPase Rab5 结合后被触发。比较 VipD 的复合形式和非复合形式表明，源自 VipD C 末端结构域的活性位点阻塞环在 Rab5 结合后重新定位，从而暴露 N 末端 PLA1 结构域内的催化袋。位于 VipD-Rab5 界面内的单个氨基酸残基的取代阻止了 Rab5 结合和 PLA1 激活，并导致 VipD 突变蛋白无法靶向细胞内富含 Rab5 的内体结构。总之，这些发现揭示了 VipD PLA1 活性的一种意想不到的远程变构调节模式，并为开发新的治疗方法提供了基础，该方法不是直接靶向酶活性位点，而是特异性干扰宿主因子介导的VipD 和相关微生物磷脂酶的激活过程。