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了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（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传导事件，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，嗜肺军团菌是一种称为军团菌病的危及生命的肺炎的病原体。 L.嗜肺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，人类经常暴露于这种生物体，免疫力低下的个体或老年人感染的风险较高。根据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。 L.嗜肺细胞是肺泡巨噬细胞，是我们肺部的专门免疫细胞。巨噬细胞清除入侵微生物的关键步骤是吞噬体成熟，这是一个含细菌的吞噬体逐渐与内体和溶酶体融合的过程，导致其内腔酸化并降解其内容物。L.嗜肺菌通过一种未知的机制绕过内体区室，并在感染的巨噬细胞内熟练地复制。内溶酶体逃避依赖于近300 L的递送。嗜肺菌效应蛋白通过Dot/Icm T4 SS进入宿主胞质溶胶。我们和其他人最近证明，几种效应物参与将感染细胞的蛋白质和膜材料重定向到含军团菌的空泡的表面，从而建立类似于宿主细胞内质网（ER）的保护区室（综述参见Neunuebel & Machner（2012）Small GTPases）。我们的研究揭示了一个显着的复杂程度，其中L。具有拮抗活性的嗜肺菌蛋白以精确协调的方式展开，以便有效地劫持宿主转运囊泡。 在过去的资助期间，我们还研究了细胞内L。嗜肺菌内体融合由宿主鸟嘌呤核苷酸结合蛋白Rab 5控制，Rab 5组装蛋白质复合物，包括栓系蛋白早期内体抗原（EEA）1和磷脂酰肌醇（PI）3-激酶hVps 34。hVps 34产生PI（3）P，其是EEA 1和其他融合因子在内体上组装所需的磷脂。我们的研究表明，L.嗜肺菌表现出磷脂酶A1（PLA 1）活性，该活性仅在与内体Rab 5或Rab 22结合时被激活。当在哺乳动物细胞内产生时，VipD定位于内体并催化PI（3）P从内体膜去除。EEA 1和其他转运和融合因子因此从内体中耗尽，使它们不能融合。因此，我们表明，在宿主细胞感染，VipD减少接触军团菌含有空泡与内体隔室，并保护其周围的空泡收购Rab 5。因此，通过以Rab 5依赖性方式催化PI（3）P消耗，VipD特异性地改变了内体的蛋白质组成，同时使其他宿主细胞器不受影响。 我们还确定了与组成型活性Rab 5复合的VipD的晶体结构。这项合作研究揭示了VipD的磷脂酶A1（PLA 1）活性是如何在与宿主细胞GTIPRab 5结合时被触发的。复合和未复合形式的VipD的比较显示，活性位点的阻碍环，其起源于C-末端域的VipD的Rab 5结合后被重新定位，从而暴露N-末端PLA 1域内的催化口袋。位于VipD-Rab 5界面内的单个氨基酸残基的取代阻止Rab 5结合和PLA 1活化，并导致VipD突变蛋白未能靶向细胞内富含Rab 5的内体结构。总之，这些发现公开了一种意想不到的VipD的PLA 1活性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，而是特异性地干扰宿主因子介导的VipD和相关微生物磷脂酶的活化过程。