Deciphering microbial virulence mechanisms during Legionella pneumophila infection

破译嗜肺军团菌感染期间微生物的毒力机制

基本信息

批准号：
10908173
负责人：
Matthias Machner
金额：
$ 175.21万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10908173
关键词：
Alveolar Macrophages Bacteria Bacterial Genes Bypass CRISPR interference Cells Contracts Dangerousness Development Disease Disease Outbreaks Drug Targeting Economic Burden Elderly Event Exposure to Foundations Fresh Water Funding Gene Combinations Gene Silencing Genes Goals Habitats Health Human Immune Immune system Individual Infant Infection Infection prevention Inhalation Investigation Knowledge Legionella Legionella pneumophila Legionnaires&apos Disease Life Lipids Lung Macrophage Membrane Molecular Personal Satisfaction Persons Phenotype Pneumonia Process Proliferating Protein translocation Proteins Reporting Research Research Personnel Respiratory Tract Infections Risk Signal Transduction Source Type IV Secretion System Pathway Vacuole Virulence Virulence Factors Virulent Water aerosolized contaminated water improved microbial novel novel therapeutic intervention novel therapeutics pathogen pathogenic bacteria pathogenic microbe prevent tool

项目摘要

The bacterium Legionella pneumophila is the causative agent of a potentially life-threatening pneumonia called Legionnaires' disease. Upon inhalation by humans, Legionella enters the lung where it can infect and replicate within alveolar macrophages, specialized immune cells. Instead of being degraded by macrophages, Legionella uses the infected cell for its intracellular replication cycle. If not treated promptly, this respiratory infection ends fatal in up to 30 percent of all cases. The number of Legionnaires' disease cases in the U.S. has increased four-fold over the past 15 years, making Legionella a significant health threat and a considerable economic burden. We are committed to studying how Legionella can bypass our immune system and cause disease so that we can develop better ways to counteract its virulence strategies. Humans are frequently exposed to Legionella since Legionella is ubiquitously found in freshwater habitats such as cooling towers, faucets, shower heads, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. Like many other microbial pathogens, Legionella bacteria have developed a variety of strategies to exploit their human host and to cause disease. They use a specialized protein translocation machine called Type IV Secretion System (T4SS) to inject an abundance of proteins, so-called effectors, into the infected host cell. The effectors modulate signaling events within the host to create conditions favorable for Legionella proliferation. Obtaining a detailed understanding of Legionella's effectors and its virulence strategy is essential for the development of novel therapeutics capable of preventing and treating this dangerous pneumonia and will profoundly improve people's lives and wellbeing. Over the past funding period, we have continued to make significant progress in deciphering the virulence strategies of Legionella pneumophila. Previous investigations of Legionella have been confounded by the fact that this bacterium produces nearly 300 effectors, which often have overlapping functions. Functional redundancy among these effectors represents a challenge to investigators to identify the most critical of these effectors the most promising drug targets. We have now developed a novel gene silencing tool in Legionella that harnesses the power of CRISPR-interference (CRISPRi) to suppress not only individual genes but entire groups of bacterial genes. Using this CRISPRi tool, we interrogated more than 200 virulence factors from Legionella pneumophila and are now observing phenotypes in an intracellular pathogen in which few had previously been reported, thus laying the foundation for decrypting the mechanisms of Legionella pneumophila virulence. More recently, we generated an improved CRISPRi tool that allows multiplexed gene silencing to look for genes that, when silenced simultaneously, render Legionella less virulent. In a proof-of-concept study, we used this approach to probe a group of highly conserved transmembrane effectors for their importance during replication of Legionella in human macrophages. Several gene combinations were identified as vital, and those hits have become the focus of our future research with the goal of developing inhibitory compounds. During infection of human immune cells, Legionella resides within a membrane-enclosed compartment, or vacuole, to his from the host cell. Yet, this 'save haven' represents a challenge when the bacteria start to replicate, as the surrounding vacuole has to be expanded as well to give space to the growing number of Legionella progeny. Our studies discovered that Legionella controls vacuole expansion using the virulence factor VpdC. VpdC catalytically modifies the lipid composition of the vacuolar membrane to promote its expansion. Too much or too little VpdC interfered with proper vacuole expansion and rendered Legionella less virulent, suggesting that blocking the coordinated expansion of their vacuole is a novel therapeutic approach to treat infections with Legionella and related pathogens.

嗜肺军团菌是一种可能危及生命的肺炎（称为军团病）的病原体。人类吸入后，军团菌进入肺部，在那里它可以感染肺泡巨噬细胞（专门的免疫细胞）并在其中复制。军团菌不会被巨噬细胞降解，而是利用受感染的细胞进行细胞内复制周期。如果不及时治疗，这种呼吸道感染在所有病例中高达 30% 会导致致命。过去 15 年来，美国的军团病病例数量增加了四倍，使军团菌成为严重的健康威胁和相当大的经济负担。我们致力于研究军团菌如何绕过我们的免疫系统并引起疾病，以便我们能够开发出更好的方法来抵消其毒力策略。人类经常接触军团菌，因为军团菌普遍存在于冷却塔、水龙头、淋浴喷头或喷泉等淡水栖息地中。当来自受污染水源的水被雾化并随后被人类吸入时，就会发生军团病的大规模爆发。免疫功能低下的个体、婴儿或老年人感染感染的风险较高。与许多其他微生物病原体一样，军团菌已经开发出多种策略来利用人类宿主并引起疾病。他们使用一种称为 IV 型分泌系统 (T4SS) 的特殊蛋白质易位机器，将大量蛋白质（所谓的效应物）注入受感染的宿主细胞中。效应器调节宿主内的信号传导事件，以创造有利于军团菌增殖的条件。详细了解军团菌的效应子及其毒力策略对于开发能够预防和治疗这种危险肺炎的新型疗法至关重要，并将深刻改善人们的生活和福祉。在过去的资助期间，我们在破译嗜肺军团菌的毒力策略方面继续取得重大进展。先前对军团菌的研究因这种细菌产生近 300 个效应器而令人困惑，这些效应器通常具有重叠的功能。这些效应器之间的功能冗余对研究人员来说是一个挑战，需要确定这些效应器中最关键的最有希望的药物靶点。我们现在在军团菌中开发了一种新型基因沉默工具，它利用 CRISPR 干扰 (CRISPRi) 的力量不仅抑制单个基因，而且抑制整个细菌基因组。利用这种CRISPRi工具，我们检测了嗜肺军团菌的200多种毒力因子，现在正在观察一种以前很少报道过的细胞内病原体的表型，从而为解密嗜肺军团菌毒力机制奠定了基础。最近，我们开发了一种改进的 CRISPRi 工具，允许多重基因沉默来寻找当同时沉默时使军团菌毒性降低的基因。在一项概念验证研究中，我们使用这种方法来探测一组高度保守的跨膜效应器，以了解它们在军团菌在人类巨噬细胞中复制过程中的重要性。几个基因组合被确定为至关重要，这些组合已成为我们未来研究的重点，目标是开发抑制化合物。在人类免疫细胞感染期间，军团菌驻留在与宿主细胞隔离的膜封闭室或液泡内。然而，当细菌开始复制时，这个“避难所”就面临着挑战，因为周围的液泡也必须扩大，以便为越来越多的军团菌后代提供空间。我们的研究发现军团菌使用毒力因子 VpdC 控制液泡扩张。 VpdC 催化改变液泡膜的脂质组成以促进其扩张。过多或过少的 VpdC 都会干扰适当的液泡扩张，并使军团菌的毒力降低，这表明阻断其液泡的协调扩张是治疗军团菌和相关病原体感染的一种新的治疗方法。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The Legionella Effector Kinase LegK7 Hijacks the Host Hippo Pathway to Promote Infection.

军团菌效应激酶 LegK7 劫持宿主河马通路以促进感染。

DOI：
发表时间：
2018
期刊：
影响因子：
30.3
作者：
Lee, Pei;Machner, Matthias P
通讯作者：
Machner, Matthias P

Host-pathogen interaction profiling using self-assembling human protein arrays.

使用自组装人类蛋白质阵列进行宿主-病原体相互作用分析。

DOI：
发表时间：
2015-04-03
期刊：
影响因子：
4.4
作者：
Yu, Xiaobo;Decker, Kimberly B;Barker, Kristi;Neunuebel, M Ramona;Saul, Justin;Graves, Morgan;Westcott, Nathan;Hang, Howard;LaBaer, Joshua;Qiu, Ji;Machner, Matthias P
通讯作者：
Machner, Matthias P

The Legionella kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation.

军团菌激酶 LegK7 利用 Hippo 通路支架蛋白 MOB1A 进行变构和底物磷酸化。

DOI：
发表时间：
2020
期刊：
影响因子：
11.1
作者：
Lee, Pei;Beyrakhova, Ksenia;Xu, Caishuang;Boniecki, Michal T;Lee, Mitchell H;Onu, Chisom J;Grishin, Andrey M;Machner, Matthias P;Cygler, Miroslaw
通讯作者：
Cygler, Miroslaw

Catch and release: Rab1 exploitation by Legionella pneumophila.

捕获并释放：嗜肺军团菌对 Rab1 的利用。