Defining cellular receptors for the Bacillus cereus hemolysin BL toxin (HBL) and the development of anti-HBL therapies

蜡样芽孢杆菌溶血素 BL 毒素 (HBL) 细胞受体的定义和抗 HBL 疗法的开发

• 批准号: 10112820
• 负责人: Shihui Liu
• 金额: $ 50.16万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-21 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10112820
• 关键词: Acute; Address; Animals; Anthrax disease; Bacillus cereus; Bacteria; Binding; Blindness; CRISPR screen; CRISPR/Cas technology; Cell Surface Receptors; Cells; Cessation of life; Child; Clinical Management; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cultured Cells; Cytolysis; Data; Development; Endophthalmitis; Genes; Gram-Positive Bacteria; Health; Hemolysin; Histology; Human; Immunocompetent; Immunocompromised Host; In Vitro; Infection; Integration Host Factors; Investigation; Investigational Therapies; Knock-out; Knockout Mice; Knowledge; Libraries; Link; Lipopolysaccharides; Mediating; Molecular; Mus; Mutagenesis; Nosocomial Infections; Pathogenesis; Pneumonia; Proteins; Receptor Gene; Reporting; Reproduction spores; Resistance; Role; TNF gene; Targeted Toxins; Testing; Therapeutic; Tissues; Toxin; Transgenic Mice; Virulence; Virulence Factors; Work; alpha Toxin; base; cell type; cytotoxicity; deep sequencing; effective therapy; expectation; foodborne outbreak; genome-wide; human pathogen; in vivo; macrophage; mouse model; nanomolar; novel therapeutics; pathogen; pathogenic bacteria; rapid diagnosis; receptor; restoration; screening; targeted treatment; therapeutically effective; whole genome

Abstract Bacillus cereus, a spore-forming, gram-positive bacterium, is a human pathogen commonly associated with hospital infections and foodborne outbreaks. Increasingly, B. cereus has been identified as a cause of acute severe infections and deaths in immunocompromised patients and children. One of B. cereus’ virulence factors is the highly potent pore-forming toxin, hemolysin BL (HBL). However, the mechanisms underlying the interactions between HBL and target cells and the identity of the HBL cellular receptor(s) remains unknown. This knowledge gap presents significant challenges for developing effective therapies in clinical management of potentially devastating B. cereus infections. Therefore, in order to develop effective therapeutics, there is a critical need to identify the toxin cellular receptor(s) and the molecular mechanisms underlying the toxin’s action. Our earlier preliminary data demonstrated that a cellular receptor is required for the cytolytic action of HBL. Based on this observation, we performed an unbiased genome-wide CRISPR screen and have identified Lipopolysaccharide-Induced Tumor Necrosis Factor-α Factor (LITAF) as the major, heretofore elusive, HBL receptor. Building on this strong preliminary data, in Aim 1, we will determine the in vivo role of LITAF in HBL pathogenesis in mouse models. To do so, we will characterize the LITAF knockout mice we have generated in this application, where our preliminary data supports the critical role of LITAF in HBL pathogenesis. We will also generate LITAF transgenic mice allowing the restoration of LITAF expression in a cell type-specific manner in a LITAF KO background. These cell type-specific LITAF-expressing mice will allow us to determine the key tissue targets responsible for HBL-induced lethality. In Aim 2, we will establish whether LITAF is a species-independent HBL receptor, perform comprehensive mutagenesis studies to identify the key residues of LITAF responsible for HBL binding, and demonstrate the utility of decoy receptors as anti-HBL therapy. Our initial CRISPR screen for HBL receptor demonstrated that CRISPR knockout of LITAF in mouse RAW264.7 macrophages resulted in complete resistance to HBL, whereas the same knockout in human HT1080 cells only yielded a 4-fold increase in resistance. This suggests additional receptor(s) may be involved in HBL-mediated cytotoxicity within certain cell types. Therefore, in Aim 3, we will delineate the full complement of host factors required for HBL action via sequential CRISPR screens. Supported by strong preliminary data, we hypothesize that this alternative receptor or additional host factors would become increasingly important when LITAF is absent, and can therefore be identified by a CRISPR screen when LITAF knockout cells are used. Together, this work will open new unbiased strategies for studying interactions between pore-forming toxins and mammalian target cells, potentially elucidating common mechanisms used by other enteropathogenic bacteria. These proposed studies will also validate the use of the unbiased stepwise CRISPR factors hijacked by other toxins in modulating bacterial pathogenesis. screens to identify host

抽象的 蜡状芽孢杆菌是一种散射形成的革兰氏阳性细菌，是一种通常与 医院感染和食源性暴发。越来越多的蜡状芽孢杆菌被确定为急性的原因 免疫功能低下的患者和儿童的严重感染和死亡。蜡状芽孢杆菌的病毒因素之一 是高潜在的孔形成毒素，血溶质蛋白BL（HBL）。但是， HBL与靶细胞之间的相互作用以及HBL细胞接收器的身份仍然未知。这 知识差距提出了开发有效疗法在临床管理中的重大挑战 可能毁灭性的蜡状芽孢杆菌感染。因此，为了开发有效的治疗，有一个 识别毒素细胞接收器和毒素作用的分子机制的迫切需要。 我们较早的初步数据表明，HBL的细胞溶解作用需要细胞接收器。 基于此观察结果，我们进行了无偏基因组CRISPR屏幕，并已经确定了 脂多糖诱导的肿瘤坏死因子-α因子（LITAF）是主要的，迄今难以捉摸的HBL 受体。在此强大的初步数据的基础上，在AIM 1中，我们将确定LITAF在HBL中的体内作用 小鼠模型中的发病机理。为此，我们将描述我们已经产生的Litaf淘汰小鼠 该应用，我们的初步数据支持LITAF在HBL发病机理中的关键作用。我们也会 生成LITAF转基因小鼠，允许以细胞类型特异性的方式恢复LITAF表达 Litaf KO背景。这些表达LITAF的小鼠这些细胞类型的小鼠将使我们能够确定钥匙组织 负责HBL引起的致死性的目标。在AIM 2中，我们将确定LITAF是否是独立于物种的 HBL接收器，进行全面的诱变研究，以确定负责LITAF的关键保留 HBL结合，并证明诱饵受体作为抗HBL疗法的实用性。 我们对HBL受体的最初CRISPR屏幕表明，鼠标RAW264.7中的LITAF的CRISPR敲除 巨噬细胞对HBL产生完全抗性，而人类HT1080细胞中相同的敲除 电阻增加了4倍。这表明HBL介导的其他受体可能涉及 某些细胞类型中的细胞毒性。因此，在AIM 3中，我们将全面列出主机因素 通过顺序CRISPR屏幕进行HBL动作所需。在强大的初步数据的支持下，我们假设 当Litaf是 缺乏，因此，当使用Litaf敲除细胞时，可以通过CRISPR屏幕识别。 这项工作将共同开设新的公正策略，以研究孔形成毒素与 哺乳动物靶细胞，可能阐明其他肠病细菌使用的常见机制。 这些拟议的研究还将验证使用公正的逐步CRISPR 其他毒素在调节细菌发病机理中劫持的因素。 屏幕以识别主机