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

摘要 蜡样芽孢杆菌是一种形成孢子的革兰氏阳性细菌，是一种常见的人类病原体， 医院感染和食源性疫情。越来越多，B。蜡样囊肿已被确定为急性 免疫功能低下的患者和儿童严重感染和死亡。B之一。蜡样芽胞杆菌毒力因子 是一种高效的成孔毒素，溶血素BL（HBL）。然而， HBL和靶细胞之间的相互作用以及HBL细胞受体的身份仍然未知。这 知识差距对在临床管理中开发有效的治疗方法提出了重大挑战。 潜在的毁灭性的B。蜡状病毒感染因此，为了开发有效的治疗方法， 迫切需要确定毒素细胞受体和毒素作用的分子机制。 我们早期的初步数据表明，HBL的细胞溶解作用需要一个细胞受体。 基于这一观察结果，我们进行了无偏的全基因组CRISPR筛选，并确定了 脂多糖诱导的肿瘤坏死因子-α因子（LITAF）是迄今为止难以捉摸的主要HBL 受体的基于这些强有力的初步数据，在目标1中，我们将确定LITAF在HBL中的体内作用 在小鼠模型中的发病机制。为了做到这一点，我们将描述我们在2010年产生的LITAF敲除小鼠的特征。 我们的初步数据支持LITAF在HBL发病机制中的关键作用。我们还将 产生LITAF转基因小鼠，其允许以细胞类型特异性方式恢复LITAF表达， LITAF KO背景。这些细胞类型特异性LITAF表达小鼠将使我们能够确定关键组织 负责HBL诱导的致死性的靶点。在目标2中，我们将确定LITAF是否是种独立的 HBL受体，进行全面的诱变研究，以确定LITAF的关键残基负责 HBL结合，并证明诱饵受体作为抗HBL治疗的效用。 我们对HBL受体的初始CRISPR筛选表明，小鼠RAW 264.7中LITAF的CRISPR敲除 巨噬细胞导致对HBL的完全抗性，而在人HT 1080细胞中相同的敲除仅导致对HBL的完全抗性。 产生了四倍的抗性。这表明另外的受体可能参与了HBL介导的 在某些细胞类型中的细胞毒性。因此，在目标3中，我们将描述宿主因子的完整补充 通过连续的CRISPR筛选进行HBL行动。在强有力的初步数据支持下，我们假设 这种替代受体或其他宿主因子将变得越来越重要， 因此，当使用LITAF敲除细胞时，可以通过CRISPR筛选来鉴定。 总之，这项工作将打开新的无偏见的战略，研究之间的相互作用孔形成毒素和 哺乳动物靶细胞，可能阐明其他肠道致病细菌使用的共同机制。 这些拟议的研究还将验证无偏逐步CRISPR的使用， 在调节细菌发病机制中被其他毒素劫持的因子。 用于识别主机的屏幕

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