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，是一种人类病原体，通常与 医院感染和食源性疾病爆发。蜡样芽胞杆菌越来越多地被确定为急性 免疫功能低下的患者和儿童出现严重感染和死亡。蜡样芽孢杆菌的毒力因子之一 是一种高效的成孔毒素，溶血素 BL (HBL)。然而，其背后的机制 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。这个 知识差距对临床管理中开发有效疗法提出了重大挑战 具有潜在破坏性的蜡样芽孢杆菌感染。因此，为了开发有效的治疗方法， 迫切需要识别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细胞受体。 基于这一观察，我们进行了无偏见的全基因组 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。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 小鼠模型中的发病机制。为此，我们将表征我们在中产生的 LITAF 基因敲除小鼠 在该应用中，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，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表达 LITAF 的小鼠将使我们能够确定关键组织 负责 HBL 诱导致死率的目标。在目标 2 中，我们将确定 LITAF 是否是与物种无关的 HBL 受体，进行全面的诱变研究，以确定负责 LITAF 的关键残基 HBL 结合，并证明诱饵受体作为抗 HBL 疗法的效用。 我们对 HBL 受体的初步 CRISPR 筛选表明，小鼠 RAW264.7 中 LITAF 的 CRISPR 敲除 巨噬细胞导致对 HBL 的完全抵抗，而相同的敲除仅在人类 HT1080 细胞中 使抵抗力增加了4倍。这表明额外的受体可能参与 HBL 介导的 某些细胞类型内的细胞毒性。因此，在目标3中，我们将描绘宿主因素的完整补充 通过连续 CRISPR 筛选进行 HBL 作用所需。在强有力的初步数据的支持下，我们假设 当 LITAF 存在时，这种替代受体或其他宿主因子将变得越来越重要 不存在，因此当使用 LITAF 敲除细胞时可以通过 CRISPR 筛选来识别。 总之，这项工作将为研究成孔毒素和成孔毒素之间的相互作用开辟新的公正策略。 哺乳动物靶细胞，可能阐明其他肠道致病细菌使用的常见机制。 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