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

摘要 蜡状芽孢杆菌是一种芽胞形成的革兰氏阳性细菌，是一种人类病原体，通常与 医院感染和食源性疫情。蜡状芽胞杆菌越来越多地被确定为急性呼吸道感染的原因之一。 免疫功能低下的患者和儿童的严重感染和死亡。蜡样芽孢杆菌的毒力因子之一 是一种高效的致孔毒素，溶血素BL(HBL)。然而，潜在的机制是 HBL与靶细胞之间的相互作用以及HBL细胞受体(S)的身份尚不清楚。这 知识差距对临床管理中开发有效的治疗方法提出了重大挑战 潜在的毁灭性蜡样芽胞杆菌感染。因此，为了开发有效的治疗方法，有一种 迫切需要确定毒素的细胞受体(S)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，我们进行了全基因组无偏CRISPR筛查，并确定了 脂多糖诱导的肿瘤坏死因子-α因子(LITAF)是迄今难以捉摸的主要HBL 受体。在这个强大的初步数据的基础上，在目标1中，我们将确定LITAF在HBL中的体内作用 小鼠模型的发病机制。为了做到这一点，我们将对我们在 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 生成LITAF转基因小鼠，允许以特定细胞类型的方式恢复LITAF表达 LITAF KO背景。这些细胞类型特异的表达LITAF的小鼠将使我们能够确定关键组织 对HBL诱导的致命性负有责任的目标。在目标2中，我们将确定LITAF是否是物种独立的 HBL受体，进行全面的突变研究，以确定负责LITAF的关键残基 HBL结合，并证明诱骗受体作为抗HBL治疗的效用。 我们对HBL受体的初步CRISPR筛查表明，CRISPR敲除了小鼠RAW264.7中的LITAF 巨噬细胞对HBL具有完全抵抗力，而同样的基因敲除仅存在于人HT1080细胞中 产生了4倍的抗药性。这提示额外的受体(S)可能参与了HbL介导的 某些细胞类型内的细胞毒性。因此，在目标3中，我们将描述宿主因素的全部补充 通过顺序CRISPR屏幕执行HBL操作所需的。在强劲的初步数据支持下，我们假设 这种替代受体或额外的寄主因子在LITAF 不存在，因此当使用LITAF基因敲除细胞时，可以通过CRISPR屏幕识别。 总之，这项工作将开辟新的无偏见的策略来研究毛孔形成毒素和 哺乳动物靶细胞，潜在地阐明了其他肠道致病细菌使用的共同机制。 这些拟议的研究也将验证无偏逐步CRISPR的使用 其他毒素劫持的因子在调节细菌发病中的作用。 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