Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

细菌及其毒素与显着的人类发病率和死亡率有关。虽然一些细菌毒素得到了很好的表征，但大多数毒素的作用机制尚未阐明，从而限制了治疗进展。其中一个例子是由革兰氏阳性病原体蜡状芽孢杆菌产生的高效成孔毒素溶血素 BL (HBL). 然而，HBL 如何发挥其作用以及它是否需要任何宿主因素尚不清楚。在这里，我们描述了一种无偏见的全基因组 CRISPR-Cas9 敲除筛选，该筛选将 LPS 诱导的 TNF-α 因子 (LITAF) 鉴定为 HBL 受体。使用 LITAF 缺陷细胞，随后的第二个全基因组 CRISPR-Cas9 筛选将 LITAF 样蛋白 CDIP1 鉴定为第二个替代受体。我们生成了 LITAF 缺陷小鼠，它们对致命的 HBL 挑战表现出明显的抵抗力。这项工作概述并验证了一种方法，该方法使用迭代全基因组 CRISPR-Cas9 筛选来识别细菌毒素利用的宿主因子的补充，以发挥其无数的生物效应。