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Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors
Jie Liu1, Zehua Zuo2, Inka Sastalla3
1Aging Institute of University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA 15219, USA; Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
Abstract:
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.
Insights
Researchers identified host factors for the potent Bacillus cereus toxin, hemolysin BL (HBL). Genome-wide screens revealed LPS-induced TNF-α factor (LITAF) and CDIP1 as HBL receptors, leading to resistant mice.
Area of Science:
- Microbiology
- Immunology
- Genetics
Background:
- Bacterial toxins cause significant human disease, but their mechanisms and host interactions are often unknown.
- Hemolysin BL (HBL), a potent toxin from Bacillus cereus, has an uncharacterized mechanism of action and host factor requirements.
- Understanding toxin-host interactions is crucial for developing targeted therapeutics.
Purpose of the Study:
- To identify host factors essential for hemolysin BL (HBL) toxin activity using unbiased screening.
- To elucidate the specific host receptors mediating HBL's cellular effects.
- To validate the identified host factors in a preclinical model.
Main Methods:
- Genome-wide CRISPR-Cas9 knockout screens were employed to identify host genes involved in HBL toxicity.
- Iterative screening was used, first in wild-type cells and then in cells deficient for the initially identified receptor.
- LITAF-deficient mice were generated to assess in vivo resistance to HBL.
Main Results:
- LPS-induced TNF-α factor (LITAF) was identified as the primary receptor for HBL.
- A subsequent screen identified CDIP1 as a secondary, alternative receptor for HBL.
- LITAF-deficient mice demonstrated significant resistance to lethal HBL challenge, validating LITAF's role.
Conclusions:
- LITAF and CDIP1 are critical host receptors for the Bacillus cereus toxin HBL.
- Iterative genome-wide CRISPR screens provide a powerful platform for discovering host factors exploited by bacterial toxins.
- This research opens avenues for therapeutic strategies targeting HBL-mediated pathogenesis.
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