Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae ß-Hemolysin/Cytolysin-Induced Cell Death.

ABSTRACT

Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT ß-hemolysin/cytolysin (ßhc). Little is understood about the cellular factors involved in ßhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to ßhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for ßhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of ßhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on ßhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that ßhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to ßhc, a toxin required for GBS virulence. These data suggest that ßhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.