IDENTIFICATION OF NOVEL HOST FACTORS THAT REDUCE CELL DAMAGE INDUCED BY THE BACTERIAL PORE‐FORMING TOXIN STREPTOLYSIN O
Authors: ESCAJADILLO TAMARA , Popov Lauren , Dahesh Samira , Carette Jan E. , Nizet Victor 
Published: June 2021

Group A Streptococcus (GAS) expresses a multitude of virulence factors with diverse properties and modes of action that act in concert to overcome host innate immune defense mechanisms. Pore forming toxin (PFT) streptolysin O (SLO) is a well‐characterized virulence factor produced by nearly all GAS clinical isolates that promotes dermal necrosis, phagocytic cell dysfunction, and increased mortality during invasive infection. To increase our understanding of how SLO modulates host cell biology, we conducted a high‐throughput genetic screen in human cells to discover novel host factors required for SLO cytotoxicity. We applied a technique that was previously used to discover a role for plekstrin‐homology domain containing protein7 (PLEKHA7) in mediating cytotoxicity of a related bacterial PFT, staphylococcal α‐toxin (Popov et al. PNAS 2015). In the present study, haploid human cells (HAP1) carrying knockout alleles in essentially all genes through insertional mutagenesis were intoxicated with SLO for the live/dead genetic screen. Several screen hits, i.e. gene deletions most enriched in the surviving cell pool, were validated using CRISPR/Cas9 gene editing to generate HAP1 knockout (Δ) subclone cell lines. Multiple gene candidates that modulate susceptibility to SLO were validated, including sterol regulatory element binding protein 2 (SREBP2), which plays a key role in cholesterol homeostasis. SREBP2 KO cells demonstrated increased survival after intoxication with purified SLO and upon live GAS infection. We proceeded to further characterize membrane alterations due to SREBP2 deletion and the effect on SLO membrane binding, and discovered an increase in membrane fluidity with concomitant reduction in cholesterol and lipid raft domains in the plasma membrane of SREBP2 KO cells, and a decrease in SLO binding to the membrane of SREBP2 KO cells. Finally, we screened for potential pharmacological inhibitors of SREBP2 that could potentially mirror the phenotype observed in the genetic knockout, and discovered two pharmacological agents, including the naturally occurring triterpenoid betulinic acid, that reproduced the phenotype in wild‐type cells. Betulinic acid has been examined in multiple therapeutic fields including cancer and immunology, and could potentially be repurposed as a drug intervention during severe GAS infection. Potential advantages of this adjunctive therapy include (a) reduced development of bacterial resistance, since it is the virulence mechanism and not the viability of the pathogen that is targeted, and (b) avoiding perturbations of the normal microflora (and their attendant health benefits), a concern of conventional broad‐spectrum antibiotic therapy.Support or Funding InformationUCSD Graduate Training Program in Cellular and Molecular Pharmacology: T32 GM007752This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.