Genomic Analysis of Variability in Delta-toxin Levels Between Staphylococcus aureus Strains

Background: The delta-toxin (delta-toxin) of Staphylococcus aureus is the only hemolysin shown to cause mast cell degranulation and is linked to atopic dermatitis, a chronic inflammatory skin disease. We sought to characterize variation in delta-toxin production across S. aureus strains and identify genetic loci potentially associated with differences between strains. Methods: A set of 124 S. aureus strains was genome-sequenced and delta-toxin levels in stationary phase supernatants determined by high performance liquid chromatography (HPLC). SNPs and kmers were associated with differences in toxin production using four genome-wide association study (GWAS) methods. Transposon mutations in candidate genes were tested for their delta-toxin levels. We constructed XGBoost models to predict toxin production based on genetic loci discovered to be potentially associated with the phenotype. Results: The S. aureus strain set encompassed 40 sequence types (STs) in 23 clonal complexes (CCs). delta-toxin production ranged from barely detectable levels to >90,000 units, with a median of >8,000 units. CC30 had significantly lower levels of toxin production than average while CC45 and CC121 were higher. MSSA (methicillin sensitive) strains had higher delta-toxin production than MRSA (methicillin resistant) strains. Through multiple GWAS approaches, 45 genes were found to be potentially associated with toxicity. Machine learning models using loci discovered through GWAS as features were able to predict delta-toxin production (as a high/low binary phenotype) with a precision of .875 and specificity of .990 but recall of .333. We discovered that mutants in the carA gene, encoding the small chain of carbamoyl phosphate synthase, completely abolished toxin production and toxicity in Caenorhabditis elegans. Conclusions: The amount of stationary phase production of the toxin is a strain-specific phenotype likely affected by a complex interaction of number of genes with different levels of effect. We discovered new candidate genes that potentially play a role in modulating production. We report for the first time that the product of the carA gene is necessary for delta-toxin production in USA300. This work lays a foundation for future work on understanding toxin regulation in S. aureus and prediction of phenotypes from genomic sequences.