Abstract
BackgroundThe delta-toxin (δ-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 δ-toxin production across S. aureus strains and identify genetic loci potentially associated with differences between strains.MethodsA set of 124 S. aureus strains was genome-sequenced and δ-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 δ-toxin levels. We constructed XGBoost models to predict toxin production based on genetic loci discovered to be potentially associated with the phenotype.ResultsThe S. aureus strain set encompassed 40 sequence types (STs) in 23 clonal complexes (CCs). δ-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 δ-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 δ-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.ConclusionsThe 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 δ-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.
Highlights
Staphylococcus aureus is a common causative agent of nosocomial and communityacquired infections, encoding a wide variety of factors that damage the host and evade immunity
Toxins contribute to important biological functions: In S. aureus, alpha-toxin is important for initial cell-to-cell contacts in biofilm formation, beta-toxin contributes to biofilm structure and growth via crosslinking, and phenol-soluble modulins (PSMs) are involved in detachment of cells for dispersal (Rudkin et al, 2017)
We showed that δ-toxin accumulation by the carA mutant could be rescued by a cloned version of the gene on an expression plasmid (71% δ-toxin production restoration compared to USA300 JE2) but not an empty vector (0% δ-toxin production restoration). carA encodes the carbamoyl-phosphate synthase small chain protein, which is involved in L-arginine biosynthesis and UMP biosynthesis and has been shown to potentially regulate nitric oxide resistance (Grosser et al, 2018) and be important for the regulation of PSMα1 expression (Hardy et al, 2019)
Summary
Staphylococcus aureus is a common causative agent of nosocomial and communityacquired infections, encoding a wide variety of factors that damage the host and evade immunity. Δ-toxin is the product of the hld gene, which is part of the Agr quorum sensing system. In a community-associated MRSA (CA-MRSA) bacteremia mouse model, PSM α and δ-toxin were shown to be important for disease severity, indicating their importance as virulence factors Δ-toxin is the only PSM shown to induce mast cell degranulation (Nakamura et al, 2013) and increase the severity of S. aureus mediated Atopic Dermatitis (AD), a chronic inflammatory skin disease, affecting 15–30% of children and 5% of adults in the US and industrialized countries (Williams & Flohr, 2006; Pustišek, VurnekŽivković & Šitum, 2016). We analyzed the performance of identified genome variants and metadata for predicting δ-toxin production
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