Dataset Information

Genomic analysis of variability in Delta-toxin levels between Staphylococcus aureus strains.

ABSTRACT: Background:The delta-toxin (?-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 ?-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 ?-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 ?-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). ?-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 ?-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 ?-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 ?-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.

SUBMITTER: Su M

PROVIDER: S-EPMC7100594 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Genomic analysis of variability in Delta-toxin levels between <i>Staphylococcus aureus</i> strains.

Su Michelle M Lyles James T JT Petit Iii Robert A RA Peterson Jessica J Hargita Michelle M Tang Huaqiao H Solis-Lemus Claudia C Quave Cassandra L CL Read Timothy D TD

PeerJ 20200324

<h4>Background</h4>The delta-toxin (δ-toxin) of <i>Staphylococcus aureus</i> 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 <i>S. aureus</i> strains and identify genetic loci potentially associated with differences between strains.<h4>Methods</h4>A set of 124 <i>S. aureus</i> strains was genome-sequenced and δ-toxin levels in stationary phase sup ...[more]

PMID: 32231873

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Project description:Streptococcus pneumoniae rapidly kills Staphylococcus aureus by producing membrane-permeable hydrogen peroxide (H2O2). The mechanism by which S. pneumoniae-produced H2O2 mediates S. aureus killing was investigated. An in vitro model that mimicked S. pneumoniae-S. aureus contact during colonization of the nasopharynx demonstrated that S. aureus killing required outcompeting densities of S. pneumoniae Compared to the wild-type strain, isogenic S. pneumoniae ΔlctO and S. pneumoniae ΔspxB, both deficient in production of H2O2, required increased density to kill S. aureus While residual H2O2 activity produced by single mutants was sufficient to eradicate S. aureus, an S. pneumoniae ΔspxB ΔlctO double mutant was unable to kill S. aureus A collection of 20 diverse methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains showed linear sensitivity (R2 = 0.95) for S. pneumoniae killing, but the same strains had different susceptibilities when challenged with pure H2O2 (5 mM). There was no association between the S. aureus clonal complex and sensitivity to either S. pneumoniae or H2O2 To kill S. aureus, S. pneumoniae produced ∼180 μM H2O2 within 4 h of incubation, while the killing-defective S. pneumoniae ΔspxB and S. pneumoniae ΔspxB ΔlctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H2O2 incubated with S. pneumoniae ΔspxB eradicated diverse S. aureus strains, suggesting that S. pneumoniae bacteria may facilitate conversion of H2O2 to a hydroxyl radical (·OH). Accordingly, S. aureus killing was completely blocked by incubation with scavengers of ·OH radicals, dimethyl sulfoxide (Me2SO), thiourea, or sodium salicylate. The ·OH was detected in S. pneumoniae cells by spin trapping and electron paramagnetic resonance. Therefore, S. pneumoniae produces H2O2, which is rapidly converted to a more potent oxidant, hydroxyl radicals, to rapidly intoxicate S. aureus strains.IMPORTANCEStreptococcus pneumoniae strains produce hydrogen peroxide (H2O2) to kill bacteria in the upper airways, including pathogenic Staphylococcus aureus strains. The targets of S. pneumoniae-produced H2O2 have not been discovered, in part because of a lack of knowledge about the underlying molecular mechanism. We demonstrated that an increased density of S. pneumoniae kills S. aureus by means of H2O2 produced by two enzymes, SpxB and LctO. We discovered that SpxB/LctO-produced H2O2 is converted into a hydroxyl radical (·OH) that rapidly intoxicates and kills S. aureus We successfully inhibited the toxicity of ·OH with three different scavengers and detected ·OH in the supernatant. The target(s) of the hydroxyl radicals represents a new alternative for the development of antimicrobials against S. aureus infections.

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Project description:Staphylococci producing exfoliative toxins are the causative agents of staphylococcal scalded skin syndrome (SSSS). Exfoliative toxin A (ETA) is encoded by eta, which is harbored on a temperate bacteriophage ΦETA. A recent increase in the incidence of SSSS in North America has been observed; yet it is largely unknown whether this is the result of host range expansion of ΦETA or migration and emergence of established lineages. Here, we detail an outbreak investigation of SSSS in a neonatal intensive care unit, for which we applied whole-genome sequencing (WGS) and phylogenetic analysis of Staphylococcus aureus isolates collected from cases and screening of healthcare workers. We identified the causative strain as a methicillin-susceptible S. aureus (MSSA) sequence type 582 (ST582) possessing ΦETA. To then elucidate the global distribution of ΦETA among staphylococci, we used a recently developed tool to query extant bacterial WGS data for biosamples containing eta, which yielded 436 genomes collected between 1994 and 2019 from 32 countries. Applying population genomic analysis, we resolved the global distribution of S. aureus with lysogenized ΦETA and assessed antibiotic resistance determinants as well as the diversity of ΦETA. The population is highly structured with eight dominant sequence clusters (SCs) that generally aligned with S. aureus ST clonal complexes. The most prevalent STs included ST109 (24.3%), ST15 (13.1%), ST121 (10.1%), and ST582 (7.1%). Among strains with available data, there was an even distribution of isolates from carriage and disease. Only the SC containing ST121 had significantly more isolates collected from disease (69%, n = 46) than carriage (31%, n = 21). Further, we identified 10.6% (46/436) of strains as methicillin-resistant S. aureus (MRSA) based on the presence of mecA and the SCCmec element. Assessment of ΦETA diversity based on nucleotide identity revealed 27 phylogroups, and prophage gene content further resolved 62 clusters. ΦETA was relatively stable within lineages, yet prophage variation is geographically structured. This suggests that the reported increase in incidence is associated with migration and expansion of existing lineages, not the movement of ΦETA to new genomic backgrounds. This revised global view reveals that ΦETA is diverse and is widely distributed on multiple genomic backgrounds whose distribution varies geographically.

Dataset Information

Genomic analysis of variability in Delta-toxin levels between Staphylococcus aureus strains.

Publications

Genomic analysis of variability in Delta-toxin levels between <i>Staphylococcus aureus</i> strains.

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