Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.