Complex regulation of gamma-hemolysin expression impacts S. aureus virulence

Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome encoded pore-forming toxin that targets the C5a receptor, similarly as the phage-encoded Panton-Valentine Leucocidin. Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in HlgC and HlgB yields between isolates. Interestingly, when testing the hypothesis that HlgCB might be associated with severe S. aureus CAP, we found that a high level of HlgCB synthesis was associated with mortality in a rabbit model of pneumonia. To decipher the molecular basis for the variation in hlgCB and hlgB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. HlgCB amount and HlgC/HlgB ratio were found to both depend on promotor activity, mRNA stability and translatability, and on the presence of an individual hlgB mRNA processed from the hlgCB transcript. Strikingly, toe-printing and in vitro translation assays revealed that a single SNP in the 5-UTR of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in HlgC but not in HlgB; the level of HlgB is likely to have been maintained by the presence of the processed hlgB mRNA. This work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect, where subtle genomic variations have a major impact on phenotype and virulence. Author SummaryThe Gram-positive bacterium Staphylococcus aureus can provoke a wide range of infections due to its ability to produce a large diversity of virulence factors, including immune evasion molecules, adhesins, and toxins. Some of these toxin-encoding genes are localized in mobile genetic elements, and are thus not present in all strains, whilst others are encoded in the core-genome and present in all strains. Gamma-hemolysin CB is a core-genome encoded toxin but its amount varies between community-acquired pneumonia isolates. The regulation mechanisms underlying this variation however, are not well characterized. Here, we show that gamma-hemolysin expression levels vary largely among clinical strains and that, when highly produced, it induces high mortality in a rabbit model of pneumonia. The molecular basis for the variation in gamma-hemolysin expression depends on multiple mechanisms including promoter strength, transcript stability and processing, and translatability (i.e. the amount of protein that is synthetized by the ribosome for a given transcript). Incredibly, all these factors rely on a subtle genetic modification. This work emphasizes the importance of the disparity in virulence factor expression among clinical isolates and points the extreme complexity of the molecular mechanisms underlying their regulation, rendering the prediction of virulence for a clinical isolate difficult.