-Hemolysin, not agrA mutation, inhibits the hemolysis of -hemolysin in Staphylococcus aureus laboratory and clinical strains

Staphylococcus aureus produces various hemolysins regulated by the Agr-QS system, except beta-hemolysin encoded by the gene hlb. A classical laboratory S. aureus strain RN4220 displays only the beta-hemolysin phenotype. It was suspected that the 8A mutation at the end of its agrA gene delayed the expressions of hla and RNAIII, then failed to express alpha- and delta-hemolysins. However, hla gene expression was detected at the later culture time without alpha-hemolysin phenotype, the reason for such a phenotype has not been clearly understood. We created hlb knockout and complementary mutants via homologous recombination in RN4220 and NRS049, two strains that normally produce beta-hemolysin and carry agrA mutation. We found interestingly that the presence or absence of alpha-hemolysin phenotype in such strains depended on the expression of beta-hemolysin instead of agrA mutations, which only inhibited delta-hemolysin expression. The hemolysis phenotype was verified by the Christie-Atkinson-Munch-Peterson (CAMP) test. Quantitative reverse transcription PCR was carried out to evaluate the relative gene expressions of hlb, hla, and RNAIII. The construction of mutants did not affect the agrA mutation status. We demonstrate that the absence of alpha-hemolysin in S. aureus RN4220 and NRS049 strains is attributed to their production of beta-hemolysin instead of agrA mutation. Our findings broaden the understanding of the molecular mechanisms that control hemolysin expression in S. aureus that is crucial for the development of new therapeutic strategies to combat S. aureus infections.