Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR

A competitive reverse transcription-PCR method was developed for the semiqu antitation of the expression of genes encoding bicomponent leucotoxins of S taphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemo lysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure i ncluded RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain re action. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10(4) CFU to 10(2) mRNA/CFU, depending o n the gene. Both specific mRNAs were more significantly expressed at the la te-exponential phase of growth, Expression was about 100-fold higher in yea st extract-Casamino Acids-pyruvate medium than in heart infusion medium. Ex pression of the widely distributed gamma-hemolysin locus in the NTCC 8178 s train was around 10-fold diminished compared with that in the ATCC 49775 st rain, Because of the lower level of hlgA expression, the corresponding prot ein, which is generally not abundant in culture supernatant, should be inve stigated for its contribution to the leucotoxin-associated virulence. The a gr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, exc ept that expression of hlgA was not affected in the ngr mutant.