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

Abstract

A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. 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 on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.

FIG. 1

Growth dependence of the transcription of lukPV (○), hlgA (●), and hlgCB (▿) in wild-type S. aureus ATCC 49775 grown in YCP medium. The expression of the genes encoding staphylococcal leucotoxins was semiquantified by RT-PCR. (A) Growth curve. (B) Semiquantitation curves of the expression of lukPV, hlgA, and hlgCB. The data represent the means ± standard deviations (error bars) from three RT-PCRs.

FIG. 2

Growth dependence of the transcription of lukED (○), hlgA (●), and hlgCB (▿) in wild-type S. aureus Newman (NTCC 8178) grown in YCP medium. The expression of the genes encoding staphylococcal leucotoxins was semiquantified by RT-PCR. (A) Growth curve. (B) Semiquantitation curves of the expression of lukED, hlgA, and hlgCB. The data represent the means ± standard deviations (error bars) from three RT-PCRs.

FIG. 3

Expression of lukPV, hlgA, and hlgCB of S. aureus ATCC 49775 (wild type) (■) and agr (●), sar (⧫), agr sar (▾) mutants of the same strain grown in YCP medium. (A) Growth curves. (B, C, and D) Semiquantitative curves specific for lukPV, hlgA, and hlgCB, respectively. The data represent means ± standard deviations (error bars) from three RT-PCR.

FIG. 4

Expression of lukED, hlgA, and hlgCB of S. aureus Newman NTCC 8178 wild-type strain (■) and agr (●), sar (⧫), and agr sar (▾) mutant strains grown in YCP medium. (A) Growth curves. (B, C, and D) Semiquantitative curves specific for lukED, hlgA and hlgCB, respectively. The data represent means ± standard deviations (error bars) from three RT-PCR analyses.