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Analysis of quorum sensing activity in staphylococci by RP-HPLC of staphylococcal delta-toxin.

Staphylococcus aureus and Staphylococcus epidermidis rank among the most important pathogens involved in nosocomial infections. Most virulence factors of these bacteria are controlled by the agr locus, which encodes a quorum-sensing system required for celldensity-dependent control of protein expression. The activity of the agr system is an important determinant of staphylococcal pathogenicity, and deletion of the agr locus results in diminished virulence. So far, measurement of agr activity has been performed by Northern analyses of RNAIII, the intracellular regulatory molecule of the agr system. RNAIII controls expression of the target pathogenicity factor genes such as α-, β-, γ-toxin, toxic shock syndrome toxin, lipase, protease, several endotoxins and protein A, to name a few (9). Here, we present a new method for qualitative and quantitative measurement of the activity of the agr system by HPLC analysis of δ-toxin. The gene for δ-toxin, which itself represents a virulence factor, is encoded within RNAIII. Determination of δ-toxin expression therefore offers an alternative to the measurement of RNAIII expression to analyze agr activity. δ-toxin is an amphiphilic molecule with surfactant-like properties. It is often used as a model substance for membrane insertion (6,13). Its amino acid sequence and three-dimensional structure have already been reported (4,8,14). Different purification procedures for δ-toxin have been described, all requiring many purification steps (3,4). We combined hydrophobic interaction chromatography using polystyrene/divinylbenzene materials with a water/acetonitrile gradient to purify and analyze δ-toxin in a one-step procedure. The most important advantage of this procedure is that pretreatment of the samples before analytical HPLC is unnecessary. Supernatants are not normally injected directly onto an analytical HPLC column, but require initial purification by precipitation or initial chromatographic steps, for example. δ-toxin was analyzed directly in the supernatants of bacterial cultures grown in a commonly used complex media [tryptic soy broth and basic medium: 1% tryptone (Difco Laboratories, Detroit, MI, USA), 0.5% yeast extract (Life Technologies, Eggenstein, Germany), 0.5% NaCl, 0.1% K2HPO4 and 0.1% glucose]. The media was centrifuged at 19 000× g for 5 min in a normal bench centrifuge. Benchmarks