great antivirulence potential in vitro, their efficacy in vivo has yet to
be determined.
Since QS inS.aureusis an excellent model system for AIP-based
signaling and as inhibition ofS.aureusQS has therapeutic potential,
methods to assess AIP production and inhibition are essential.
Although AIPs can be detected directly in culture supernatants by
LC-MS, the cost of equipment and the required technical expertise
can be prohibitive [14]. Here, the use of simple and relatively
inexpensivelux-based bio-reporters, genetically engineered so that
they emit light in a concentration-dependent manner only when
provided with an exogenous AIP (Fig.2)[15], is described. These
assays can be used to identify theagrgroup to which a given strain
belongs, as well as to assess the kinetics of AIP production and the
concentration of AIP produced by a givenS.aureusstrain.2 Materials
2.1 Culture Medium 1. Brain heart infusion broth (BHI).
- Brain heart infusion agar: BHI broth plus 1.5% (wt/vol) agar.
- Antibiotics: Chloramphenicol 10 mg/ml in ethanol (1000
stock concentration).
Fig. 2Schematic representation ofS. aureusreporter strain ROJ48 harboring the expression construct
pSKermP2-agrC1A. Incubation with exogenously supplied AIP-1 (red chevron) from either filtered culture
supernatant or a synthetic supply drives AgrA phosphorylation. Phosphorylated AgrA binds to and further
increases expression from both the P2 and P3 promoters resulting in the production of bioluminescence
Detection of AIPs Produced byS. aureus 91