Abstract
The staphylococcal accessory gene regulatory (agr) operon is a well-characterised global regulatory element that is important in the control of virulence gene expression for Staphylococcus aureus, a major human pathogen. Hence, accurate and sensitive measurement of Agr activity is central in understanding the virulence potential of Staphylococcus aureus, especially in the context of Agr dysfunction, which has been linked with persistent bacteraemia and reduced susceptibility to glycopeptide antibiotics. Agr function is typically measured using a synergistic haemolysis CAMP assay, which is believe to report on the level of expression of one of the translated products of the agr locus, delta toxin. In this study we develop a vesicle lysis test (VLT) that is specific to small amphipathic peptides, most notably delta and Phenol Soluble Modulin (PSM) toxins. To determine the accuracy of this VLT method in assaying Agr activity, we compared it to the CAMP assay using 89 clinical Staphylococcus aureus isolates. Of the 89 isolates, 16 were designated as having dysfunctional Agr systems by the CAMP assay, whereas only three were designated as such by VLT. Molecular analysis demonstrated that of these 16 isolates, the 13 designated as having a functional Agr system by VLT transcribed rnaIII and secreted delta toxin, demonstrating they have a functional Agr system despite the results of the CAMP assay. The agr locus of all 16 isolates was sequenced, and only the 3 designated as having a dysfunctional Agr system contained mutations, explaining their Agr dysfunction. Given the potentially important link between Agr dysfunction and clinical outcome, we have developed an assay that determines this more accurately than the conventional CAMP assay.
Highlights
Staphylococcus aureus is a Gram-positive bacterial pathogen that plays a major role in human disease
Erythrocytes lysis occurs after incubation with 10 mg/ml of individual Phenol Soluble Modulin (PSM) [42], whereas our vesicles require in the range of 1.5–2.5 mM depending on the peptide toxins, both systems requiring incubation at 37uC for 30 min
If we compare this to the results of PSM interaction with POPC vesicles used by Duong et al we see a stark difference in the concentration of PSMs used cells subjected to selected concentrations of purified PSM3a toxin
Summary
Staphylococcus aureus is a Gram-positive bacterial pathogen that plays a major role in human disease It is an opportunistic pathogen which inhabits the moist squamous epithelium of the anterior nares, permanently colonising approximately 20% of the population and found intermittently in another 50% [1]. The P3 dependent regulatory RNA, RNAIII, is the effector molecule of this regulon and is involved in the up - and down - regulation of specific genes as well as encoding the delta toxin This complex alteration in expression of virulence genes occurs in conjunction with many global regulators namely the DNA-binding Sar family of proteins [11,12], the alternative sigma factor [13] and other twocomponent system such as SaeRS [14] and ArlRS [15]
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