Ne another to exert GLPG0187 biological activity precise control over toxin gene expression. It is becoming clear that while there are dominant inputs, the leucocidins display differential regulatory patterns that still require substantial investigation. Semiquantitative reverse transcriptase PCR (semi-qRT-PCR) studies confirmed previous studies indicating that YCP medium increases leucocidin expression compared to that in BHI medium (291). These transcript studies also demonstrated growth phase-dependent expression of the leucocidins, with greater transcript abundances of hlgA, hlgCB, lukED, and lukSF-PV as the bacteria entered late exponential and early stationary phases (291). Such growth phasedependent gene expression is suggestive of regulatory input imparted by the accessory gene regulatory (Agr) system in S. aureus (Fig. 7) (292). The Agr system (composed of 4 genes, agrBCDA) is a quorum-sensing system that becomes activated upon reaching ahigh bacterial density (292). agrC and agrA encode a histidine kinase and a response regulator, respectively, like other canonical two-component regulatory systems. AgrC is activated upon reaching a high bacterial density due to the increased effective concentration of a small PP58 cost peptide ligand processed from AgrD by AgrB (293?95). This small peptide is known as an autoinducing peptide (AIP) that interacts with AgrC to activate the AgrCA twocomponent system (294, 296). Agr activation leads to the increased transcription of two RNAs, RNAII (encoding AgrBCDA) and RNAIII, a regulatory RNA that inhibits and/or promotes the translation of multiple gene products, including a transcription factor known as repressor of toxins (Rot) (292, 297?00). Rot is so named due to its ability to act as a repressor at S. aureus toxin promoters; however, it is known to also activate promoters of other virulence genes (Fig. 7) (301?04). Rot is negatively regulated at the translational level by RNAIII, such that when the Agr system is activated, RNAIII inhibits Rot translation due to antisense binding to the rot transcript, thereby increasing the expression levels of toxins (305?07). Thus, on a global level, the activation of the Agr system leads to increased expression levels of majorJune 2014 Volume 78 Numbermmbr.asm.orgAlonzo and TorresS. aureus virulence factors, including proteases, cytotoxins, and cytolytic peptides. Indeed, it was found that when agr is deleted, S. aureus expresses lower overall levels of leucocidin transcripts (291, 300). This equates to largely reduced leucocidin abundance at the protein level, although some toxins are more dramatically influenced than others. For example, in strain Newman, levels of LukED and Hla are dramatically reduced in an agr mutant background, while the abundances of LukAB/HG and HlgCB are reduced but to a lesser degree (47). As described above, the primary input responsible for the reduced production of toxins in an agr mutant is believed to be increased repression of gene expression imparted by Rot, which is produced in a greater abundance in an agr mutant (in an agr mutant, RNAIII levels are low, and thus, Rot translation is derepressed). Indeed, the generation of an agr rot double mutant restores most leucocidins to levels similar to or greater than those of wild-type S. aureus (47). This suggests that the Agr-Rot axis is critical for optimal leucocidin regulation, although it is not the only regulatory input dictating leucocidin gene expression. In addition to Agr and Rot, the staphylococcal acc.Ne another to exert precise control over toxin gene expression. It is becoming clear that while there are dominant inputs, the leucocidins display differential regulatory patterns that still require substantial investigation. Semiquantitative reverse transcriptase PCR (semi-qRT-PCR) studies confirmed previous studies indicating that YCP medium increases leucocidin expression compared to that in BHI medium (291). These transcript studies also demonstrated growth phase-dependent expression of the leucocidins, with greater transcript abundances of hlgA, hlgCB, lukED, and lukSF-PV as the bacteria entered late exponential and early stationary phases (291). Such growth phasedependent gene expression is suggestive of regulatory input imparted by the accessory gene regulatory (Agr) system in S. aureus (Fig. 7) (292). The Agr system (composed of 4 genes, agrBCDA) is a quorum-sensing system that becomes activated upon reaching ahigh bacterial density (292). agrC and agrA encode a histidine kinase and a response regulator, respectively, like other canonical two-component regulatory systems. AgrC is activated upon reaching a high bacterial density due to the increased effective concentration of a small peptide ligand processed from AgrD by AgrB (293?95). This small peptide is known as an autoinducing peptide (AIP) that interacts with AgrC to activate the AgrCA twocomponent system (294, 296). Agr activation leads to the increased transcription of two RNAs, RNAII (encoding AgrBCDA) and RNAIII, a regulatory RNA that inhibits and/or promotes the translation of multiple gene products, including a transcription factor known as repressor of toxins (Rot) (292, 297?00). Rot is so named due to its ability to act as a repressor at S. aureus toxin promoters; however, it is known to also activate promoters of other virulence genes (Fig. 7) (301?04). Rot is negatively regulated at the translational level by RNAIII, such that when the Agr system is activated, RNAIII inhibits Rot translation due to antisense binding to the rot transcript, thereby increasing the expression levels of toxins (305?07). Thus, on a global level, the activation of the Agr system leads to increased expression levels of majorJune 2014 Volume 78 Numbermmbr.asm.orgAlonzo and TorresS. aureus virulence factors, including proteases, cytotoxins, and cytolytic peptides. Indeed, it was found that when agr is deleted, S. aureus expresses lower overall levels of leucocidin transcripts (291, 300). This equates to largely reduced leucocidin abundance at the protein level, although some toxins are more dramatically influenced than others. For example, in strain Newman, levels of LukED and Hla are dramatically reduced in an agr mutant background, while the abundances of LukAB/HG and HlgCB are reduced but to a lesser degree (47). As described above, the primary input responsible for the reduced production of toxins in an agr mutant is believed to be increased repression of gene expression imparted by Rot, which is produced in a greater abundance in an agr mutant (in an agr mutant, RNAIII levels are low, and thus, Rot translation is derepressed). Indeed, the generation of an agr rot double mutant restores most leucocidins to levels similar to or greater than those of wild-type S. aureus (47). This suggests that the Agr-Rot axis is critical for optimal leucocidin regulation, although it is not the only regulatory input dictating leucocidin gene expression. In addition to Agr and Rot, the staphylococcal acc.
