Essory regulatory protein (SarA) and the S. aureus exoprotein (Sae) two-component system has also been shown to positively regulate the expression of a number of S. aureus cytotoxins at the transcriptional level (Fig. 7) (300, 308?10). SarA has been shown to positively regulate the gamma-hemolysin genes hlgCB and the gene encoding alpha-hemolysin, hla, as determined by microarray analysis (300). Like Agr, SarA is a PP58 web global regulator with influences on the expression of many S. aureus virulence genes, including the Agr system itself (311?14). Thus, SarA may impose significant indirect influences on leucocidin gene expression via its positive modulation of the Agr-Rot axis. In contrast to the presumed indirect influences of SarA on leucocidin gene expression, the S. aureus exoprotein regulatory system (SaePQRS) is known to play a direct role in the regulation of a number of S. aureus leucocidin genes (Fig. 7) (309, 315). SaeRS is a two-component system that responds to host environmental cues, including PMNs, to upregulate the expression of a diverse repertoire of S. aureus order MLN1117 secreted proteins, many of which are major virulence factors (304, 309, 310, 316?21). SaeR binds to a consensus sequence within virulence factor promoters to induce gene expression (309, 321). SaeR binding sites have been identified within the promoter regions of nearly all leucocidin genes, including lukSF, hlgA, hlgCB, lukED, and lukAB (lukHG) (309, 321). Experimentally, an sae deletion mutant of S. aureus has significantly reduced transcript levels of all known leucocidins and is dramatically attenuated in murine infection models (308?10, 315). In addition to the Agr, SarA, and Sae regulatory inputs, the master regulator of iron acquisition, Fur, provides additional regulatory control over leucocidin gene expression (322). S. aureus fur mutants significantly upregulate the production of LukED and HlgCB in broth culture (322). Whether the regulation of leucocidins by Fur occurs via direct or indirect mechanisms is yet to be determined. Nevertheless, the induction of LukED and HlgCB in a fur mutant links leucocidin production with nutrient acquisition and suggests that leucocidins may be responsive to other forms of metabolic and nutritional signals. In summary, while we have gained a better appreciation for the fundamental regulatory inputs that influence leucocidin gene expression, the diverse signalsand mechanistic details underlying the optimal activity of these complex regulatory pathways remain to be uncovered.Regulation at the Posttranslational LevelWhile understanding leucocidin gene regulation at the transcriptional level has been a primary research focus, a small number of studies have assessed the possibility that posttranslational modifications may serve to modulate leucocidin activity. Two studies by Kamio and colleagues suggest that HlgC is phosphorylated in a protein kinase A-dependent manner and that this phosphorylation event is required for the activity of HlgCB on host cells (164, 165). There is a predicted consensus phosphorylation sequence (KRST) within the C terminus of HlgC that is believed to be recognized by protein kinase A (164). In initial studies, it was determined that the threonine at position 246 could be phosphorylated in the presence of protein kinase A but only when HlgC was first denatured (164). When this threonine at position 246 was mutated to an alanine, phosphorylation of denatured HlgC did not occur, and the toxin had no cytoly.Essory regulatory protein (SarA) and the S. aureus exoprotein (Sae) two-component system has also been shown to positively regulate the expression of a number of S. aureus cytotoxins at the transcriptional level (Fig. 7) (300, 308?10). SarA has been shown to positively regulate the gamma-hemolysin genes hlgCB and the gene encoding alpha-hemolysin, hla, as determined by microarray analysis (300). Like Agr, SarA is a global regulator with influences on the expression of many S. aureus virulence genes, including the Agr system itself (311?14). Thus, SarA may impose significant indirect influences on leucocidin gene expression via its positive modulation of the Agr-Rot axis. In contrast to the presumed indirect influences of SarA on leucocidin gene expression, the S. aureus exoprotein regulatory system (SaePQRS) is known to play a direct role in the regulation of a number of S. aureus leucocidin genes (Fig. 7) (309, 315). SaeRS is a two-component system that responds to host environmental cues, including PMNs, to upregulate the expression of a diverse repertoire of S. aureus secreted proteins, many of which are major virulence factors (304, 309, 310, 316?21). SaeR binds to a consensus sequence within virulence factor promoters to induce gene expression (309, 321). SaeR binding sites have been identified within the promoter regions of nearly all leucocidin genes, including lukSF, hlgA, hlgCB, lukED, and lukAB (lukHG) (309, 321). Experimentally, an sae deletion mutant of S. aureus has significantly reduced transcript levels of all known leucocidins and is dramatically attenuated in murine infection models (308?10, 315). In addition to the Agr, SarA, and Sae regulatory inputs, the master regulator of iron acquisition, Fur, provides additional regulatory control over leucocidin gene expression (322). S. aureus fur mutants significantly upregulate the production of LukED and HlgCB in broth culture (322). Whether the regulation of leucocidins by Fur occurs via direct or indirect mechanisms is yet to be determined. Nevertheless, the induction of LukED and HlgCB in a fur mutant links leucocidin production with nutrient acquisition and suggests that leucocidins may be responsive to other forms of metabolic and nutritional signals. In summary, while we have gained a better appreciation for the fundamental regulatory inputs that influence leucocidin gene expression, the diverse signalsand mechanistic details underlying the optimal activity of these complex regulatory pathways remain to be uncovered.Regulation at the Posttranslational LevelWhile understanding leucocidin gene regulation at the transcriptional level has been a primary research focus, a small number of studies have assessed the possibility that posttranslational modifications may serve to modulate leucocidin activity. Two studies by Kamio and colleagues suggest that HlgC is phosphorylated in a protein kinase A-dependent manner and that this phosphorylation event is required for the activity of HlgCB on host cells (164, 165). There is a predicted consensus phosphorylation sequence (KRST) within the C terminus of HlgC that is believed to be recognized by protein kinase A (164). In initial studies, it was determined that the threonine at position 246 could be phosphorylated in the presence of protein kinase A but only when HlgC was first denatured (164). When this threonine at position 246 was mutated to an alanine, phosphorylation of denatured HlgC did not occur, and the toxin had no cytoly.
