The accessory growth regulator (Agr)-like quorum sensing (QS) system of controls the production of many toxins including beta toxin (CPB). faster signaling than the 5-mer linear peptide. Strain-related variations in sensing these peptides were detected with CN3685 sensing PTC124 (Ataluren) the synthetic peptides more strongly than CN1795. Consistent with those synthetic peptide results Transwell coculture experiments showed that CN3685 exquisitely senses native AIP signals from other isolates (types A B C and D) while CN1795 barely senses even its own AIP. Finally a AgrD sequence-based peptide with a 6-amino-acid thiolactone ring interfered with CPB production by several strains suggesting potential therapeutic applications. These results indicate that AIP signaling sensitivity and responsiveness vary among strains and suggest GATA6 prefers a 5-mer AIP to initiate Agr signaling. IMPORTANCE possesses an Agr-like quorum sensing (QS) system that regulates virulence sporulation and toxin production. The current study used synthetic peptides to identify the structure-function relationship for the signaling peptide that activates this QS system. We found that a 5-mer peptide induces optimal signaling. Unlike other Agr systems a linear version of this peptide (in addition to thiolactone and lactone versions) could induce signaling. Two strains were found to vary in sensitivity to these peptides. We also found that a PTC124 (Ataluren) 6-mer peptide can inhibit toxin production by some strains suggesting therapeutic applications. INTRODUCTION In humans and livestock is a major cause of histotoxic infections such as human gas gangrene and intestinal infections including enteritis and enterotoxemias (1 2 The versatility of this Gram-positive anaerobic bacterium is largely attributable to its ability to produce ～17 different toxins (3 -5). However individual strains produce only portions of this toxin repertoire; this toxin production variability is used in a classification system that assigns isolates to one of five types (A to E) based upon their production of alpha (CPA) beta (CPB) epsilon (ETX) and iota toxins (4 5 Each type is associated with certain diseases (1 4 during vegetative growth is PTC124 (Ataluren) controlled at least in part by a two-component regulatory system named VirS/VirR (6 -8). In some cases regulatory RNAs are involved in VirS/VirR-mediated regulation although production of perfringolysin O (PFO) is directly regulated by the binding of phosphorylated VirR to DNA sequences named VirR boxes that are located directly upstream of the gene (6 9 In contrast the production of the enterotoxin (CPE) during sporulation is mediated by sporulation-specific alternative sigma factors (4 10 11 The accessory growth regulator (Agr)-like quorum sensing (QS) system was first discovered in Agr-like QS system was shown to regulate the production of ETX PFO and CPA in type D strain CN3718 (14) the production of CPB PFO and CPA (but not ETX) in type B strains CN1793 and CN1795 (15) the production of CPA CPB and PFO in type C strain CN3685 (16) and the production of CPE during sporulation by CPE-positive type A strain F5603 (17). In addition the Agr-like system was found to be essential for type C strain CN3685 to cause necrotic enteritis in PTC124 (Ataluren) a rabbit small intestinal loop model or lethal enterotoxemia in a mouse oral challenge model (16). This requirement for a functional Agr-like QS system in CN3685 virulence was determined to involve the QS system controlling the intestinal production of CPB (16) which is essential for the virulence of type C strains (18). Both similarities and differences exist between the Agr-like QS system of and the well-characterized paradigm Agr system of (8 12 13 19 -21). For example the Agr-like PTC124 (Ataluren) QS system includes an operon encoding AgrD and AgrB where AgrD is the precursor peptide for the autoinducing peptide (AIP) that mediates Agr QS signaling and AgrB is the integral membrane endopeptidase that is involved in processing AgrD to the active AIP. However lacks the AgrA/AgrC two-component system that responds to the AIP in (6 8 19 20 Some evidence suggests that the VirS/VirR two-component regulatory system may sometimes be the functional equivalent of AgrA/AgrC in gene is regulated by the Agr-like QS system yet expression is not affected by inactivation of the operon (6 8 12 14 Another difference is the absence of an identifiable RNAIII in Agr QS system (6 8 19 20 There is considerable variation in the nature of the AIPs that activate the Agr systems of various Gram-positive bacteria (22). In is only a 5-mer thiolactone ring with no.