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Structural and Functional Dissection of the Heterocyclic Peptide Cytotoxin Streptolysin S

2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês

10.1074/jbc.m900802200

1083-351X

Douglas A. Mitchell, Shaun W. Lee, Morgan A. Pence, Andrew L. Markley, Joyce D. Limm, Victor Nizet, Jack E. Dixon,

Antimicrobial Resistance in Staphylococcus

The human pathogen Streptococcus pyogenes secretes a highly cytolytic toxin known as streptolysin S (SLS). SLS is a key virulence determinant and responsible for the β-hemolytic phenotype of these bacteria. Despite over a century of research, the chemical structure of SLS remains unknown. Recent experiments have revealed that SLS is generated from an inactive precursor peptide that undergoes extensive post-translational modification to an active form. In this work, we address outstanding questions regarding the SLS biosynthetic process, elucidating the features of substrate recognition and sites of posttranslational modification to the SLS precursor peptide. Further, we exploit these findings to guide the design of artificial cytolytic toxins that are recognized by the SLS biosynthetic enzymes and others that are intrinsically cytolytic. This new structural information has ramifications for future antimicrobial therapies. The human pathogen Streptococcus pyogenes secretes a highly cytolytic toxin known as streptolysin S (SLS). SLS is a key virulence determinant and responsible for the β-hemolytic phenotype of these bacteria. Despite over a century of research, the chemical structure of SLS remains unknown. Recent experiments have revealed that SLS is generated from an inactive precursor peptide that undergoes extensive post-translational modification to an active form. In this work, we address outstanding questions regarding the SLS biosynthetic process, elucidating the features of substrate recognition and sites of posttranslational modification to the SLS precursor peptide. Further, we exploit these findings to guide the design of artificial cytolytic toxins that are recognized by the SLS biosynthetic enzymes and others that are intrinsically cytolytic. This new structural information has ramifications for future antimicrobial therapies. Streptolysin S (SLS) 4The abbreviations used are: SLS, streptolysin S; WT, wild type. 4The abbreviations used are: SLS, streptolysin S; WT, wild type. is secreted by the human pathogen Streptococcus pyogenes, the causative agent of diseases ranging from pharyngitis to necrotizing fasciitis (1Nizet V. Trends Microbiol. 2002; 10: 575-580Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). SLS is a potent cytolysin that is ribosomally synthesized, extensively posttranslationally modified, and exported to exert its effects on the target cell (2Datta V. Myskowski S.M. Kwinn L.A. Chiem D.N. Varki N. Kansal R.G. Kotb M. Nizet V. Mol. Microbiol. 2005; 56: 681-695Crossref PubMed Scopus (136) Google Scholar, 3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). The expression of SLS promotes virulence in animal models of invasive infection and accounts for the hall-mark zone of β-hemolysis surrounding colonies of these bacteria grown on blood agar (2Datta V. Myskowski S.M. Kwinn L.A. Chiem D.N. Varki N. Kansal R.G. Kotb M. Nizet V. Mol. Microbiol. 2005; 56: 681-695Crossref PubMed Scopus (136) Google Scholar, 4Nizet V. Beall B. Bast D.J. Datta V. Kilburn L. Low D.E. De Azavedo J.C. Infect. Immun. 2000; 68: 4245-4254Crossref PubMed Scopus (173) Google Scholar). An intriguing feature of SLS is its nonimmunogenic nature (5Robinson J. J. Immunol. 1951; 66: 661-665PubMed Google Scholar). This characteristic is likely due to its small size and its capacity to lyse cells involved in both innate and adaptive immunity (6Ofek I. Bergner-Rabinowitz S. Ginsburg I. Infect. Immun. 1972; 6: 459-464Crossref PubMed Google Scholar, 7Hryniewicz W. Pryjma J. Infect. Immun. 1977; 16: 730-733Crossref PubMed Google Scholar). The β-hemolytic phenotype of S. pyogenes has been studied since the early 1900s, but the molecular structure of SLS has remained elusive (8Marmorek A. Ann. Inst. Pasteur. 1895; 9: 593-620Google Scholar). In the last decade, transposon mutagenesis studies identified the gene encoding the SLS toxin precursor (sagA, for SLS-associated gene) and eight additional genes in an operon required for toxin maturation and export (9Betschel S.D. Borgia S.M. Barg N.L. Low D.E. De Azavedo J.C. Infect. Immun. 1998; 66: 1671-1679Crossref PubMed Google Scholar). Targeted mutagenesis of the sag operon yields nonhemolytic S. pyogenes mutants with markedly diminished virulence in mice (2Datta V. Myskowski S.M. Kwinn L.A. Chiem D.N. Varki N. Kansal R.G. Kotb M. Nizet V. Mol. Microbiol. 2005; 56: 681-695Crossref PubMed Scopus (136) Google Scholar). More recently, it was demonstrated that the protein products of sagA–D are sufficient for the in vitro reconstitution of cytolytic activity (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). The first gene product, SagA, serves as a structural template that after a series of tailoring reactions matures into the active SLS metabolite (see Fig. 1A). A trimeric complex of SagBCD catalyzes these tailoring reactions, which results in the conversion of cysteine, serine, and threonine residues to thiazole, oxazole, and methyloxazole heterocycles, respectively (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). A DNA gyrase inhibitor, microcin B17, is produced by an orthologous biosynthetic cluster (mcb) found in a subset of Escherichia coli strains (10Yorgey P. Davagnino J. Kolter R. Mol. Microbiol. 1993; 9: 897-905Crossref PubMed Scopus (47) Google Scholar, 11Yorgey P. Lee J. Kordel J. Vivas E. Warner P. Jebaratnam D. Kolter R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4519-4523Crossref PubMed Scopus (150) Google Scholar, 12Li Y.M. Milne J.C. Madison L.L. Kolter R. Walsh C.T. Science. 1996; 274: 1188-1193Crossref PubMed Scopus (250) Google Scholar). Microcin B17 contains four thiazole and four oxazole heterocycles, which are indispensable for biological activity. By analogy to microcin B17 and the lantibiotics, the heterocycles of SLS are formed on the C terminus of SagA, whereas the N terminus serves as a leader peptide (13de Vos W.M. Kuipers O.P. van der Meer J.R. Siezen R.J. Mol. Microbiol. 1995; 17: 427-437Crossref PubMed Scopus (157) Google Scholar, 14Madison L.L. Vivas E.I. Li Y.M. Walsh C.T. Kolter R. Mol. Microbiol. 1997; 23: 161-168Crossref PubMed Scopus (56) Google Scholar, 15Roy R.S. Kim S. Baleja J.D. Walsh C.T. Chem. Biol. 1998; 5: 217-228Abstract Full Text PDF PubMed Scopus (49) Google Scholar). The installation of thiazole and (methyl)-oxazole heterocycles restricts backbone conformational flexibility and provides microcin B17 and SLS with rigidified structures. The SLS heterocycles are formed via two distinct steps; SagC, a cyclodehydratase, generates thiazoline and (methyl)-oxazoline heterocycles, whereas SagB, a dehydrogenase, removes two electrons to afford the aromatic thiazole and (methyl)-oxazole (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar, 16Milne J.C. Roy R.S. Eliot A.C. Kelleher N.L. Wokhlu A. Nickels B. Walsh C.T. Biochemistry. 1999; 38: 4768-4781Crossref PubMed Scopus (76) Google Scholar, 17Roy R.S. Gehring A.M. Milne J.C. Belshaw P.J. Walsh C.T. Nat. Prod. Rep. 1999; 16: 249-263Crossref PubMed Scopus (274) Google Scholar). SagD is proposed to play a role in trimer formation and regulation (see Fig. 1A). The final genes in the genetic cluster encode a predicted leader peptidase/immunity protein (SagE), a membrane-associated protein of unknown function (SagF), and three ABC transporters (SagGHI). It is now appreciated that many other prokaryotes harbor similar genetic clusters for the synthesis of thiazole and (methyl)-oxazole heterocycles (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar, 18Cotter P.D. Draper L.A. Lawton E.M. Daly K.M. Groeger D.S. Casey P.G. Ross R.P. Hill C. PLoS Pathog. 2008; 4: e1000144Crossref PubMed Scopus (156) Google Scholar, 19Donia M.S. Ravel J. Schmidt E.W. Nat. Chem. Biol. 2008; 4: 341-343Crossref PubMed Scopus (216) Google Scholar). Additional important mammalian pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum, contain sag-like gene clusters that produce SLS-like cytolysins. These toxins are expected to promote pathogen survival and host cell injury during infection, but this has only been conclusively shown for S. pyogenes and L. monocytogenes (2Datta V. Myskowski S.M. Kwinn L.A. Chiem D.N. Varki N. Kansal R.G. Kotb M. Nizet V. Mol. Microbiol. 2005; 56: 681-695Crossref PubMed Scopus (136) Google Scholar, 18Cotter P.D. Draper L.A. Lawton E.M. Daly K.M. Groeger D.S. Casey P.G. Ross R.P. Hill C. PLoS Pathog. 2008; 4: e1000144Crossref PubMed Scopus (156) Google Scholar). Like E. coli, many other prokaryotes harbor a sag-like genetic cluster but are not known to produce cytolysins. Some examples are the goadsporin-producing organism, Streptomyces sp. TP-A0584 and cyanobactin producers such as Prochloron didemni (20Igarashi Y. Kan Y. Fujii K. Fujita T. Harada K. Naoki H. Tabata H. Onaka H. Furumai T. J. Antibiot. (Tokyo). 2001; 54: 1045-1053Crossref PubMed Scopus (51) Google Scholar, 21Onaka H. Tabata H. Igarashi Y. Sato Y. Furumai T. J. Antibiot. (Tokyo). 2001; 54: 1036-1044Crossref PubMed Scopus (77) Google Scholar, 22Schmidt E.W. Nelson J.T. Rasko D.A. Sudek S. Eisen J.A. Haygood M.G. Ravel J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7315-7320Crossref PubMed Scopus (467) Google Scholar). The molecular targets of these secondary metabolites remain to be elucidated, but it is known that goadsporin exhibits antibiotic activity, and the cyanobactin, patellamide D, reverses multiple drug resistance in a human leukemia cell line (23Williams A.B. Jacobs R.S. Cancer Lett. 1993; 71: 97-102Crossref PubMed Scopus (84) Google Scholar). Because genetic loci containing sagBCD-like genes have been widely disseminated in prokaryotes (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar), nature appears to have found a preferred route to synthesizing such secondary metabolites. In this work, we build upon our initial report on the in vitro reconstitution of SLS biosynthesis to uncover the requisite features of substrate selectivity and cytolytic activity. The impetus for defining substrate tolerance arose from earlier results showing that SagBCD accepts alternate substrates in vitro (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar), as evidenced by two key experiments. First, SagBCD converted a noncognate substrate, ClosA (C. botulinum), into a cytolytic entity. Second, mass spectrometry revealed heterocycle formation on the McbA (E. coli) peptide after SagBCD treatment (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). Here, we dissect the N-terminal leader peptide and C-terminal protoxin of SagA to define the residues necessary for conversion into SLS. In Vitro Synthetase Reactions—Protein preparation and synthetase reactions employing maltose-binding protein-tagged substrate and SagBCD were performed as described earlier (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). Membrane damage was quantified by the erythrocyte lysis assay. In every case, omission of the substrate or the SagBCD synthetase resulted in no detectable hemolytic activity (data not shown). Cytolytic Activity Assay—The in vitro and genetic reconstitution assays described below were performed at least three times for each substrate tested. Because of lot-to-lot variation in commercial blood sources and prep-to-prep variation in the specific activity of the synthetase complex, we have elected to report activity semi-quantitatively. In vitro hemolytic activity equal to wild type SagA treated with SagBCD is reported as three plus signs (+++). Cytolytic activity that is ∼30–70% of wild type SagA is given as (++). Detectable activity that is less than 30% of SagA is thus a single plus sign (+), and nondetectable activity is a single minus sign (-). The cytolytic activity of mutant substrates tested via genetic reconstitution was scored in an analogous manner. All of the assays were internally normalized, and the base line was adjusted using two positive controls (Triton X-100 and wild type SagA treated with SagBCD) and two negative controls (substrate and SagBCD alone). Transformation and Verification of S. pyogenes M1 sagA Mutants—The sagA allelic exchange mutant of S. pyogenes M1 was made electrocompetent using a previously published glycine/sucrose method (2Datta V. Myskowski S.M. Kwinn L.A. Chiem D.N. Varki N. Kansal R.G. Kotb M. Nizet V. Mol. Microbiol. 2005; 56: 681-695Crossref PubMed Scopus (136) Google Scholar). Maxiprepped pDCerm constructs (3 and 12 μg) were incubated with electrocompetent S. pyogenes M1 ΔsagA and electroporated using an Eppendorf 2510 electroporator set to 1.5 kV. These cells (50 and 150 μl) were then plated on Todd-Hewitt agar plates supplemented with 2 μg/ml erythromycin. Typically, 5–15 colonies would appear ∼40 h post-transformation. The insert size was evaluated by screening transformants by colony PCR. Clones harboring an appropriately sized insert were initially screened for cytolytic activity by streaking bacteria on blood agar plates (Hardy Diagnostics). All of the clones were verified to be S. pyogenes (Group A Streptococcus) by using the BBL Streptocard Enzyme Latex Test (BD Diagnostics). Cytolytic Assay of Genetically Reconstituted Mutants—Because of the possibility that nonphysiological concentrations could lead to artifactual activity in vitro, the cytolytic activity of the peptide substrates were also tested using genetic reconstitution. This method requires that endogenous SagBCD accept the substrate. The method described below does not involve lysing the bacteria. Therefore, mutant substrates must also be proteolytically processed and accepted by the SagGHI export apparatus (ABC transporters). Because of toxicity in E. coli and transformation difficulties, intrinsically lytic SagA mutants were not tested by genetic reconstitution. Extracts containing bovine serum albumin-stabilized SLS were prepared in the following manner. Overnight cultures (10 ml) of S. pyogenes M1 ΔsagA containing pDCerm-sagA plasmids were grown to A600 ∼0.6 in Todd Hewitt broth containing 2 μg/ml erythromycin. The cultures were treated with bovine serum albumin (10 mg/ml) for 1 h at 37 °C and then centrifuged (6,000 × g, 10 min) before passing the supernatant through a 0.2-μm acrodisc syringe filter (Pall Corporation). These samples were centrifuged again (6,000 × g, 10 min), and the supernatants were assayed for hemolytic activity by addition to defribinated sheep blood (in V-bottom microtiter plates at 1:25 and 1:50 dilutions). The blood was treated for 2–4 h before assessing hemolytic activity as previously reported (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). Assessment of SLS Mutants in a Murine Skin Infection Model— The experiments were performed using models reported previously (4Nizet V. Beall B. Bast D.J. Datta V. Kilburn L. Low D.E. De Azavedo J.C. Infect. Immun. 2000; 68: 4245-4254Crossref PubMed Scopus (173) Google Scholar, 9Betschel S.D. Borgia S.M. Barg N.L. Low D.E. De Azavedo J.C. Infect. Immun. 1998; 66: 1671-1679Crossref PubMed Google Scholar, 24Humar D. Datta V. Bast D.J. Beall B. De Azavedo J.C. Nizet V. Lancet. 2002; 359: 124-129Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). S. pyogenes M1 ΔsagA complemented with sagA-WT, sagA-S39A, and sagA-7C/S were grown to log phase, harvested by centrifugation, washed, resuspended in phosphate-buffered saline, and mixed 1:1 with Cytodex beads (1 mg/ml; Sigma). An inoculum of (1 × 107 colony-forming units in 100 μl) of S. pyogenes ΔsagA complemented with either sagA-S39A or the sagA-7C/S mutant was then injected subcutaneously into the right flank of 4-week-old male hairless crl: SKH1(hrhr)Br mice (n = 8/group). S. pyogenes ΔsagA complemented with sagA-wt was injected into the left flank of the same animal for identical comparison. The animals were monitored daily for development of necrotic ulcers. At 4 days post-infection, all of the animals were sacrificed. Biopsies were performed on injection sites for histopathologic assessment (hematoxylin/eosin staining) after measuring the size of tissue ulcers. Generation of [35S]Met-SagBCD—Radioactive synthetase was prepared using maxiprepped pET28-maltose-binding protein-SagBCD and in vitro transcription/translation under T7 promoter control. Rabbit reticulocyte extract (Promega) gave superior yield and purity to wheat germ and S30 extracts. A typical 50-μl reaction was set up as follows: 1–3 μg of plasmid DNA was added to a master mix containing 2 μl of transcription/translation buffer, 25 μl of rabbit reticulocyte extract, 1 μl of RNAsin, 1 μl of minus Met amino acid mix, 1 μl of T7 RNA polymerase, 10 μCi of [35S]Met (PerkinElmer Life Sciences), and DNase/RNase-free water. A small aliquot (0.5 μl) of the radiolabeled product was separated by SDS-PAGE, dried, and visualized by autoradiography using a Kodak BioMax low energy isotope intensifying screen (20 h, -80 °C). Peptide Array—Immobilized peptides were synthesized on cellulose membranes using a MultiPep Autospot synthesis robot following the manufacturer's instructions (Intavis AG). Irradiation with 254 nm (UV) light gave an indication of the relative amount of peptide per spot. [35S]Met-SagB, -C, and -D were allowed to bind to the array for 15 h at 4 °C in Tris-buffered saline/Tween 20 (0.1% v/v) supplemented with 2.5% nonfat milk and 2.5% bovine serum albumin (both w/w). The array was then extensively washed with Tris-buffered saline/Tween (4 × 10 min, 23 °C) before exposing film with a Kodak BioMax low energy isotope intensifying screen (20 h, -80 °C). Under these conditions, SagB and SagD did not bind tightly to the array, as indicated by a weak radioactive signal, even after longer exposure times (4 days). Using SagBCD together led to a substantial amount of "radioactive precipitation" on areas of the cellulose membrane that did not contain peptide. Therefore, later experiments were carried out using SagC alone. The SagA Leader Peptide Provides Substrate Recognition— The precursor peptide from C. botulinum, ClosA, shares significant amino acid sequence similarity with SagA (62%) and was converted to a cytolysin by recombinant SagBCD (3Lee S.W. Mitchell D.A. Markley A.L. Hensler M.E. Gonzalez D. Wohlrab A. Dorrestein P.C. Nizet V. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5879-5884Crossref PubMed Scopus (161) Google Scholar). The microcin B17 precursor, McbA, is only moderately similar (32%) but was also accepted as a substrate by SagBCD. Therefore, we hypothesized that SagBCD would accept numerous noncognate substrates (Fig. 1B). To demonstrate that the permissive behavior of SagBCD on ClosA was not limited to in vitro biochemical studies with purified proteins, genetic complementation studies were performed using a sagA deletion mutant produced in an M1 serotype strain of S. pyogenes (ΔsagA). By transforming this strain with a plasmid encoding the desired peptide substrate, the in vivo selectivity of endogenous SagBCD was probed. β-Hemolysis on blood agar was restored when either the wild type sagA or closA gene was provided to the S. pyogenes ΔsagA mutant bacteria. To increase sensitivity, we adopted a liquid phase hemolysis assay. Here, the SLS is extracted from the S. pyogenes lipoteichoic acid layer using bovine serum albumin as a carrier/stabilizer. This sample is mixed with sheep blood, and the amount of hemoglobin released from lysed erythrocytes is quantified by absorbance. Using this assay, we confirmed that ClosA was converted to a cytolysin by endogenous SagBCD but calculated the level of activity to be reduced by ∼⅓ relative to that of SagA (Fig. 1B). Interestingly, the related peptide substrates from S. aureus (StaphA) and L. monocytogenes (ListA) did not exhibit detectable hemolytic activity with purified recombinant proteins or in genetically complemented S. pyogenes ΔsagA (Fig. 1B). Cotter et al. (18Cotter P.D. Draper L.A. Lawton E.M. Daly K.M. Groeger D.S. Casey P.G. Ross R.P. Hill C. PLoS Pathog. 2008; 4: e1000144Crossref PubMed Scopus (156) Google Scholar) have recently shown that the sag-like genetic cluster from L. monocytogenes synthesizes an SLS-like cytolytic factor. Therefore, the lack of activity in our assays must originate at the level of substrate recognition or enzymatic tolerance. Previous work from the Walsh laboratory has shown that the N-terminal leader peptide of McbA is required for substrate recognition (14Madison L.L. Vivas E.I. Li Y.M. Walsh C.T. Kolter R. Mol. Microbiol. 1997; 23: 161-168Crossref PubMed Scopus (56) Google Scholar). The most important residues implicated in this process are McbA-F8 and -L12, which are proposed to lie on the same face of an α-helix (15Roy R.S. Kim S. Baleja J.D. Walsh C.T. Chem. Biol. 1998; 5: 217-228Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Alignment with other substrates shows that a similar motif (FXXXB, where X is any amino acid and B is a branched chain amino acid) is found in the leader peptides of SagA and ClosA but is lacking in StaphA and ListA (Fig. 1B). This finding prompted the construction of a double alanine mutant of the FXXXB motif (SagA-FIA) and chimeric substrates comprised of the SagA leader peptide fused to the StaphA and ListA C terminus (Fig. 1C). If the SagA leader peptide contains adequate substrate recognition information, then activity should be restored upon treating the chimera with purified SagBCD. Furthermore, if the FXXXB motif is required for substrate recognition, then cytolytic activity should be reduced for similarly treated SagA-FIA. A full restoration of in vitro cytolytic activity was observed for both the SagA-ListA and SagA-StaphA chimera. In contrast, genetic complementation revealed that only SagA-StaphA retained activity, indicating that either the endogenous rules of cytolytic conversion are more restrictive or the SagA-ListA substrate is not efficiently exported. Enzymatic promiscuity could theoretically be amplified in vitro by the presence of large amounts of purified SagBCD. This is supported by the observation that SagA-FIA has detectable activity using purified enzymes but not under genetic reconstitution conditions (Fig. 1C). Unnatural SagA analogs were prepared to shed light on the positional requirements of heterocycle formation. We first designed an artificial substrate, SagX, in an attempt to assess the minimum features for the formation of a cytolysin. Four criteria were used to design this potential substrate: (i) the SagA leader peptide to provide recognition, (ii) a stretch of contiguous heterocyclizable residues adjacent to the leader peptide cleavage site, (iii) 30% glycine evenly distributed through the C-terminal half, and (iv) a serine flanked by two glycine residues. Akin to SagA-ListA, this artificial substrate gave activity equal to SagA in the in vitro assay but no activity upon complementation of the ΔsagA mutant of S. pyogenes. The region of SagA between the cysteine-rich region and the last heterocyclizable residue (33FSIA...GSYT50) was further probed by designing two additional unnatural substrates. SagA inverse simply inverted residues 33–50 of SagA, whereas in SagA scramble this region was reordered in a randomized fashion. Despite having the highest protoxin similarity to wild type SagA, the SagA inverse substrate did not yield lytic activity in either assay. SagA scramble was active in vitro but did not complement hemolytic activity in S. pyogenes ΔsagA. These data show that the leader peptide, especially the FXXXB motif, is important for substrate acceptance and that heterocyclizable positions must be available at precise positions to allow creation of an SLS-like cytolytic factor. Additionally, these data indicate that even though SagBCD accepts artificial substrates, there is a limit to the enzymatic promiscuity. These limits are more pronounced during genetic complementation experiments most likely because substrate recognition and enzymatic tolerance are bypassed to an extent when using purified SagBCD. An alternative explanation is that the unnatural substrates are secreted from S. pyogenes with differing efficiencies. Analysis of SagA Leader Peptide Binding Requirements—Because the above activity assays measure both substrate binding and heterocycle formation in a simultaneous and indirect fashion, peptide arrays were synthesized to directly evaluate binding. Based on a perceived importance of the FXXXB motif in SagBCD substrate acceptance, the first array consisted of a panel of leader peptides that contain the FXXXB motif. Next to each wild type (WT) sequence, the FXXXB double alanine mutation was synthesized for SagA, ClosA, McbA, and a substrate from Pyrococcus furiosus, designated PagA (Fig. 2A). Equal peptide loading was confirmed by irradiating the array with UV (254 nm) light. To assess binding, [35S]Met-labeled SagBCD was prepared by in vitro transcription/translation (supplemental Fig. S1) and allowed to interact with the peptide array as described under "Experimental Procedures." Initially, the SagBCD complex was tested for binding in approximately a 1:1:1 ratio, as judged by the number of methionines and the intensity of exposure. Unfortunately, this led to a substantial amount of precipitation that could not be removed from the cellulose membrane and rendered the binding information unreliable. Therefore, SagB, SagC, and SagD were tested individually for binding. SagB and SagD did not bind tightly to the leader peptide array and were washed off before exposing the film (data not shown). SagC, however, bound with high affinity to the array. For the WT sequences, SagC binding was highest to SagA, followed by ClosA and PagA (Fig. 2A). Binding to McbA was the weakest, as measured by autoradiographic intensity. Importantly, binding to each FXXXB mutant was reduced in comparison with the corresponding WT leader peptide. The FXXXB Leader Motif Is Necessary but Not Sufficient for SagC Recognition—Although the FXXXB motif is an important determinant for directing SagC substrate binding, it does not contribute all of the interaction energy. This is illustrated by the observation that SagC bound to the SagA-WT leader peptide more efficiently than the other WT peptides (Fig. 2A). Furthermore, simple incorporation of FXXXB into the StaphA and ListA leader peptide did not provide sufficient affinity to detect SagC binding by this method (data not shown). These observations led to the hypothesis that additional residues of SagA contribute to SagC binding. To elucidate these binding determinants, a second array was synthesized that consisted of an alanine scan of the SagA leader peptide (Fig. 2B). Each residue of SagA that is not naturally found as alanine was individually mutated to alanine and tested for SagC binding as above. As expected, mutation to alanine did not disrupt SagC affinity at every location. However, the binding levels were significantly reduced when residues comprising the FXXXB motif were mutated (Phe4 and Ile8). It was also found that residues adjacent to the FXXXB sequence (Leu2, Lys3, and Leu9) and another binding site (Thr17, Gln18, and Val19) were also critical for SagC binding (Fig. 2B). An additional array was used to ascertain the contribution of each interaction site found in the leader peptide and to also assess whether the C-terminal region of SagA plays a role in binding. A panel of fourteen 10-mer peptides were included in the array that scan the full-length of SagA, except for the oxidatively prone, and synthetically challenging, cysteine-rich region (Fig. 2C). After binding SagC to this array, it was found that only the first peptide was capable of providing enough binding information to retain SagC through the washing steps. Peptide 1 comprises residues 1–10 of SagA and not only contains the FXXXB motif but also the important adjacent residues (Leu2, Lys3, Phe4, Ile8, and Leu9). Peptide 5 contains Ile8 and Leu9 but only the first residue of the TQV site (The17). This peptide is insufficient at retaining SagC. Taken together, these findings demonstrate that separate sites within the N-terminal leader peptide synergize to provide SagC with a high affinity binding site. Binding parameters for the SagA-WT, SagA-FIA (FXXXB double alanine mutant), and SagA-TQV (triple alanine mutant) leader peptides with SagC were then measured by surface plasmon resonance to quantify the contribution of each binding motif. Using a C-terminal hexahistidine tagged version of SagA-WT, -