Molecular Analysis of Streptococcus Anginosus -Derived Saga Peptides
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ANTICANCER RESEARCH 34: 4627-4632 (2014) Molecular Analysis of Streptococcus anginosus -derived SagA Peptides YUKI KAWAGUCHI1, ATSUSHI TABATA2, HIDEAKI NAGAMUNE2 and KAZUTO OHKURA1,3* 1Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Chiba, Japan; 2Department of Biological Science and Technology, Life System, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Tokushima, Japan; 3Graduate School of Pharmaceutical Sciences, Suzuka University of Medical Science Graduate School, Suzuka, Mie, Japan Abstract. Background: SagA1 and SagA2 molecules and gastrointestinal tract (4). It is generally considered that produced from beta-hemolytic Streptococcus anginosus they have relatively low pathogenic potential compared to subsp. anginosus are composed of a leader peptide and a other streptococci, in particular members of the Pyogenic propeptide, and their mature form has hemolytic activity as a group streptococci (PGS) such as S. pyogenes (SPy, also well-known Streptococcal peptide toxin, streptolysin. The designated as Group A streptococci, GAS). However, SAA function of these SagA molecules is thought to be dependent is being increasingly recognized as a pathogen that is able to on intra-molecular heterocycle formation. In this study, we cause a wide range of purulent infections that commonly examined the heterocycle-involved molecular features of manifest as abscess formation, and SAA presence has been SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing detected in esophageal cancer (5, 6). The awareness of the on their heterocycle formation. Materials and Methods: clinical importance of SAA has increased, but the molecular Molecular models of SagA1, SagA2, and SPySagA were basis of the pathogenicity of this species has not been clearly constructed using a molecular modeling technique. determined. It is known that several strains of AGS, Molecular dynamics and molecular mechanic analyses of the including SAA, exhibit beta-hemolysis on blood agar, and it modeled SagA molecules were performed to obtain their has been assumed that a beta-hemolytic reaction indicates energy profiles. Results: Total energy of the modeled SagA1, production of cytolytic factors thought to be important for SagA2, and SPySagA decreased with heterocycle formation, their pathogenicity. However, the beta-hemolytic factor of and the border between the leader peptide and propeptide AGS examined was only in a human-specific cholesterol- was clearly observed after heterocycle formation. dependent cytolysin, intermedilysin, secreted from S. Conclusion: The flexibility of SagA molecules was changed intermedius (7). There are no reports describing other factors by intramolecular heterocycle formation, and their function conferring beta-hemolytic capability on beta-hemolytic SAA (e.g. hemolytic activity) seems to be regulated by structural and other beta-hemolytic AGS except for S. intermedius. transition with heterocycle formation. We examined beta-hemolysis factors in SAA-type strain NCTC10713T using a random gene-knockout approach (8). Streptococcus anginosus subsp. anginosus (SAA) is a The genes responsible for the production of the beta- member of the Anginosus group streptococci (AGS) (1-3). hemolytic factor were found to be a homologue of sag SAA is an opportunistic pathogen and forms part of the operon gene clusters including sagA encoding the cytolytic normal flora in the human oral cavity, genitourinary tract, toxin streptolysin S (SLS) present in PGS such as S. pyogenes. A significant difference in the sag operon homologue of beta-hemolytic SAA was observed around the sagA gene, and two sagA homologues (designated as sagA1 Correspondence to: Professor Kazuto Ohkura, Graduate School of and sagA2) existed in tandem upstream of the sagB gene. No Pharmaceutical Sciences, Suzuka University of Medical Science such tandem structure was found in the sag operon of PGS Graduate School, 3500-3 Minamitamagaki-cho, Suzuka, Mie 513- with a single sagA gene (8). The alignment of the deduced 8670, Japan. Tel: +81 593400611, Fax: +81 593681271, e-mail: amino acid sequences of sagA1 and sagA2 product, SagA1 [email protected] and SagA2, shows that the primary structure of these SagA Key Words: Cytolysin, hemolysis, Streptococcus anginosus, SagA, molecules are highly conserved (8). They have a leader heterocycle formation. peptide and propeptide region, and the amino acid sequence 0250-7005/2014 $2.00+.40 4627 ANTICANCER RESEARCH 34: 4627-4632 (2014) Figure 1. Alignment of amino acid sequences of SagA molecules for SagA1 and SagA2 of Streptococcus anginosus subsp. anginosus NCTC10713T and typical SagA of S. pyogenes MGAS5005. The alignment analyses were conducted using ClustalX. The amino acids deduced to contribute to heterocycle formation in SagA1, SagA2, and SagA are underlined. Arrowhead indicates the cleavage site of SagA molecules for maturation, which divides the molecule into leader peptide and propeptide. alignment of SagA1, SagA2, and SagA of S. pyogenes NCTC10713T (SagA1 and SagA2), and SPySagA from S. (designated hereafter as SPySagA) revealed a conserved pyogenes MGAS5005 were investigated especially for intra- sequence, especially in the leader peptide among these molecular heterocycle formation. Total energy of modeled molecules (Figure 1) (8). For the amino acids potentially SagA1 molecule gradually decreased during the whole MD contributing to heterocycle formation, the number and simulation period (500 ps) and the average was 1830.8 location of candidate amino acids concerned with kcal/mol (Figure 2A). In heterocycle-formed SagA1, the oxazoline/thiazoline formation are suspected to vary among total energy decreased smoothly from the start of MD these SagA molecules (Figure 1) (8). In the present study, simulation at 100 ps and the energy average (1784.7 we examined the structural features of these SagA kcal/mol) (Figure 2D) was lower than that of pre- molecules, and the role of heterocycle formation in their heterocycled SagA1. The total energy of SagA2 gradually maturation and function. converged during the MD analysis period (500 ps) and the average was 1574.9 kcal/mol (Figure 2B). The total energy Materials and Methods of heterocycle-formed SagA2 decreased within 100 ps of the start of simulation and the average (1538.0 kcal/mol) Molecular modeling of SagA molecules. Molecular models of SagA (Figure 2E) was lower than that of the pre-heterocycled T molecules from SAA NCTC10713 (SagA1 and SagA2) and SagA SagA2 molecule. The SagA2 molecule was lower in total from S. pyogenes MGAS5005 (SPySagA) were constructed using energy than SagA1, and SagA2 was more stable than insightII-discover (Accelrys Inc., San Diego, CA, USA) as previously described (9). SagA1 has four heterocycle formable sites SagA1. For SPySagA, the convergence of total energy (31C-32C, 33F-34S, 44G-45S, 46T-47T), SagA2 has three formable indicates a similar tendency between pre- and post- sites (31C-32C, 33F-34S, 44G-45S) and SPySagA has five formable heterocycle formation, but the total energy of post- sites (31C-32C, 33F-34S, 38G-39S, 45G-46S, 47G-48S) (underlined heterocycle formation was lower than that before formation. in Figure 1). The heterocycle-formed model of SagA molecules The total energy of SPySagA decreased with heterocycle were constructed using builder module and their structures were formation and the average energy decreased from 1799.3 to optimized under Consistence Valence Forcefield (CVFF). The molecular mechanics (MM) and molecular dynamics (MD) analysis 1777.5 kcal/mol (Figure 2C and 2F). of modeled SagA molecules (with/without heterocycles) were The molecular structure containing the leader peptide and performed by discover 3 module under CVFF (10). propeptide in modeled SagA1, SagA2, and SPySagA were compared pre- and post-heterocycle formation (Figure 3). Energy profile analysis of SagA molecules. The kinetic energy and Before heterocycle formation, the border between leader potential energy of modeled SagA molecules (with/without peptide (dark gray line) and propeptide (light gray line) was heterocycles) during simulation period (500 ps) were monitored, and unclear (Figure 3B, D and F). After heterocycle formation, the total energy (= kinetic energy + potential energy) profile was determined (10). After MD simulation period (500 ps), the the conformation of these SagA1, SagA2, SPySagA electrostatic potential fields of modeled SagA molecules molecules was significantly changed. In SagA1 and SagA2, (with/without heterocycles) were examined using insightII-discover the leader peptide domain (dark gray) was enclosed in the as previously described (11). inner part of the molecule (Figure 3A and 3C). In SPySagA, the leader peptide domain (dark gray) was bundled with the Results propeptide domain (Figure 3E). These results suggest that the heterocyclic structure is involved not only in the cytolytic Molecular features of SagA molecules. The differences of activity of these molecules but also in the proper processing molecular features among SagA molecules from SAA to convert them into their active form. 4628 Kawaguchi et al: Molecular Αnalysis of SagA Figure 2. Total energy profile of SagA molecules. The total energy of SagA1 (A, D) and SagA2 (B, E) from S. anginosus subsp. anginosus NCTC10713T, and SagA (C, F) from S. pyogenes MGAS5005 were calculated for before (A, B, C) and after (D, E, F) heterocycle formation. Broken lines indicate the average total energy (kcal/mol) during the simulation period