ANTICANCER RESEARCH 34: 4627-4632 (2014)

Molecular Analysis of 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

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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 (500 ps), for which the values are shown.

The distribution of the electrostatic potential (ESP) field compared for each four ring-formable sites, and the energy (an index of reactivity) also changed markedly due to of the 33F-34S heterocycled molecule was the lowest heterocycle formation (Figure 3G, I and K). In the pre- (1772.3 kcal/mol in Table I) among these sites [1789.3 heterocycled SagA1 molecule, a negative ESP field (dark kcal/mol (31C-32C), 1801.7 kcal/mol (46T-47T), 1828.0 gray cloud) covered the whole molecule (Figure 3H). After kcal/mol (44G-45S)]. The lowest total energy of the heterocycle formation, the propeptide region of SagA1 was second, third, and fourth ring-formed SagA1 molecule was covered with a negative ESP field (Figure 3G). For the 1762.1 kcal/mol (46T-47T heterocycled SagA1), 1755.0 SagA2 molecule, positive (light gray cloud) and negative kcal/mol (31C-32C heterocycled SagA1), and 1784.7 (dark gray cloud) ESP fields covered the whole molecule kcal/mol (44G-45S heterocycled SagA1), respectively. with and without heterocycle formation (Figure 3I and J). In SagA2 has three heterocycle formable sites (31C-32C, 33F- the heterocycled and pre-heterocycled SPySagA molecule, 34S, 44G-45S). The order of heterocycle formation in the ESP distribution pattern was similar to that of SagA1 SagA2 was examined as well as SagA1, and the first ring (Figure 3K and L), respectively. These results indicate that was suggested to form at 31C-32C (1617.6 kcal/mol). The the reactivity of these SagA molecules with their target second and third rings were suggested to form at 33F-34S molecule(s) is extremely different pre- and post-heterocycle (1623.9 kcal/mol) and 44G-45S (1538.0 kcal/mol), formation. respectively. SPySagA has five heterocycle formable sites (31C-32C, 33F-34S, 38G-39S, 45G-46S, 47G-48S), and the Order of heterocycle formation in SagA molecules. SagA1 order of heterocycle formation was determined by MM-MD has four heterocycle formable sites (31C-32C, 33F-34S, energy simulation as follows: first (31C-32C: 1811.9 44G-45S, 46T-47T), and the order of heterocycle ring kcal/mol), second (33F-34S: 1796.9 kcal/mol), third (38G- formation was examined using MM-MD energy simulation. 39S: 1803.8 kcal/mol), fourth (45G-46S: 1798.0 kcal/mol), The total energy of the first ring-formed SagA1 was an fifth (47G-48S: 1777.5 kcal/mol).

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Figure 3. Molecular structure and electrostatic potential field of SagA molecules. MM-MD-simulated structure before (B, D, F)- and after (A, C, E)- heterocycle formation of SagA1 (A, B) and SagA2 (C, D) from S. anginosus subsp. anginosus NCTC10713T and SagA (E, F) from S. pyogenes MGAS5005. Dark gray line indicates the leader peptide (A-F). Arrowheads indicate the heterocycle-formable sites and the amino acid numbers are shown. The distribution pattern of electrostatic potential field (negatively charged field: dark gray cloud, positively charged field: light gray cloud) were also overlaid onto SagA1 (G, H), SagA2 (I, J), and SagA (K, L). In each electrostatic potential field figure (G - L), the results for before and after heterocycle formation are shown.

Table I. Total average energy of SagA molecules during heterocycle ring formation. The values indicate the total energy (kcal/mol) of heterocycled SagA molecules in each step. Values in the parentheses indicate the amino acid positions (see Figure 1) for heterocycle ring formation.

Step

Molecule 0 1st 2nd 3rd 4th 5th

SAA SagA1 1830.8 1772.3 (33F-34S) 1762.1 (46T-47T) 1755.0 (31C-32C) 1784.7 (44G-45S) 1789.3 (31C-32C) 1805.1 (31C-32C) 1766.6 (44G-45S) 1801.7 (46T-47T) 1828.8 (44G-45S) 1828.0 (44G-45S) SAA SagA2 1574.9 1617.6 (31C-32C) 1623.9 (33F-34S) 1538.0 (44G-45S) 1630.8 (33F-34S) 1624.8 (44G-45S) 1635.9 (44G-45S) S. pyogenes SagA 1799.3 1811.9 (31C-32C) 1796.9 (33F-34S) 1803.8 (38G-39S) 1798.0 (45G-46S) 1777.5 (47G-48S) 1815.8 (33F-34S) 1817.9 (45G-46S) 1806.2 (47G-48S) 1803.6 (47G-48S) 1818.1 (45G-46S) 1824.1 (38G-39S) 1815.4 (45G-46S) 1823.9 (47G-48S) 1830.8 (47G-48S) 1827.5 (38G-39S)

4630 Kawaguchi et al: Molecular Αnalysis of SagA

Discussion with intra-molecular ring formation. Moreover, the control of the molecular mechanism by intramolecular heterocycle ring The beta-hemolytic peptide called streptolysin-S is well- formation can be also applied to the analysis of drug- known and has been investigated in PGS such as S. pyogenes, excreting molecules (e.g. SagG, SagH, SagI) related to anti- and this beta-hemolytic peptide was found to be encoded by bacterial and anticancer drug resistance. sagA gene in the sag operon. Recently, the genes encoding twin streptolysin S-homologous peptides (SagA1 and SagA2) References were found to exist in the sag operon homolog of beta- hemolytic SAA strains, and each is responsible for the beta- 1 Whiley RA and Beighton D: Emended descriptions and hemolysis of beta-hemolytic SAA (8). In the present study, recognition of Streptococcus constellatus, Streptococcus these SagA molecules from beta-hemolytic SAA and from S. intermedius, and Streptococcus anginosus as distinct species. Int J Syst Bacteriol 41: 1-5, 1991. pyogenes were modeled and their molecular features were 2 Whiley RA, Hall LMC, Hardie JM and Beighton D: A study of analyzed. These SagA molecules had 3-5 heterocycle formable small colony, β-hemolytic, Lancefield group C streptococci sites (Figure 1), and their structural features were expected to within the anginosus group: description of Streptococcus change according to heterocycle formation. The total energy constellatus subsp. pharynges subsp. nov., associated with the of SagA1, SagA2 and SPySagA decreased with heterocycle human throat and pharyngitis. Int J Syst Bacteriol 49: 1443- formation during MD simulation period (Figure 2), and these 1449, 1999. molecules were thought to be stabilized by intramolecular 3 Jensen A, Hoshino T and Kilian M: of the Anginosus group of the genus Streptococcus and description of heterocycle formation. In heterocycle-formed SagA molecules, Streptococcus anginosus subsp. whileyi subsp. nov. and the total energy converged in a short time (100 ps in Figure Streptococcus constellatus subsp. viborgensis subsp. nov. Int J 2D-F). From these results, it was thought that the flexibility Syst Evol Microbiol 63: 2506-2519, 2013. of SagA molecules changed by intramolecular heterocycle 4 Whiley RA, Beighton D, Winstanley TG, Fraser HY and Hardie formation. It seemed that the change in this molecular JM: Streptococcus intermedius, Streptococcus constellatus, and flexibility affected the separation process between the leader Streptococcus anginosus (the Streptococcus milleri group): peptide and propeptide (protoxin) region. Heterocycle association with different body sites and clinical infections. J Clin Microbiol 30: 243-244, 1992. formation is an important event not only for the functional 5 Sasaki M, Yamaura C, Ohara-Nemoto Y, Tajika S, Kodama Y, appearance of SagA molecules but also for their maturation. Ohya T, Harada R and Kimura S: Streptococcus anginosus In heterocycle-formed SagA molecules, the boundary infection in oral cancer and its infection route. Oral Dis 11: 151- between leader peptide and propeptide was plain (Figure 3A, 156, 2005. C and E). For instance, it existed with the leader peptide 6 McKenzie TJ, Lillegard JB, Grotz TE, Moir CR and Ishitani region contained in the propeptide region in SagA1 (Figure MB: Pyogenic liver abscess secondary to Streptococcus 3A) and SagA2 (Figure 3C). The heterocycle formation of anginosus in an adolescent. J Pediatr Surg 45: E15-E17, 2010. 7 Nagamune H, Whiley RA, Goto T, Inai Y, Maeda T, Hardie JM SagA molecules is also suggested to contribute to the and Kourai H: Distribution of the intermedilysin gene among the compartmentalization of the leader peptide and propeptide, anginosus group streptococci and correlation between and then the leader peptide dividing from the propeptide intermedilysin production and deep-seated infection with region, and the later process for the maturation of SagA Streptococcus intermedius. J Clin Microbiol 38: 220-226, 2000. molecules might thus be enhanced. 8 Tabata A, Nakano K, Ohkura K, Tomoyasu T, Kikuchi K, The whole molecule of SagA1 and SPySagA was covered Whiley RA and Nagamune H: Novel twin Streptolysin S-Like with negatively-charged ESP field before heterocycle peptides encoded in the sag operon homologue of beta-hemolytic Streptococcus anginosus. J Bacteriol 195: 1090-1099, 2013. formation (Figure 3H and L). After heterocycle formation, the 9 Ohkura K, Nagamune H and Kourai H: Structural analysis of negatively-charged ESP field was observed in the propeptide human specific cytolysin intermedilysin aiming application to region of SagA1 and SPySagA (Figure 3G and K). In the cancer immunotherapy. Anticancer Res 24: 3343-3354, 2004. SagA2 molecule, the distribution pattern of positive and 10 Ohkura K, Hori H and Nagamune H: Molecular dynamics of negative ESP fields was significantly changed by heterocycle human-specific cytolysin: Analysis of membrane binding motif formation (Figure 3I: heterocycled, 3J: pre-heterocycled). The for therapeutic application. Anticancer Res 26: 4055-4062, 2006. change of the distribution of the ESP field according to 11 Ohkura K, Hori H and Shinohara Y: Role of C-terminal region of yeast ADP/ATP carrier 2 protein: dynamics of flexible C- heterocycle formation might take part in dividing the leader terminal arm. Anticancer Res 29: 4897-4900, 2009. peptide region. We have found so far that the ESP field distribution changed the functional appearance of general transport carriers, such as the mitochondrial ATP/ADP carrier (11). The idea of a functional control mechanism by Received April 4, 2014 heterocycle formation seen in the SagA family can be applied Revised June 9, 2014 to the structural analysis of SagB, SagC and SagD molecules Accepted June 10, 2014

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