Abstract
Background: SagA1 and SagA2 molecules produced from beta-hemolytic Streptococcus anginosus subsp. anginosus are composed of a leader peptide and a propeptide, and their mature form has hemolytic activity as a well-known Streptococcal peptide toxin, streptolysin. The function of these SagA molecules is thought to be dependent on intra-molecular heterocycle formation. In this study, we examined the heterocycle-involved molecular features of SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing on their heterocycle formation. Materials and Methods: Molecular models of SagA1, SagA2, and SPySagA were constructed using a molecular modeling technique. Molecular dynamics and molecular mechanic analyses of the modeled SagA molecules were performed to obtain their energy profiles. Results: Total energy of the modeled SagA1, SagA2, and SPySagA decreased with heterocycle formation, and the border between the leader peptide and propeptide was clearly observed after heterocycle formation. Conclusion: The flexibility of SagA molecules was changed by intramolecular heterocycle formation, and their function (e.g. hemolytic activity) seems to be regulated by structural transition with heterocycle formation.
Streptococcus anginosus subsp. anginosus (SAA) is a member of the Anginosus group streptococci (AGS) (1-3). SAA is an opportunistic pathogen and forms part of the normal flora in the human oral cavity, genitourinary tract, and gastrointestinal tract (4). It is generally considered that they have relatively low pathogenic potential compared to other streptococci, in particular members of the Pyogenic group streptococci (PGS) such as S. pyogenes (SPy, also designated as Group A streptococci, GAS). However, SAA is being increasingly recognized as a pathogen that is able to cause a wide range of purulent infections that commonly manifest as abscess formation, and SAA presence has been detected in esophageal cancer (5, 6). The awareness of the clinical importance of SAA has increased, but the molecular basis of the pathogenicity of this species has not been clearly determined. It is known that several strains of AGS, including SAA, exhibit beta-hemolysis on blood agar, and it has been assumed that a beta-hemolytic reaction indicates production of cytolytic factors thought to be important for their pathogenicity. However, the beta-hemolytic factor of AGS examined was only in a human-specific cholesterol-dependent cytolysin, intermedilysin, secreted from S. intermedius (7). There are no reports describing other factors conferring beta-hemolytic capability on beta-hemolytic SAA and other beta-hemolytic AGS except for S. intermedius.
We examined beta-hemolysis factors in SAA-type strain NCTC10713T using a random gene-knockout approach (8). The genes responsible for the production of the beta-hemolytic factor were found to be a homologue of sag operon gene clusters including sagA encoding the cytolytic 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 and sagA2) existed in tandem upstream of the sagB gene. No such tandem structure was found in the sag operon of PGS with a single sagA gene (8). The alignment of the deduced amino acid sequences of sagA1 and sagA2 product, SagA1 and SagA2, shows that the primary structure of these SagA molecules are highly conserved (8). They have a leader peptide and propeptide region, and the amino acid sequence alignment of SagA1, SagA2, and SagA of S. pyogenes (designated hereafter as SPySagA) revealed a conserved sequence, especially in the leader peptide among these molecules (Figure 1) (8). For the amino acids potentially contributing to heterocycle formation, the number and location of candidate amino acids concerned with oxazoline/thiazoline formation are suspected to vary among these SagA molecules (Figure 1) (8). In the present study, we examined the structural features of these SagA molecules, and the role of heterocycle formation in their maturation and function.
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.
Materials and Methods
Molecular modeling of SagA molecules. Molecular models of SagA molecules from SAA NCTC10713T (SagA1 and SagA2) and SagA from S. pyogenes MGAS5005 (SPySagA) were constructed using insightII-discover (Accelrys Inc., San Diego, CA, USA) as previously described (9). SagA1 has four heterocycle formable sites (31C-32C, 33F-34S, 44G-45S, 46T-47T), SagA2 has three formable sites (31C-32C, 33F-34S, 44G-45S) and SPySagA has five formable sites (31C-32C, 33F-34S, 38G-39S, 45G-46S, 47G-48S) (underlined in Figure 1). The heterocycle-formed model of SagA molecules were constructed using builder module and their structures were optimized under Consistence Valence Forcefield (CVFF). The molecular mechanics (MM) and molecular dynamics (MD) analysis of modeled SagA molecules (with/without heterocycles) were performed by discover 3 module under CVFF (10).
Energy profile analysis of SagA molecules. The kinetic energy and potential energy of modeled SagA molecules (with/without heterocycles) during simulation period (500 ps) were monitored, and the total energy (= kinetic energy + potential energy) profile was determined (10). After MD simulation period (500 ps), the electrostatic potential fields of modeled SagA molecules (with/without heterocycles) were examined using insightII-discover as previously described (11).
Results
Molecular features of SagA molecules. The differences of molecular features among SagA molecules from SAA NCTC10713T (SagA1 and SagA2), and SPySagA from S. pyogenes MGAS5005 were investigated especially for intra-molecular heterocycle formation. Total energy of modeled SagA1 molecule gradually decreased during the whole MD simulation period (500 ps) and the average was 1830.8 kcal/mol (Figure 2A). In heterocycle-formed SagA1, the total energy decreased smoothly from the start of MD simulation at 100 ps and the energy average (1784.7 kcal/mol) (Figure 2D) was lower than that of pre-heterocycled SagA1. The total energy of SagA2 gradually converged during the MD analysis period (500 ps) and the average was 1574.9 kcal/mol (Figure 2B). The total energy of heterocycle-formed SagA2 decreased within 100 ps of the start of simulation and the average (1538.0 kcal/mol) (Figure 2E) was lower than that of the pre-heterocycled SagA2 molecule. The SagA2 molecule was lower in total energy than SagA1, and SagA2 was more stable than SagA1. For SPySagA, the convergence of total energy indicates a similar tendency between pre- and post-heterocycle formation, but the total energy of post-heterocycle formation was lower than that before formation. The total energy of SPySagA decreased with heterocycle formation and the average energy decreased from 1799.3 to 1777.5 kcal/mol (Figure 2C and 2F).
The molecular structure containing the leader peptide and propeptide in modeled SagA1, SagA2, and SPySagA were compared pre- and post-heterocycle formation (Figure 3). Before heterocycle formation, the border between leader peptide (dark gray line) and propeptide (light gray line) was unclear (Figure 3B, D and F). After heterocycle formation, the conformation of these SagA1, SagA2, SPySagA molecules was significantly changed. In SagA1 and SagA2, the leader peptide domain (dark gray) was enclosed in the inner part of the molecule (Figure 3A and 3C). In SPySagA, the leader peptide domain (dark gray) was bundled with the propeptide domain (Figure 3E). These results suggest that the heterocyclic structure is involved not only in the cytolytic activity of these molecules but also in the proper processing to convert them into their active form.
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 (an index of reactivity) also changed markedly due to heterocycle formation (Figure 3G, I and K). In the pre-heterocycled SagA1 molecule, a negative ESP field (dark gray cloud) covered the whole molecule (Figure 3H). After heterocycle formation, the propeptide region of SagA1 was covered with a negative ESP field (Figure 3G). For the SagA2 molecule, positive (light gray cloud) and negative (dark gray cloud) ESP fields covered the whole molecule with and without heterocycle formation (Figure 3I and J). In the heterocycled and pre-heterocycled SPySagA molecule, the ESP distribution pattern was similar to that of SagA1 (Figure 3K and L), respectively. These results indicate that the reactivity of these SagA molecules with their target molecule(s) is extremely different pre- and post-heterocycle formation.
Order of heterocycle formation in SagA molecules. SagA1 has four heterocycle formable sites (31C-32C, 33F-34S, 44G-45S, 46T-47T), and the order of heterocycle ring formation was examined using MM-MD energy simulation. The total energy of the first ring-formed SagA1 was compared for each four ring-formable sites, and the energy of the 33F-34S heterocycled molecule was the lowest (1772.3 kcal/mol in Table I) among these sites [1789.3 kcal/mol (31C-32C), 1801.7 kcal/mol (46T-47T), 1828.0 kcal/mol (44G-45S)]. The lowest total energy of the second, third, and fourth ring-formed SagA1 molecule was 1762.1 kcal/mol (46T-47T heterocycled SagA1), 1755.0 kcal/mol (31C-32C heterocycled SagA1), and 1784.7 kcal/mol (44G-45S heterocycled SagA1), respectively. SagA2 has three heterocycle formable sites (31C-32C, 33F-34S, 44G-45S). The order of heterocycle formation in SagA2 was examined as well as SagA1, and the first ring was suggested to form at 31C-32C (1617.6 kcal/mol). The second and third rings were suggested to form at 33F-34S (1623.9 kcal/mol) and 44G-45S (1538.0 kcal/mol), respectively. SPySagA has five heterocycle formable sites (31C-32C, 33F-34S, 38G-39S, 45G-46S, 47G-48S), and the order of heterocycle formation was determined by MM-MD energy simulation as follows: first (31C-32C: 1811.9 kcal/mol), second (33F-34S: 1796.9 kcal/mol), third (38G-39S: 1803.8 kcal/mol), fourth (45G-46S: 1798.0 kcal/mol), an fifth (47G-48S: 1777.5 kcal/mol).
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.
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.
Discussion
The beta-hemolytic peptide called streptolysin-S is well-known and has been investigated in PGS such as S. pyogenes, and this beta-hemolytic peptide was found to be encoded by sagA gene in the sag operon. Recently, the genes encoding twin streptolysin S-homologous peptides (SagA1 and SagA2) were found to exist in the sag operon homolog of beta-hemolytic SAA strains, and each is responsible for the beta-hemolysis of beta-hemolytic SAA (8). In the present study, these SagA molecules from beta-hemolytic SAA and from S. pyogenes were modeled and their molecular features were analyzed. These SagA molecules had 3-5 heterocycle formable sites (Figure 1), and their structural features were expected to change according to heterocycle formation. The total energy of SagA1, SagA2 and SPySagA decreased with heterocycle formation during MD simulation period (Figure 2), and these molecules were thought to be stabilized by intramolecular heterocycle formation. In heterocycle-formed SagA molecules, the total energy converged in a short time (100 ps in Figure 2D-F). From these results, it was thought that the flexibility of SagA molecules changed by intramolecular heterocycle formation. It seemed that the change in this molecular flexibility affected the separation process between the leader peptide and propeptide (protoxin) region. Heterocycle formation is an important event not only for the functional appearance of SagA molecules but also for their maturation.
In heterocycle-formed SagA molecules, the boundary between leader peptide and propeptide was plain (Figure 3A, C and E). For instance, it existed with the leader peptide region contained in the propeptide region in SagA1 (Figure 3A) and SagA2 (Figure 3C). The heterocycle formation of SagA molecules is also suggested to contribute to the compartmentalization of the leader peptide and propeptide, and then the leader peptide dividing from the propeptide region, and the later process for the maturation of SagA molecules might thus be enhanced.
The whole molecule of SagA1 and SPySagA was covered with negatively-charged ESP field before heterocycle formation (Figure 3H and L). After heterocycle formation, the negatively-charged ESP field was observed in the propeptide region of SagA1 and SPySagA (Figure 3G and K). In the SagA2 molecule, the distribution pattern of positive and negative ESP fields was significantly changed by heterocycle formation (Figure 3I: heterocycled, 3J: pre-heterocycled). The change of the distribution of the ESP field according to heterocycle formation might take part in dividing the leader 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 heterocycle formation seen in the SagA family can be applied to the structural analysis of SagB, SagC and SagD molecules with intra-molecular ring formation. Moreover, the control of the molecular mechanism by intramolecular heterocycle ring formation can be also applied to the analysis of drug-excreting molecules (e.g. SagG, SagH, SagI) related to anti-bacterial and anticancer drug resistance.
- Received April 4, 2014.
- Revision received June 9, 2014.
- Accepted June 10, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.