Structural, Functional and Evolutionary study of In silico Three Dimensional Model of Pneumolysin.
Summary: Streptococcus pneumoniae, a gram-positive cocci shaped bacteria, is the major human pathogen, causing diseases like septic meningitis, otitis media, sinusitis, pneumonia and septicemia. The objective of present study is to gain more knowledge about the function of important domain of the toxin pneumolysin. This study aims to analyze the structural and functional features of pneumolysin and to investigate the residues involved in its pathogenicity.The major virulence factor of this bacterium is a protein, pneumolysin, which is the member of thiol-activated cytolysins. From the three dimensional homology model of the present study, it was found that pneumolysin has four domains, out of which domain 4 is of great importance. It was observed that Cys 428 and Trp 433 of pneumolysin are of great importance and any mutation in this region highly reduces its cytotoxicity.
Cys 428 forms hydrophobic contact with Ala 373 and Trp 436 of the conserved region, while Trp 433 is bonded with Trp 436 and Arg 426 through hydrogen interactions .The particular cysteine residue is present at position 428 and is also sandwiched between b-sheet and Trp 436. In pneumolysin, the undecapeptide or the Trp-rich loop spans the region (amino acid 427 to 437) and several single amino acid substitutions within this region reduce the cytolytic activity of pneumolysin by up to 99.9% as reported previously. The primary structure of pneumolysin has a total eight tryptophan residues and one cysteine. The undecapeptide region has three tryptophan and one cysteine residue containing 11 amino acid sequence i.e ECTGLAWEWWR. Cysteine 428 of pneumolysin present in trp-rich motif is responsible to act on cholestrol.
Pairwise alignment reveals that pneumolysin do not have the N-terminus signal peptide sequence which is present in the template i.e. perfringolysin. This shows that pneumolysin is an intracellular protein and released only upon cell lysis.
Keywords: Protein; Pneumolysin; Perfringolysin; Streptococcus pneumoniae; Toxin
The anaerobic, gram-positive and alpha hemolytic organism Streptococcus pneumoniae is known to cause life threatening diseases like bacteraemia, otitis media, septicemia and community acquired pneumonia . S. pneumoniae colonizes the upper respiratory tract of the host from where it can spread to the lower respiratory tract and can cause infection which leads to a condition known as pneumonia. Pneumonia refers to the inflammatory condition of the lungs . Community-acquired pneumonia (CAP) is one of the major causes of death with mortality rate of 10- 15% in hospitalized patients . Individuals with weak immune system are most susceptible to the infections caused by this bacterium, especially children, elderly people and patients with immunodeficiency disorders.
Treatment and immunization procedures against influenza virus and S. pneumoniae are less effective which has increased the risk of respiratory infections particularly in developing countries. Not only in developing countries but it is one of the leading causes of death in developed countries like in the United States [1, 4]. S. pneumoniae causes infection by producing virulence factors or toxins. Among these toxins pneumolysin is the most lethal even in nanogram quantities. It is a 53 kDa protein belonging to the group of thiol-activated or cholesterol-dependent cytolysins (CDC).
Pneumolysin generally intervene host cell by binding to membrane cholesterol and then oligomerize to form macromolecular pores in the host cell membrane which leads to cell death . Pneumolysin also interfere with specific functions of cells of the immune system such as the respiratory burst in polymorphonuclear leukocytes, chemotaxis, and phagocytosis of bacteria . As this toxin causes damage to the cells of the immune system and also interferes with their activity; therefore it is also known as a bifunctional toxin .
Nowadays efforts are being made to investigate virulence genes coding for pneumolysin (ply) and autolysin (lytA), as these two antigens have been linked up with virulence of S. pneumoniae in experimental animals .
The primary aim of the current study was to predict the homology model of pneumolysin and to evaluate its important domains in secondary structure, analysis of the predicted properties and functions, interactions of important domains of the toxin and to analyze its phylogenetic relationship with other family members.
Experimental Materials and Methods Retrieving primary sequence
Target sequence of pneumolysin (PLY) was retrieved from Expert Protein Analysis System (ExPASY) proteomic server (www. expasy. org) and was checked for sequence similarity against Protein Data Bank (PDB)  by using Basic Local Alignment Search Tool (BLAST) . The suitable template selected is perfringolysin (PDB ID 1PFO), produced by Clostridium perfringens, thus its crystal structure coordinates was obtained from PDB.
Target sequence of pneumolysin (PLY) was retrieved from Expert Protein Analysis System (ExPASY) proteomic server (www.expasy.org). This sequence to be modelled should be aligned with the database of known 3D structures using the programe Basic Local Alignment Search Tool (BLAST) , which estimates the degree of similarity of sequences and perform sequence alignment. On the basis of BLAST result, the suitable template selected was, perfringolysin (PDB ID 1PFO), produced by Clostridium perfringens. Crystal structure coordinates of template structure was obtained from Protein Data Bank (PDB) .
Three-dimensional model building and evaluation
Homology model of pneumolysin (PLY) was constructed using the modeller (9v7) program . This program has an alignment and python files which were used in this study after making few changes in it according to the target and template structures and ten energy minimized models were built. For the assessment of quality of the newly built models stereochemistry, the Procheck software  was used for the best model selection, which was further evaluated using the ProSA program . For visualization and superposition of the protein structures the Discovery Studio Visualizer (DS Viewer) program was used  while the interactions in these proteins were studied through Ligplot .
The sequence of pneumolysin (PLY) was compared with other thiol-activated cytolysins. Multiple sequence alignment was subjected to clustal X program  to analyze its phylogenetic relationship with other family members.
Results and Discussion
Sequence comparison/BLAST result
The accession number of pneumolysin (P0C2J9) was used to retrieve its sequence from SwissProt database . It consists of 471 amino acid residues and is present in FASTA format. The three dimensional structure of Perfringolysin (PDB ID 1PFO) was selected as a template because it is the thiol-activated cytolysin whose structure showed better homology with the target sequence (PLY). Both of these proteins belong to the same cholesterol dependent cytolysin family .The amino acid sequence of Perfringolysin (P0C2E9) was retrieved from Swissprot database.
The BLAST result Fig. 1 showed that PFO is homologous to PLY with one gap. There are 216 identities or identical residues and 316 positives having similar biochemical properties which accounts for 47% and 69% respectively.
Three-dimensional model building and evaluation
The amino acid sequence of pneumolysin (PLY) was subjected to modeller (9v7)  software to construct its three dimensional structure. Ten energy minimized models were constructed along with Het atoms (water) and all of them were analyzed by Procheck software. Best model was selected for further analysis according to Procheck result (Ramachandran plot). Three dimensional model consists of 471 residues among which 384 (90.6%) were present in favored regions, 38 (9%) and 2 (0.5%) were present in additional allowed and generously allowed regions respectively, whereas there were no residues in disallowed region (0%) Fig. 2. Few differences were observed between its template structure and the target. PLY has an elongated structure with four domains named as domain 1, 2, 3, and 4 Fig. 3. Domain 1 is situated on the N-terminus and has a/b topology.
Domain 2 is mainly composed of b-sheets while domain 3 has a/b/a topology. The most important domain 4 is consists of eight b-sheets that are present in parallel and antiparallel fashion. Domain 2 and 4 are linked by a linker residue asparagine-360 leading towards the fourth domain.
Phylogenetic analysis was carried out using Clustal X program to track the evolutionary history of PLY with other homologous proteins. Pairwise alignment Fig. 4 showed the relative homology between the two sequences. Pneumolysin contains 471 residues and the template perfringolysin contains 500 residues whereas it also contains a 27 amino acid signal peptide at the N-terminus. Pneumolysin do not have this signal peptide sequence indicating the fact that it is an intracellular protein. Multiple sequence alignment of 13 different thiol-activated cytolysins was retrieved and showed that N-terminus regions of these proteins are markedly variable whereas the C-terminus region showed higher homology Fig. 5. Domain 4, the most important domain that contains the undecapeptide region also lies towards the C-terminus of thiol-activated cytolysins and is conserved in most of them.
The phylogram shows that pneumolysin is evolutionary related with intermedilysin and they share a common ancestry. The relative distances of different proteins are also shown in the phylogram Fig. 6.
C-terminus conserved region and hydrophobic nature
All cholesterol binding toxins (CBT's) shows similarity in their primary amino acid sequence and identity in the so called undecapeptide region having tryptophan-rich loop that contains 11 amino acids, which is nearest to the C-terminus of the polypeptide chain . This structural motif is rich in hydrophobic residues and is connected to the functionality of CBTs . All CBTs contain this identical trp-rich region except intermedilysin which shows some variation in this region.
In PLY, the undecapeptide region spans the region (amino acid 427 to 437) is critical for its cytotoxicity. The particular cysteine 428 and tryptophan 433 of pneumolysin are of great importance and any mutation in this region reduce its cytotoxicity to a greater extent e.g. two pneumolysin (PLY) mutations i.e. Cys 428-Gly and Trp 433-Phe leads to the production of a toxin with only 0.001 cytolytic activity as reported by Saunders et al., .
Present study shows that Cys 459 of PFO is in hydrophobic contact with Gln 405 and Trp 467 of the conserved region Fig. 7. The nitrogen atom (N) attached to a-carbon (CA) of Cys is making hydrogen bond (2.89A) with the oxygen atom (O) of Val 403 whereas Glu 458 and Thr 460 are present in the nearby vicinity. This Cys 459 is located near the tip of domain 4 and is sandwiched between a b-sheet and Trp 467 as reported by Anne et al., also . The functionality of PFO mainly depends on this hydrophobic loop as it triggers cholesterol binding and membrane insertion. The overall conformation of this Trp-rich loop changes if the modification of cysteine occurs. These changes also have adverse effects on the cytotoxicity of the toxin .
In the in-silico three dimensional (3D) model of pneumolysin (PLY), cysteine of this conserved region is present at position 428 and is in hydrophobic contact with Ala 373 and Trp 436 of the conserved region. The nitrogen atom (N) attached to the a-carbon (CA) of Cys 428 is making hydrogen bond (3 A) with the oxygen atom (O) of Val 372 whereas Glu 427 and Thr 429 are present nearby vicinity Fig. 8.
Comparison of template and target 3D structures reveals that only the glutamine contact of perfringolysin (PFO) is replaced by alanine, which is mildly hydrophobic in its chemical nature in pneumolysin (PLY) , whereas all other contacts are almost same in both proteins indicating that both of them would have almost the same function. In template PFO, Trp 464 is bonded with Glu 407 and Arg 457 through hydrogen interactions which have got the ability as a dragger for penetration into cholesterol rich membranes as reported by Alouf and Geoffroy .
In present study, it was observed that Trp 433 of PLY is bonded with Trp 436 and Arg 426 through hydrogen interactions Fig 9 a, b. The arginine contact is present but the acidic residue of template involved in contact, is replaced by Trp which is biochemically hydrophobic in nature. Therefore it can be hypothesized that the dragging and penetration ability of PLY into cholesterol containing membranes would be greater as compared to PFO.
Cholesterol dependent cytolysins (CDCs) have been identified in five different genera of Gram positive bacteria . They exhibit a number of unique features that includes an absolute dependence on cholesterol rich membranes and the formation of transmembrane pores. More than 20 members of this family have been identified so far. The crystal structure of one CDC i.e. PFO from Clostridium perfringens has been identified  which revealed that CDCs are elongated and rod shaped molecules, composed of four domains and are mainly rich in b-sheets.
Almost all CDCs have a conserved undecapeptide region. The cytotoxicity of the CDCs is sensitive to the modifications in this region, especially mutations of the tryptophan or the cysteine residues highly affect its binding capacity, for example, intermedilysin (ILY) secreted by Streptococcus intermedius has an alanine residue instead of cysteine of the conserved region, resulting in an oxidation-resistant toxin and a proline residue is present in place of second conserved tryptophan . Similarly, in PLY the undecapeptide or the Trp-rich loop spans the region (amino acid 427 to 437) and several single amino acid substitutions within this region reduce the cytolytic activity of pneumolysin by up to 99.9% [18, 19].
The cholesterol binding sites of PFO lies in its conserved cysteine residue, sandwiched between a b-sheet and Trp 467 [25, 26]. Another supporting reason is that the particular cysteine residue has a potential for the formation of amphiphilic helix . In PLY, the particular cysteine residue is present at position 428 and is also sandwitched between b-sheet and Trp 436 Fig. 10.
The primary structure of PLY has a total eight tryptophan residues and 1 cysteine. The undecapeptide region has three tryptophan and one cysteine residue. The cysteine and tryptophan of this particular region is of great importance. From the high degree of sequence similarity, similar conformation and position of Trp residues in Trp-rich loop, we propose the same mechanism of action and the formation of amphiphilic helix by PLY. These amphipatic beta-hairpins of cytolysins from membrane binding beta-barrel with hydrophilic inner and hydrophobic outer surfaces .
The model built (pneumolysin) was superimposed on the template 3D structure and was found that it superimposed well on the template. There were no evident differences found between the two Fig. 11.
On the basis of present research study it was observed that pneumolysin belongs to the group of protein named as thiol-activated cytolysin. These proteins have the property of damaging cholestrol containing membranes and cause lysis of the host cell. On the basis of this study, it was found that pneumolysin has four domains, out of which domain 4 is mainly important. The conserved undecapeptide region also called as trp-rich loop is present in domain 4 and contain 11 amino acid sequence i.e ECTGLAWEWWR. This region is hydrophobic in nature which would affect the penetration ability of the toxin. Cholesterol in cell membrane acts as a receptor for the toxin to recognize and attack on the host cell.
Cysteine 428 of pneumolysin present in trp-rich motif is responsible to act on cholestrol. Pairwise alignment reveals that pneumolysin do not have the N-terminus signal peptide sequence which is present in the template i.e perfringolysin. This shows that pneumolysin is an intracellular protein and released only upon cell lysis.
Authors are thankful to the Centre of Biotechnology and Microbiology, University of Peshawar, for provision of research facilities. All authors have no conflict of interest.
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Apr 30, 2017|
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