1. Introduction

Streptococcus pneumoniae is a major pathogen that causes diseases such as pneumoniae, meningitis and sepsis ^[1]. World Health Organization (WHO) estimated that 476000 global annual deaths among children less than 5 years of age were caused by pneumococcal infections ^[2]. S. pneumoniae serotype 19F is among main pneumococcal serotypes that cause invasive pneumococcal disease in children less than 5 years of age in the world. Pneumococcal vaccines have been used for protection against pneumococcal infections. Capsular polysaccharides of pneumococci are main antigenic components of these vaccines. However, the capsule elicits weak immunogenicity in children and is not able to induce immune memory. In order to overcome these shortcomings, the capsule is chemically conjugated to a proteinaceous carrier. WHO recommends the inclusion of pneumococcal conjugate vaccines (PCVs) in national immunization programs for children ^[3]. However, high manufacturing costs of PCVs limit their implementation in developing countries ^{[1, 2]}. Development of protein based vaccines against pneumococci offers a more affordable protective strategy against pneumococcal infections. Cell surface proteins are key factors in infectious processes of pathogens and can be considered as vaccine candidates ^{[4, 5]}. LPxTG motif containing proteins are a group of cell surface proteins in gram positive bacteria and are first synthesized as a preprotein containing an N-terminal signal peptide and a C-terminal cell wall anchor domain. The anchor domain is attached to the cell wall by a sortase enzyme ^{[5, 6]}. Lipoproteins constitute another group of cell surface proteins in gram positive bacteria. These proteins are lipid modified proteins produced as secretory precursors with a signal peptide linked to their amino termini. The C-terminal region of their signal peptide contains a well conserve lipobox motif of four amino acid residues and invariably, the last residue is cysteine. The covalent binding of lipoproteins to the cell membrane is achieved by a diacylglyceryl transferase ^{[7, 8]}. Choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall via a conserved choline binding domain. LPxTG motif containing proteins, lipoproteins and CBPs are among main groups of pneumococcal cell surface proteins ^[5].

Conventional experimental approaches for vaccine discovery cultivate pathogens under laboratory conditions, dissecting them into their main component proteins. Antigens offering protective immunity are then identified by testing individual components. The antigens also need to be produced on a large scale, making the process extremely time consuming ^[9]. However, bioinformatic approaches for identification of vaccine candidates offer the ability to undertake a rapid and comprehensive assessment of a microorganism's surface proteins ^[10]. To our knowledge, there is no report on bioinformatic analysis of surface proteins of a serotype 19F S. pneumoniae. In this study therefore, we aim to determine appropriate vaccine candidates among the above mentioned three groups of surface proteins in S. pneumoniae serotype 19F to develop protein based vaccines against this pneumococcal serotype. The pneumococcal surface proteins were therefore determined using bioinformatic tools. Then suitability of the proteins as vaccine candidates was evaluated in terms of their antigenicity and the absence of autoimmunity induction.

2. Materials and Methods

Protein BLAST was used to search for LPxTG consensus sequence in the proteome data of S. pneumoniae 19F-14. CW-PRED tool was used to determine the cell surface anchoring domain in these proteins. Cellular localization of the proteins and the signal peptide presence were specified using PSORTb v.3.0 program and Signal P 4.1 tool respectively.

The predicted lipoproteins of S. pneumoniae D39, S. pneumoniae R6 and S. pneumoniae TIGR4 available in PRED-LIPO were used to specify conserved motifs of lipobox. Protein BLAST was used to search for these motifs in proteome data of S. pneumoniae 19F-14. The PRED-LIPO tool was used to verify the presence of lipoprotein specific signal peptide in these proteins.

Protein BLAST was done to find proteins of S. pneumoniae 19F-14 containing the consensus sequence of the choline binding domain (CBD) (TGWVKINGNWYYFDSNGVM) available in Pfam. The cellular localization of these proteins was determined by PSORTb v.3.0.

VaxiJen v.2.0 was used to evaluate the antigenicity of the proteins. Blast against human proteome was carried out to exclude the possibility of inducing autoimmunity by the proteins.

3. Results and Discussion

Search for LPxTG motif in proteome data of S. pneumoniae 19F-14 demonstrated the presence of 25 proteins containing this motif. LPxTG motif in 20 of these proteins resides in the C-terminal region and it is in the N-terminal region of the rest of the proteins. The LPxTG motif in cell surface proteins has been reported to reside in the C-terminal region. Therefore, we continued our analyses with the 20 proteins possessing this status. Among these proteins, the presence of cell surface anchoring domain was confirmed in 12 proteins. PSORTb v.3.0 results demonstrated the cell wall localization for these proteins. The Signal P 4.1 analysis indicated the presence of signal peptides belonging to YSIRK family of signal peptides in 7 proteins (Table 1). Signal peptides direct proteins towards secretory pathways and are located in amino terminal regions of these proteins. Evaluation of antigenicity of these proteins using VaxiJen v.2.0 demonstrated their capabilities as protective antigens. However, cell wall surface anchor family protein under accession number YP_002741626.1 showed the highest antigenic probability.

Table 1. Pneumococcal cell surface proteins containing LPxTG motifs

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Antigenic similarity of either amino acid sequences or the conformational structure between antigens of pathogenic agents and host tissues might induce an immune response against the shared determinant. Therefore, the host tolerance to these antigens (autoantigens) breaks down and the elicited autoantigen specific immune response cross reacts with host structures to cause tissue damage and disease ^[11]. As a result, we excluded the possibility for induction of autoimmunity by analysis of homology between pneumococcal antigens and human proteome via BLAST. The cell wall surface anchor family protein under accession number YP_002741626.1 showed no homology to human proteome. The homolog proteins of this protein found in other pneumococcal strains belonging to serotype 19F including S. pneumoniae A026, S. pneumoniae G54, and S. pneumoniae ST556 with homology percentages in the range of 98-100%. The predicted function for the cell wall surface anchor family protein obtained via BLAST was fibronectin binding. Our results therefore demonstrated that this protein can be considered as an appropriate vaccine candidate for development of protein based vaccines against serotype 19F pneumococci.

Conserved motifs of pneumococcal lipobox were found in 61 proteins of S. pneumoniae 19F-14. Results of PRED-LIPO analysis indicated that 34 proteins among them contain lipoprotein signal peptide and are thus considered as lipoproteins. PSORTb v.3.0 demonstrated the extracytoplasmic localization for 31 proteins among the predicted lipoproteins and analysis with VaxiJen v.2.0 showed that only 20 proteins were probable protective antigens (Table 2). The two most probable antigens were foldase protein PrsA and D-alanyl-D-alanyl-carboxy peptidase. BLAST results showed that homology percentages of foldase protein PrsA and D-alanyl-D-alanyl-carboxy peptidase with the human proteome were 28% and 0% respectively. The homolog proteins of D-alanyl-D-alanyl-carboxy peptidase were available in other pneumococcal serotype 19F strains including S. pneumoniae A026, S. pneumoniae G54, and S. pneumoniae ST556 with homology percentages in the range of 88-100%. These results demonstrated that D-alanyl-D-alanyl-carboxy peptidase is an appropriate protein based vaccine candidate. This protein is involved in the control of peptidoglycan crosslinking extents ^[12].

Table 2. Pneumococcal antigenic lipoproteins

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The consensus sequence of CBD was found in 100 proteins of S. pneumoniae 19F-14. Extracellular, cell wall and extracytoplasmic localizations were demonstrated by PSORTb v.3.0 for 7, 5, and 8 proteins respectively (Table 3). Thirteen proteins among them were antigenic. However, only 5 proteins did not show similarity to the human proteome and homologs of just three proteins among them including autolysin, surface protein PspC, and choline binding protein D (CBPD) were available in other pneumococcal serotype 19F strains including S. pneumoniae A026, S. pneumoniae G54, and S. pneumoniae ST556 with homology percentages of more than 70%. The antigenicity of PspC was more than that of CBPD. However, its homology percentage to the similar protein in the above mentioned three pneumococcal strains (71-90%) was less than that of CBPD (99-100%). PspC promotes uptake of pneumococci into nasopharyngeal epithelial cells and interferes with components of the innate immune systems ^[5]. Vaccination with surface protein PspC derived from S. pneumoniae serotype 2 has shown to be protective against sepsis in mice caused by the same pneumococcal serotype ^[13]. Our results also indicated the suitability of surface protein PspC and CBPD as antigenic candidates for development of proteinaceous vaccines against S. pneumoniae serotype 19F infection. CBPD is a key component of competence induced DNA release process of pneumococci ^[14].

4. Conclusions

Among 186 proteins entered our bioinformatic based analysis for selection of vaccine candidates against serotype 19F pneumococcal infection, four proteins including cell wall surface anchor family protein under accession number YP_002741626.1, D-alanyl-D-alanyl-carboxy peptidase, surface protein PspC, and CBPD were appropriate candidates. For future studies, these proteins will be expressed in suitable hosts and their protective abilities against serotype 19F pnemococci will be evaluated experimentally.

Table 3. Extracytoplasmic pneumococcal choline binding proteins

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References

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