In Silico Design of a Chimeric Protein Containing Antigenic Fragments of Helicobacter pylori; A Bioinformatic Approach

INTRODUCTION

H. pylori is a gram negative microaerophilic bacterium which inhabits the mucus layer overlying gastric epithelial cell [1-3]. The resulting infection may cause mild to severe complications including gastritis, peptic ulcer, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma (MALT) [4, 5]. This microorganism infects half of the world's population and is considered as a type I carcinogen by the World Health Organization [6]. Although antibiotic therapy is effective in the majority of patients with gastrointestinal symptoms, current therapies such as the use of proton pump inhibitors combined with antibiotics, has several disadvantages including low patient compliance, high cost and increased antibiotic resistance. Consequently, the treatment by antibiotics aren’t ideal [7]. Therefore, an alternative approach is needed to overcome the problems of the conventional treatment of H. pylori infection. Synthetic vaccines can offer long-term solutions to numerous important infectious diseases. So an effective vaccine would require to prohibit H. pylori-induced gastric disease. For achieving the mentioned therapeutic goals, the subunit vaccine is a promising way to increase the safety. But the development of such vaccines depends on the identification of antigens able to confer protection against H. pylori. Numerous protein antigens of H. pylori were evaluated including Urease [8-13] , heat-shock proteins [14, 15], VacA cytotoxin [16], thiolperoxidase (Tpx) [17], OipA [18], HpaA [19], NapA [20], and CagA [16, 21]. Previous studies illustrated that fusion proteins, a class of novel engineered biomolecules, have multi-functional properties derived from each of their component moieties [22, 23], therefore, combining various antigens participating in different aspects of colonization and pathogenesis having enough potential to elicit a board of cellular or humoral immune response that may be more effective than a single recombinant antigen inducing insufficient immunity with limited protective effect [24-27]. There is an increasing interest to determine the potential of therapeutic or protective effect of such fusion proteins. In this regard, some chimeric proteins obtained from different components of H.pylori have been proposed including HpaA, UreB and CagA as a fusion protein stimulating CD₄⁺ T cell response [28]. Furthermore, in another study three protective antigens including UreB, VacA and CagA were used to construct a new fusion protein expressed in live attenuated Salmonella showing increased the levels of IgG_2a, mucosal IgA and induced Th₁ phenotype response [29]. However, how some antigenic fragment can be combined as they show their highest immunogenicity and protectivity effect would be a problem. Bioinformatics is an interdisciplinary area combining statistics, computer science, mathematics, and engineering to evaluate and interpret of data in order to improve methods and software tools for understanding biological data. Fortunately, by using such interdisciplinary approach, we would have the chance to understand the function of a designed chimeric protein in vivo. Then, bioinformatic approach would help us effectively design a chimeric protein with more immunogenicity and protectivity.

In this study, we postulated that combination of FliD with antigenic fragments of well-known H.pylori proteins including UreB, VacA and CagL may enhance the cumulative antigenicity of antigens.

FliD protein (685aa) is an essential element in assembling of the functional flagella and a FliD mutant strain is completely non-motile. Flagellin plays a central role in bacterial motility and is necessary for colonization and persistence of H. pylori infection [30]. Interestingly, It has been shown that FliD protein reacts approximately with 97 percent of sera obtained from patients infected with H.pylori [31].

Urease B has been widely investigated as a potential antigen for the development of prophylactic and therapeutic vaccines against H. pylori infection [32, 33]. UreB(327-334) is considered as a good B cell epitope and has been found to be protective in mice [34, 35].

Another well-characterized H. pylori protein is the vacuolating cytotoxin (VacA). It induces large cytoplasmic vacuoles and apoptosis which are partly responsible for H. pylori induced epithelial cell damage [36, 37]. It suggests that 744-805aa of VacA includes MHC Class-II binding regions which efficiently stimulates CD⁴⁺ T-cells responses [29].

CagL protein is a versatile type IV secretion system (T4SS) surface protein equipped with at least two motifs to help binding to integrins causing the deviant signaling within host cells and facilitating translocation of CagA into host cells [38]. The critical role of CagL in attaching to the epithelial cells and starting pathogencity of H. pylori [39] may make it a good candidate for further studies.

To test our postulate, we designed a new structural model containing most dominant epitopes of FliD, UreB, VacA and CagLthat may provide a proper and safe candidate for prophylaxis against H.pylori infection. In this regard, FliD, UreB, VacA and CagL, were bound together by three hydrophobic linkers. Then, chimeric protein structure was analyzed through a bioinformatic approache.

MATERIALS AND METHODS

RESULTS

Design and Construction of Chimeric Gene

No redundant gene and related protein sequences of H.pylori virulence factors (FliD, UreB , VacA and CagL) were obtained from GenBank and analyzed with the ClustalW software (www.ebi.ac.uk/Tools/msa/clustalw2/) [67]. The nominated sequences for designing of chimeric construct were FliD (1-600), UreB (327-334), VacA (744-805) and CagL (51-100). These fragments were fused together using a repeat of hydrophobic amino acid linker. Four repeated GGGGS sequences were introduced between different domains for more flexibility and efficient separation. Schematic diagram of protein domain structures, their junctions and linker sites are shown in Fig. (1). A synthetic sequence encoding the chimeric protein was reverse translated into the nucleotide sequence. The Percentage of codons having a high-usage frequency distribution (91-100%) which was improved to 64 % in the optimized gene sequence (Fig. 2a). The codon usage bias in E.coli BL21 was increased by upgrading the codon adaption index (CAI) to 0.84 (Fig. 2b). The overall GC (guanine plus cytosine) content was enhanced to 50.33 in our antigenic sequence (Fig. 2c), which is a measurement of transcriptional and translational efficiency influenced as a compositional bias on the codon usage of a gene of interest. The Eco RI and HindIII restriction sites for cloning in prokaryotic vectors were successfully introduced at the N and C terminal of sequence, respectively.

Prediction of mRNA Secondary Structure

The evaluation of minimum free energy for 37 structures of chimeric mRNA, unoptimized and optimized sequences carried out by the ‘Mfold’server. The results showed that ΔG of the best predicted structure for the optimized construct was ΔG = -745.80 kcal/mol. The first nucleotides at 5' did not have a long stable hairpin or pseudoknot. Therefore, the binding of ribosomes to the translation initiation site and the following translation process can be readily accomplished in the target host. These outcomes were in the agreement with data obtained from the ‘RNAfold’web server (Fig. 3). Thermodynamic features of the optimized sequence are shown in Table 1.

Secondary Structure Prediction

The secondary structure of the chimeric protein was predicted by several online programs, and the best result was achieved by GOR-IV as shown in Fig. (4). Results indicated total residues of 785 which were made up 140 strands (17.83%), 299 helices (38.09%) and 346 random coils (44.08%). No predicted signal peptide was identified in the initial region of the protein sequence.

Evaluation of Model Stability

The predicted model was subjected to energy minimization by application of the Swiss-PdbViewer. The amount -25414.018 kcal/mol indicated that the synthetic chimeric protein would have an acceptable stability compared the original structure of each moiety. The structure validity of the chimeric protein was assessed for its reliability and structural quality by the computation of a Ramachandran plot. Results of RAMPAGE showed that 65.6 % resides clustered in the favored, 24.5 % in the allowed and only 9.9% in the outlier (Fig. 6). The PROCHECK server verified the RAMPAGE results.

DISCUSSION

H.pylori is a microorganism that has been classified as class I human carcinogen and has been linked to diseases such as gastritis and peptic ulcers. It has a high incidence of infection, especially in developing countries and the antibiotic therapy is often not successful [6]. Taking these issues into account, the recombinant subunit vaccine may have a potential preventive strategy in H.pylori infection which would defiantly help to reduce the risk of such infection. In the current study, different bioinformatic approaches were employed for predicting B and T cell epitopes by using only those peptides that possess high immunogenicity instead of the whole proteins. Based on our finding, a chimeric protein including immunodominant epitopes from different antigenic proteins such as FliD (1-600), UreB(327-334), VacA (744-805) and CagL (51-100) would likely induce strong comprehensive protective immunity. These amino acid sequences were checked and found that these epitopes highly conserved among different H. pylori strains. Suitable linker to attach the multi-domain proteins is important for desired component protein and accurate function [68]. These different domains are fused together using a repeat of GGGGS (Gly-Gly-Gly-Gly-Ser) linker. Two GGGGS linkers are placed between FliD and UreB, entailing two GGGGS moieties between UreB and VacA, CagL. Flexible GS spacers could provide structural flexibility improving protein stability and increasing biological activity [23]. Additionally, it has been reported that suitable linker peptides might be more effective than independent domains to improve the biological activity or increase the protein stability [68-70]. The physico-chemical parameters of chimeric protein were analyzed. The theoretical isoelectric point (pI) values of the fusion protein, as calculated by the Prot-Param were detected to be at 5.01. Thus, the protein is considered to be stable and compact at pI. Molecular weight was 84717.8Da which provides information on the full-length of protein expression, expression of modified, cleavage products, splice variants and processed protein. In order to improve the transcription efficiency and transcript stability, codon optimization is the starting point to the chimeric creation of a gene sequence that would be introduced into a specific host organism. The codon optimization was achieved by improving codon adaptation index (CAI) which is a measurement of the relative adaptiveness of the codon usage bias for a particular DNA or RNA sequence [71], the frequency of optimal codon (FOP) used to display the optimization level of the synonymous codon choice in each gene during the translation process [72], the overall GC content of the gene and eliminating negative components that may incorporate unfavorable secondary structures on mRNA. A codon adaptation index of 0.84 was achieved by the optimized gene sequence. At its ideal, a maximal CAI design would show a value of 1.0 and a CAI of >0.8 is considered as good although no natural bacterial gene has such theoretical value [73].The percentage distribution of codons was 64%. A low GC content and disapproving peaks that could cause extremely quick mRNA turnover were also transformed and distribution was balanced to 50.33%. Furthermore, rare codons, destabilizing elements and repetitive sequences that may be detrimental to protein expression levels in E.coli BL21 [64] were eliminated. According to the energy minimization, the predicted chimeric protein would have the acceptable stability with the lowest energy (-25414.018 kcal/mol). The Mfold server was used for the prediction of RNA secondary structure. A theoretically tractable DP algorithm which is employed by the Mfold software can find the minimum ΔG structure within its thermodynamic model and the high ability to predict true positive base pairs. These results showed that the mRNA was stable enough for the efficient translation in the prokaryotic host. Five models generated by I-TASSER server for the synthetic construct and the chemical features of the modeled structure were assessed by Ramachandran plot. As described in the result section, 65.6 % resides clustered in the favored, 24.5 % in the allowed regions indicated that the backbone of the structure, φ and ψ, were reasonably accurate. Therefore, results of RAMPAGE and PROCHECK servers for the chimeric fusion protein showed that 90.1% of the residues were totally located in the favored and allowed regions and only 9.9% of the total protein were in the outlier, it is worth nothing that these negligible residues were not a part of the conformational B-cell or T-cell epitopes under investigation and could possibly be due to the presence of chimeric junctions. Consequently, it seems that outliers would not have adverse effects on the chimeric protein quality. The data of Z, C, and TM scores and RMSD for accuracy of predicted 3D models by I-TASSER and the result of model quality confirmation by the ProSA-web showed that synthetic chimeric protein has features which are the characteristics of native structures. Regarding protein structure prediction, the fusion protein designed four domains that were separated by three main coil moieties which can help the fusion protein to form a final structure. The two GS linkers which are placed between FliD and UreB domains are likely to facilitate the folding of multimeric proteins. These coil structures are associated with special amino acid sequences, residues 600-610, 668-673 and 735-740, inserted between each segment. These fragments would support the stable structure of the fusion protein which contained four domains. The effectiveness of a vaccine significantly depends on stimulating a strong immune response containing both memory B and T cells [74]. The recombinant subunit vaccines which consist of epitopes can induce a more effective response than the immunity conferred by whole protein vaccines [75]. B-cell epitopes for the chimeric protein could be predicted on the basis of the solvent accessibility, antigenicity, flexibility, hydrophilicity and secondary structure analyses [64]. Only epitopes with high prediction scores as continuous and discontinuous B-cell epitopes were selected by ABCpred and discotope servers, respectively. T-cell epitopes were selected with high potential to interact with human HLA alleles. Regarding to the solubility importance, the presence of amino acids with the high accessible value such as LYS, SER, ASP, ASN, GLY and ALA would possibly result in the chimeric protein expressed in soluble form. Consequently, all epitopes would be possibly explored to immune system in in vivo that is very important to develop of a recombinant vaccine [75].

CONCLUSION

Our data showed that the possibility of successful production of a large chimeric protein composing of four domains FliD (1-600), UreB(327-334), VacA (744-805) and CagL (51-100) in the prokaryotic host. The epitopes of the synthetic chimeric protein can induce both humoral and cellular immune responses. These findings will intensify efforts to develop a protective vaccine against H.pylori infection and may also suggest this synthetic chimeric protein could help to diagnosis of the H. pylori infection. Further studies are required to stablish these notions which are the theme of our future researches.

FINANCIAL DISCLOSURE

This work was supported by grants from Iran National Science Foundation: INSF (grant No. 93034823) and Kashan University of Medical Sciences (grant no.93194).

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

REFERENCES