In silico design of a novel chimeric shigella IpaB fused to C terminal of clostridium perfringens enterotoxin as a vaccine candidate.

This study aimed to design a novel chimeric protein in silico to serve as a serotype-independent vaccine candidate against Shigella. The chimera contains amino acid residues 240-460 of Shigella invasion plasmid antigen B (IpaB) and the C-terminus of Clostridium perfringens enterotoxin (C-CPE). Amino acid sequences of 537 peptide linkers were obtained from two protein linker databases. 3D structures of IpaB-CPE290-319, IpaB-CPE184-319, IpaB-CPE194-319 and 537 newly designed IpaB-linker-CPE290-319 constructs with varying linker regions were predicted. These predicted 3D structures were merged with the 3D structures of native IpaB240-460, CPE194-319, CPE184-319 and CPE290-319 to select the structure most similar to native IpaB and C-CPE. Several in silico tools were used to determine the suitability of the selected IpaB-C-CPE structure as a vaccine candidate. None of the 537 linkers was capable of preserving the native structure of CPE290-319 within the IpaB-linker-CPE290-319 structure. In silico analysis determined that the IpaB-CPE194-319 3D structure was the most similar to the 3D structure of the respective native CPE domain and that it was a stable chimeric protein exposing multiple B-cell epitopes. IpaB-CPE194-319 was designed for its capability to bind to human intestinal epithelial and M cells and to accumulate on these cells. The predicted B-cell epitopes are likely to be capable of inducing a mucosal antibody response in the human intestine against Shigella IpaB. This study also showed that the higher binding affinities of CPE184-319 and CPE194-319 to claudin molecules than those of CPE290-319 is the result of preserving the 3D structures of CPE184-319 and CPE194-319 when they are linked to the C-termini of other proteins.

Introduction

Shigellosis, a disease caused by Shigella, is endemic in developing countries due to a lack of hygienic drinking water sources, malnutrition and the high cost of antibiotic therapy. There are more than 40 Shigella serotypes, which are classified based on O antigen polysaccharide diversity. S. flexneri and S. dysentery are the causative agents of endemic and epidemic dysentery, particularly in children under 5 y old in developing countries. Shigellosis often causes severe symptoms and may lead to death. An estimated 80–160 million annual cases of dysentery occur worldwide, which lead to approximately one million deaths annually. To date, no effective vaccine prevents the disease caused by Shigella. In addition, the rapid and broad global spread of antibiotic resistance has been reported among different serotypes of Shigella. Thus, the World Health Organization (WHO) has prioritized the development and production of an efficient vaccine to globally protect people against Shigella. Immunogenic Shigella surface proteins that are conserved among different serotypes are thought to be capable of inducing broad-spectrum protection against multiple Shigella serotypes. Invasion plasmid antigen B (IpaB) is a Shigella surface protein located at the very top of its type three secretory system (TTSS), and it is a key virulence factor of Shigella. This protein alone may not be capable of inducing a protective immune response against Shigella, but it has been recently used, along with other Shigella proteins, to generate effective vaccine candidates. Since 2012, the combinations of IpaB/IpaD and IpaB/GroEL have been reported aspotential vaccine candidates capable of inducing protection against experimental Shigella infections in mice.

Clostridium perfringens enterotoxin (CPE) consists of two functional domains: a cytotoxic N-terminal domain and a non-toxic C-terminal domain (C-CPE). The C-terminal domain of CPE is known to be capable of binding to claudin-3 and claudin-4 molecules at the surface of some mammalian cells. The strong attachment of the C-CPE to claudin-3 and -4 on human intestinal epithelial cells causes this peptide to aggregate on the luminal sides of intestinal epithelial cells, allowing it to act as a biological and mucosal adjuvant for orally administered drugs and vaccines. Previous studies have indicated that these attachments eventually lead to the transfer of the C-CPE, and likely any protein fused to it, into the cytoplasm of intestinal epithelial cells through receptor mediated endocytosis.

Claudin-3 and -4 molecules belong to the claudin receptor molecule family. Claudin-3 is localized on the apical surfaces of villi and cryptic epithelial cells of the human small and large intestines. Moreover, these molecules are localized on the entire apical surfaces of follicle associated epithelia (FAEs) in the human intestinal tract. However, claudin-4, another receptor for the C-CPE, is found more frequently on the basolateral surfaces of epithelial cells in the intestinal villi. Claudin-4 is also found on the apical surfaces of FAEs in Peyer's patches and on the apical surfaces of cecal patches in the human large intestine.

The aim of the current study is to design a chimeric IpaB-C-CPE protein for use in the development of a vaccine to combat Shigella infections. To the best of our knowledge, this report is the first to design a chimeric IpaB-C-CPE for any biological or medical purpose.

Results

The amino acid sequences of 537 chimeric IpaB-linker-CPE290–319 variants generated using 537 different peptide linkers to connect IpaB240–460 to CPE290–319 were obtained using an in silico sequence conversion tool. Furthermore, the amino acid sequences of IpaB-CPE184–319 and IpaB-CPE194–319 were obtained successfully using the abovementioned software. All the obtained amino acid sequences were analyzed by phyre2 software, and 3D structures of the recombinant proteins were downloaded as 539 PBD files. The 3D structures of 537 chimeric proteins were compared with the 3D structures of native IpaB240–460 and CPE290–319 using TM-align software, and the rate of similarity index between these proteins and recombinant IpaB-linker-CPE290–319 was calculated by measuring the TM-score. The obtained results showed that all 537 linkers had impaired CPE290–319 and IpaB240–460 3D structures. Using TM-align software from the Zhang lab, the 3D structures of CPE184–319 and CPE194–319 were found to exhibit considerable similarity to their corresponding domains in IpaB-CPE184–319 (TM-score: 0.97) and IpaB-CPE194–319 (TM-score: 0.99), respectively (Fig. 1).

Figure 1.

Protein 3D structure alignments generated by the UCSF Chimera package. (A) Structural alignment of IpaB-CPE194–319 (in white) with native CPE194–319 (in black). (B) 3D structure of IpaB-CPE184–319 (in white) aligned with the 3D structure of native CPE184–319 (in black).

IpaB-CPE194–319 was predicted to be a stable 38.5 KDa protein with an estimated half-life of more than 10 hours in E. coli. The aliphatic and grand average of hydropathicity (GRAVY) indices of IpaB-CPE194–319 were predicted by ExPASy ProtParam online software to be 109.66 and 0.117, respectively. The results obtained from IpaB-CPE194–319 secondary structure analysis revealed a high percentage of alpha helices and low percentages of beta strands and disordered regions. In addition to these secondary structures, a moderate percentage of transmembrane helices was predicted (residues 72–184).

In total, 150 B-cell lineal epitopes (5–20 amino acids) exhibiting FBCpred scores greater than 0.7 were predicted within the IpaB-CPE194–319 structure (Supplementary Table 1). Among all 150 lineal epitopes within the chimeric protein, 62 B-cell epitopes fit within residues 1–221, which correspond to the IpaB240–460 structure. Moreover, 28 B-cell conformational epitope residues were predicted by the DiscoTope server within full-length IpaB-CPE194–319 (Table 1). Among all predicted conformational epitope residues, 13 epitope residues were predicted within residues 1–221.

Table 1.

IpaB-CPE194–319 B-cell conformational epitopes predicted by the DiscoTope server. In total, 28 epitope residues were identified within IpaB-CPE194–319.

Epitope position	Epitope sequence	Contact number	DiscoTope score
1–4	MQKS	1, 8, 6, 0	−0.257, −1.600, −2.288, −2.433
25	N	0	−2.840
45	K	11	−3.585
47	E	11	−2.467
152	G	0	−2.651
154	D	5	−3.627
217–218	SQ	5, 5	−2.878, −3.606
220–224	DDLDI	6, 4, 9, 0, 3	−2.641, −1.886, −3.211, −1.327, −2.379
248–249	PA	0, 9	−1.307, −2.740
262	P	5	−3.416
309–312	DGVK	5, 4, 0, 4	−2.702, −1.851, −1.469, −2.795
334–338	SYSGN	1, 20, 0, 5, 9	−1.462, −2.922, 0.607, −0.292, −1.866

Codon optimization of ipaB-c-cpe (encoding IpaB-CPE194–319) was performed using OPTIMIZER according to an E. coli codon usage table provided by the Kazusa codon usage database. The codon adaptation indices (CAIs) of the codon-optimized and non-optimized recombinant genes were analyzed by both the OPTIMIZER and GenScript rare codon analysis tools. The results showed a significant increase in the CAI following codon optimization. The CAI value determined by OPTIMIZER following codon optimization was 1, whereas the CAI values calculated by the GenScript rare codon analysis tool were 0.62 and 0.94 prior to and following codon optimization, respectively (Fig. 2).

Figure 2.

Codon adaptation index (CAI) values of ipaB-c-cpe. (A) A CAI of 0.62 was calculated by the GenScript rare codon analysis tool before codon optimization. (B) A CAI of 0.94 was calculated by the GenScript rare codon analysis tool after codon optimization.

mRNA secondary structure prediction of ipaB-c-cpe showed that the most stable mRNA structure had a ΔG equal to −311.2 kcal/mol, and no stable hairpins or pseudo knots were observed in this predicted structure (Fig. 3).

Figure 3.

Prediction of ipaB-c-cpe mRNA secondary structure. No long stable hairpins or pseudo knots were observed at the 5′ of the predicted structure.

Discussion

Currently, there is no available licensed vaccine against Shigella; however, several ongoing studies are focusing on Shigella surface proteins that are common among different serotypes of this microorganism as potential vaccine candidates. IpaB, one of these surface proteins, has been shown to be capable of inducing an antibody response in humans. Moreover, IpaB-specific antibodies have been shown to be negatively correlated with shigellosis severity in humans. Recently, a Lactococcus lactis bacterium-like particles (BLP) vaccine candidate harboring IpaB and IpaD was successfully designed and used in adult and newborn mouse studies. Protective immune responses against S. flexneri and S. sonnei were generated in 90% and 80% of the adult mice and 90% and 44% of newborn mice, respectively, following immunization with BLP-IpaB/D. IpaB fused to GroEL (IpaB-GroEL) has been investigated in a more recent study as a vaccine candidate against shigellosis. Interestingly, the immunization of mice with chimeric IpaB-GroEL resulted in the protection of 90–95% of mice following lethal challenges with S. flexneri, S. boydii and S. sonnei. The present study focuses on the design of a novel chimeric protein consisting of a variant of IpaB, IpaB240–460, which is an immunogenic surface protein conserved among different serotypes of Shigella, fused to the C-terminus of the Clostridium perfringens enterotoxin.

Residues 240–280 of IpaB are involved in protein stability and folding, while residues 313–333 and 399–419 form two large globular domains located at the Tip Complex (TC) of the TTSS that are likely responsible for any contact of IpaB with the host cell as well as the penetration of this protein into the host cell membrane. Our designed chimeric protein contains amino acid residues from IpaB, as well as their respective domains, that are mostly in contact with host cells and the environment and are essential for the initiation of Shigella invasion into epithelial cells. Therefore, the neutralization of these structures and domains by the induction of mucosal immunity likely leads to the inhibition of Shigella invasion and the prevention of the disease caused by these bacteria.

The presence of the C-CPE in the structure of our designed chimeric protein makes this chimeric protein better able to induce a mucosal immune response, as the C-CPE exhibits adjuvant properties when administered on mucosal surfaces. Several C-CPE variants, such as CPE168–319, CPE171–319, CPE184–319, CPE194–319 and CPE290–319, have been shown to be capable of binding to claudin family proteins. Among these variants, CPE290–319 is the most frequently studied. This variant is sufficient for binding to some claudin proteins with an affinity comparable to that of native CPE. Thus, the first chimeric protein designed in this study was IpaB-CPE290–319.

To preserve the native conformations and functions of IpaB240–460 and CPE290–319 in the structure of our first designed chimeric protein, different peptide linkers were placed between IpaB240–460 and CPE290–319. Then, the 3D structures of 537 recombinant IpaB-linker-CPE290–319 proteins differing in their peptide linkers were analyzed by TM-align software to determine the linker sequence resulting in the highest TM-score. The TM-score varies between 0 and 1. A TM-score of 1 indicates perfect similarity between the 3D structures of two proteins. As TM-align software compares uploaded protein data bank (PDB) files to the 3D structures of different proteins and peptides in several protein databases, and there is no 3D structure related to full-length IpaB or the C-terminus of IpaB, TM-align software was unable to determine any valid TM-scores for the comparisons of the IpaB-linker-CPE290–319 variants to IpaB240–460. However, we successfully used this software to compare our designed protein with CPE290–319. The similarity between IpaB-linker-CPE290–319 and CPE290–319 3D structures was determined to be random, with a TM-score of 0.39, and all the analyzed linkers were determined to cause significant disorganization in the 3D structure of CPE290–319.

In addition to the efforts described above, the 3D structures of IpaB-CPE290–319, IpaB-CPE184–319 and IpaB-CPE194–319 were compared to the 3D structures of CPE290–319, CPE184–319 and CPE194–319, respectively. The highest TM-score, 0.99, was achieved for the alignment of the IpaB-CPE194–319 and CPE194–319 3D structures, showing a high degree of similarity between these two 3D structures. Bioinformatic analysis also showed excellent similarity between the 3D structures of IpaB-CPE184–319 and CPE184–319, with a TM-score of 0.97.

Both CPE194–319 and CPE184–319 have been shown to be capable of high-affinity specific binding to claudin-3 and -4 at the tight junctions of intestinal epithelial cells without inducing cytotoxicity. Although CPE290–319 is known as the claudin-binding domain of the CPE, this variant of the C-CPE is not able to bind claudin-4 without the presence of its N-terminal domain. However, CPE184–319 and CPE194–319, containing longer N-terminal regions than CPE290–319, are capable of binding to claudin-4 more strongly, likely because they contain more of the functional domains required to bind their receptor molecules and claudins. According to previous results and the in silico analysis performed in the current study, the N-terminal domains of CPE184–319 or CPE194–319 are likely involved in the folding of these proteins, as they are necessary for the formation of a strong attachment to claudin-4.

Based on our findings, many natural and synthetic peptide linkers with different physical and chemical properties were unable to provide the physicochemical interactions necessary to correctly fold CPE290–319, which is a requirement for it to bind claudin-4 molecules. Thus, although CPE290–319 has been frequently used as a natural ligand for the attachment of other molecules to targeted cells, The results of this study indicate that CPE194–319 and CPE184–319 are better suited to this purpose. Both of these C-CPE variants have been successfully tested as absorption enhancers for delivering biologically active peptides to pulmonary, nasal and jejunal tissues. CPE184–319 is capable of enhancing the mucosal absorption of dextran, a model drug, approximately 400–fold more than sodium caprate, a common clinical absorption enhancer. CPE194–319 was shown to have similar absorption enhancing properties, while CPE289–319 was shown to have no enhancing effect on the absorption of dextran. However, the low solubility of CPE184–319 limits its application as a mucosal absorbent. The solubility of CPE194–319 has been shown to be 30–fold greater than that of CPE184–319, making it more suitable for use as a mucosal absorbent than CPE184–319.

The physical and chemical properties of chimeric IpaB-CPE194–319 were predicted by the ExPASy ProtParam server. The isoelectric point (pI) value was predicted to be lower than 7, which indicates the intrinsic acidity of the designed protein. The approximate low molecular weight of the designed protein decreases the probability of inclusion body formation consequent to its expression in E. coli. In addition, its estimated half-life of more than 10 hours in E. coli suggests an enhanced probability of the large-scale production and purification of this protein in E. coli without significant loss of its structure and activity. The high aliphatic index of chimeric IpaB-CPE194–319 shows the high stability of this protein across a wide range of temperatures. The positive predicted GRAVY value indicates the hydrophobic nature of this designed protein.

As a vaccine candidate against Shigella infections, IpaB-CPE194–319 is designed to induce mucosal antibody production to prevent IpaB-mediated Shigella invasion. The B-cell epitope prediction conducted in this study determined 150 lineal B-cell epitopes (5–20 amino acids in length) and 28 conformational B-cell epitope residues in the chimeric IpaB-CPE194–319. Among these, 62 lineal B-cell epitopes and 13 conformational B-cell epitope residues fit within the IpaB domain of the IpaB-CPE194–319 chimeric protein. According to these B-cell epitope prediction results, we assume that IpaB-CPE194–319 is theoretically capable of inducing a mucosal antibody response in the human intestinal tract against several IpaB epitopes. The abundance of predicted IpaB epitopes enhances the probability that chimeric IpaB-CPE194–319 can induce a protective humoral immune response.

Codon optimization of the recombinant protein was conducted by OPTIMIZER to enhance its further expression levels in E. coli. To calculate the CAI of the designed ipaB-c-cpe gene, two web-based programs, OPTIMIZER and the GenScript rare codon analysis tool, were used. The CAI is associated with the expression level of the desired protein and varies between 0 and 1. Genes with CAI values close to 1.0 and G/C contents between 30% and 70% are predicted to be ideally expressed. The CAI value of non-optimized ipaB-c-cpe was calculated to be 0.62 by the GenScript rare codon analysis tool. The values for the codon-optimized ipaB-c-cpe were 1.00 and 0.94, as calculated by OPTIMIZER and the GenScript rare codon analysis tool, respectively. The G/C contents of ipaB-c-cpe were also optimized to 51.61% and 42.95%, respectively, using OPTIMIZER. The CAI and G/C content values of the optimized genes indicate an enhanced probability of the high-yield expression of IpaB-CPE194–319 in E. coli.

Its predicted mRNA secondary structure indicates that the ipaB-c-cpe gene will be transcribed into a stable mRNA. The predicted mRNA structure does not include any translation inhibitory structures such as pseudo knots or long stable hairpins. Therefore, the predicted mRNA is theoretically capable of being translated into IpaB-CPE194–319 in E. coli cells.

This study describes the successful in silico design of a novel IpaB-CPE194–319 chimeric protein that is a potential vaccine candidate against Shigella infections. The obtained results show that, in comparison to CPE290–319, two other variants of the C-CPE, CPE184–319 and CPE194–319, are better able to preserve their natural 3D structures and to form stronger attachments to their natural ligands when they are linked to the C-terminal regions of other proteins. This ability may be a consequence of extra domains (184–289 and 194–289) that are involved in correct folding by CPE, CPE184–319 and CPE194–319. However, more in-depth studies are required to validate the results obtained in this study from the in silico analyses of different chimeric proteins containing different variants of the C-CPE. Furthermore, in vitro expression and purification of the designed IpaB-CPE194–319, followed by in vitro and in vivo evaluation of the immune response to this protein are proposed to determine the potential of this newly designed protein as a vaccine against shigellosis.

Materials and methods

Sequence analysis

To design the IpaB-C-CPE encoding gene, nucleotides 718–1380 of the full-length ipaB gene (NCBI accession number NC_004851.1) and a 5′ fragment of the cpe gene (NCBI accession number NC_008262.1) were used. The 5′ fragment of the cpe gene was chosen from the sequences encoding CPE290–319, CPE194–319 and CPE184–319 as is described in the construct design section. All amino acid sequences were obtained by converting their respective nucleotide sequences using an in silico sequence conversion tool (http://in-silico.net/tools/biology/sequence_conversion).

Construct design

To construct a chimeric protein resembling the native folding and function of the respective regions of IpaB and the C-CPE, 537 derivatives of chimeric IpaB-linker-CPE290–319 constructs varying in their linker moieties were analyzed in silico. The amino acid sequences of all 537 natural and synthetic linkers were adapted from Klein et al., 2014 (for a review, see ref. 31) and two protein linker databases [(http://www.ibi.vu.nl/programs/linkerdbwww) and (http://parts.igem.org/Protein_domains/Linker)] (Supplementary Table 2). In addition to these 537 derivatives of IpaB-linker-CPE290–319, three other variants of IpaB-C-CPE were designed: IpaB-CPE290–319, IpaB-CPE184–319 and IpaB-CPE194–319.

structure analysis

Three dimensional structures of 537 different IpaB-linker-CPE290–319 variants were obtained as PDB files by introducing the amino acid sequences of all 537 chimeric proteins to the Protein Homology/analogY Recognition Engine (Phyre2) (http://www.sbg.bio.ic.ac.uk/∼phyre2/html/page.cgi?id=index). In addition, PDB files containing 3D structures of IpaB240–460, CPE184–319, CPE194–319, CPE290–319, IpaB-CPE290–319, IpaB-CPE184–319 and IpaB-CPE194–319 without any linkers were created. Afterward, residue to residue alignments of the 3D structures of the chimeric proteins with native IpaB240–460 and CPE290–319 were performed using TM-align to analyze the similarity of the 3D structures of the relevant proteins based on their TM-scores (http://zhanglab.ccmb.med.umich.edu/TM-align). The 3D structures of IpaB-CPE184–319 and IpaB-CPE194–319 were also compared to CPE184–319 and CPE194–319, respectively. The chimeric protein most closely resembling the native structure of the C-CPE was selected for further analysis.

Physical and chemical parameters and secondary structure

The physical and chemical properties of IpaB-CPE194–319, including its theoretical pI, estimated half-life, instability index, aliphatic index and GRAVY, were predicted using ExPASy ProtParam online software (http://web.expasy.org/protparam/). Secondary structures and probable transmembrane helices of IpaB-CPE194–319 were predicted using Phyre2 web-based software.

B-cell epitope prediction

IpaB-CPE194–319 continuous/lineal B-cell epitopes (5–20 amino acids) were predicted through flexible length epitope prediction (FBCpred) using the BCPREDS server (http://ailab.ist.psu.edu/bcpred/predict.html). IpaB-CPE194–319 discontinuous/conformational B-cell epitopes were analyzed by uploading the 3D structure of chimera to the Disco Tope server (http://www.cbs.dtu.dk/services/DiscoTope/).

Codon optimization

The amino acid sequence of IpaB-CPE194–319 was then converted back to a nucleotide sequence using an in silico sequence conversion tool (http://in-silico.net/tools/biology/sequence_conversion). The nucleotide sequence of recombinant ipaB-c-cpe was codon optimized to be expressed in E. coli using OPTIMIZER (http://genomes.urv.es/OPTIMIZER). An E. coli codon usage table was adapted from a codon usage database (www.kazusa.or.jp/codon). The CAI, which is directly related to the expression level of the desired gene, was analyzed by both OPTIMIZER and the GenScript rare codon analysis tool (http://www.genscript.com/cgibin/tools/rare_codon_analysis) to determine the expression efficiency of the recombinant gene in E. coli.

mRNA secondary structure

The secondary structure of ipaB-c-cpe messenger RNA was predicted using the RNAstructure Web Server (http://rna.urmc.rochester.edu/RNAstructureWeb), and the presence of hairpins and pseudo knots at the 5′ end of the mRNA were analyzed.