In silico analysis of a chimeric fusion protein as a new vaccine candidate against Clostridium perfringens type A and Clostridium septicum alpha toxins

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacteria. It causes anaerobic cellulitis, myonecrosis (gas gangrene), enteritis necroticans, and food poisoning in humans and gastrointestinal and enterotoxemic diseases in other animals (Dwivedi et al. 2015). C. perfringens based on the production of 4 major lethal toxins such as alpha, beta, epsilon, and iota is classified into 5 toxinotype (A, B, C, D, E) (Petit et al. 1999). One of the most important lethal and dermonecrotic toxins is alpha toxin (phospholipase C) produced in different amounts by all C. perfringens types (A–E), the predominant product in C. perfringens type A, stated as a primary virulence factor implicated in the necrotic enteritis and gas gangrene (Sakurai et al. 2004). The C. perfringens type A alpha toxin gene(cpa) is located on a chromosome and is 1197 bp in length, and translated into a mature protein containing 398 aa, with a molecular weight of 43 kDa (Takahashi et al. 1974; Saint-Joanis et al. 1989). Alpha toxin has phospholipase C(PLC) activity, is a zinc metalloenzyme, and can connect to target cell membranes in the presence of Ca²⁺ ions. The three-dimensional structure of the toxin by X-ray crystallography displays a two-domain protein. The N-terminal domain (residues 1–246) is a predicted structure similar to Bacillus cereus phospholipase C, and the C-terminal (residues 256–370) is a structure similar to eukaryotic Ca²⁺-binding C2 domains and also a linker fragment (residues 247–255) that binds the 2 domains (Naylor et al. 1998). During the twentieth century, several researchers showed that immunization could be used to prevent myonecrosis (Dwivedi et al. 2015). In 2012, Langroudi et al. investigated in silico analysis of a fusion protein against C. perfringens type D and B toxins and showed that the fusion protein is functional (Langroudi et al. 2012). C. septicum is a Gram-positive, anaerobic, spore-forming, motile bacteria. It is the causative agent of traumatic gas gangrene and non-traumatic gas gangrene in patients with various diseases that affect the colon, necrotizing enterocolitis, and pericarditis (Gordon et al. 1997). Several toxins such as alpha, beta, gamma, and delta are produced by C. septicum. One of the most important lethal virulence factors of C. septicum is alpha toxin (AT) and responsible for myonecrosis. This toxin utilizes its pathogenesis by pore formation on the host cell surface and also by a range of effects on the target cell (Tweten 2001). Secreted AT is an inactive protoxin and proteolytic hydrolysis is required for its activation. Protoxin located on the target cell membrane and connected to the GPI-anchored proteins (Ballard et al. 1993; Knapp et al. 2010). The Clostridium septicum alpha toxin gene (csa) is located on a chromosome and is 1332 bp in length and translated into an inactive protoxin protein containing 443 aa with a molecular weight of 48 kDa. The 48 kDa toxin via a carboxy-terminal cleavage site (located 4 kDa from the carboxy-terminus) was activated by trypsin (Imagawa et al. 1994; Ballard et al. 1993). The three-dimensional structure of the AT toxin by X-ray crystallography shows that AT structure, and sequence is similar to large lobe (D2-D4) of aerolysin. Because aerolysin is divided into 2 lobe proteins where the small lobe is domain D1 and the large lobe is domains D2–D4, respectively (Melton-Witt et al. 2006). In 2018, a protein fusion was developed against C. perfringens type D epsilon and C. septicum alpha toxins (Kamalirousta and Pilehchian 2018). The objective of current research was in silico analysis of a chimeric fusion protein against C. perfringens type A and C. septicum alpha toxins.

Recombinant DNA technical methods have permitted fusions of genes in a simple way. The fusion of cpa and csa genes and its cloning in E. coli TOP10 has been reported (unpublished). In our work, in silico analysis of a chimeric fusion protein against C. perfringens type A and C. septicum alpha toxins is described. According to the newest findings, this is the 1st time that alpha-alpha fusion gene is designed for producing alpha-alpha fusion protein. It could be as a proper candidate for recombinant vaccine development. In our search, the complete cpa sequence containing its signal peptide for the proper secretion of the fusion protein and the csa sequence lacking signal peptide were used. A linker fragment “A(EAAAK)2A” was designed to link both genes (Langroudi et al. 2011). The InterPro program using several databases such as PRINTS, PROSITE, PFam-A, TIGRFAM, PROFILES, and PRODOM shows that the fusion protein consists of C. perfringens alpha toxin/phospholipase C/P1 nuclease domain superfamily and PLAT/LH2 domain superfamily, the family member which is combined with C. septicum alpha toxin/Aerolisin/ETX pore-forming (Jones et al. 2014). In 1989, Titball et al. showed that the nucleotide sequences of the cpa gene are 1197 bp in length (at base 1327–2523) and signal peptide sequences of the cpa gene are 84 bp in length (at base 1327–1410) and about 28 aa of that Trp may be the first amino acid of the mature activated toxin (Titball et al. 1989). In another search csa gene of C. septicum, BX96 was cloned and expressed in E. coli. The nucleotide sequences of the csa gene are 1332 bp in length (at base 561–1892) and signal peptide sequences of the csa gene are 93 bp in length (at base 561–653) about 31 aa (Ballard et al. 1995). The nucleotide sequences of the fusion protein displayed that the signal peptide site is located at amino acid number 1 to 28 (Fig. 4). For verification of this finding, the secondary structure and signal peptide of the fusion protein were predicted by proMotif of proFunc server (Laskowski et al. 2005a, b) and signalP (Petersen et al. 2011) online program, respectively. The data displayed similar features of each of the C. perfringens and C. septicun alpha toxin fragments comprising of alpha-alpha fusion protein constructions. The finding, which is shown in Fig. 6, verifies that the first residue of the fusion protein is amino acid Trp number 29. Based on the newest finding, for the 1st time, the designed fusion gene was constructed and cloned into pUC57 vector and then transformed into cloning host cell (E. coli TOP10) (unpublished). The deposited accession number for the sequence of the fusion gene in the GenBank is MK908396, and also, in GenBank protein ID QDK65251.1, there is translation of the fusion gene that would produce a 777 amino acid alpha-alpha fusion protein (Fig. 6). At the 5′ end of cpa and the 3′ end of csa were added NdeI and XhoI restriction sites and their flanking regions respectively, which are necessary for insertion of the fusion gene into pET22b (+) expression vector; they are in the synthetic construct alpha-alpha fusion gene. The InterPro results were verified by the findings of tertiary structure prediction of the fusion gene by I-TASSER server. Findings revealed that the fusion protein consists of two main domains linked together with a linker fragment. The secondary structure prediction results were verified by tertiary structure prediction findings of cpa complete alpha toxin, csa-activated alpha toxin, and five models of fusion proteins. The results displayed that the secondary structure characteristics of both toxins are present in model 1 of tertiary structure of fusion protein with the best C-score = − 2.68, TM-score = 0.41 ± 0.14 and RMSD = 15.1 ± 3.5 (Fig. 6). Different parts of the tertiary structure of the synthetic construct fusion protein were predicted by I-TASSER and are shown in (Fig. 6). The linker fragment between two domains of the fusion protein can provide proper flexibility and separation, and the fusion protein after expression can provide proper collection in the periplasmic space of suitable host cell, because a signal peptide is present at the N-terminal of fusion protein, which will permit it to cross the cytoplasmic membrane. Thus, the fusion protein will be secret to the culture media and inclusion body formation would not occur (Langroudi et al. 2012). I-TASSER server predicted models of protein by combining the methods of threading, structural refinement, and ab initio modeling (Roy et al. 2010; Zhang 2008; Zhang 2009). The final model in the procedure is created as PDB format. The model to the proFunc server upload and the server uses sequence and structure-based methods to determine the likely function of a protein from its 3D structure (Laskowski et al. 2005a, b).

ProtParam software was used to identify the physicochemical characteristics of the fusion protein sequence (Gasteiger et al. 2005). phyre² software predicted the fusion protein binding sites (Kelley et al. 2015; Wass et al. 2010) and also Ramachandran plot and ProSA software results showed that the synthetic alpha-alpha fusion protein model is valid (Lovell et al. 2003; Wiederstein and Sippl 2007; Sippl 1993). In silico analysis is the most important approach to understand protein structure and functions. In conclusion, the designed fusion protein is valid and functional. Thus, the fusion gene could be used for clone and expression in a proper prokaryotic cell and also as a recombinant vaccine candidate.