Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies

Abstract

Background: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the Cterminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. Results: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. Conclusions: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Molecular Characterization of Clostridium perfringens Strains Isolated in Italy

“…The lecithinase activity of CPA toxin expressed to the different C. perfringens strains could be tested, in egg yolk agar well diffusion turbidity (EYDT) in vitro assay. The culture supernatants of this type E cpe+aty-cpb2 strain were tested in EYDT in a parallel study conducted to detect the biological activity of rBacCPA250–363H6 compared to that of the native, full length phospholipase C (PLC), which was used as a standard [ 52 ]. Preliminary results provided evidence that while the non-toxic rBacCPA250–363H6 protein did not exhibit any lecithinase activity on TSC agar, this type E cpe+aty-cpb2 strain showed increased CPA activity (in preparation for submission).…”

Crystal Structure of Fungal Nonspecific Phospholipase C Unveils a Distinct Catalytic Mechanism

“…perfringens (PDB ID: 1QM6), which possesses an additional C-terminal domain that plays a key role in the Ca 2+ -mediated recognition of membrane phospholipids (Figure S2, Supporting Information). However, our structure of RePLC, the first structure derived from fungi, did not resemble any of the known bacterial phospholipase structures. Instead, it exhibited similarity to a very recently solved crystal structure of NPC4 (PDB ID: 8HAW), originating from the A.…”

“…One is exemplified by the NPC derived from B. cereus (PDB ID: 1AH7), with the overall structure consisting of only nine α-helices and three Zn ions, 39 and the other is represented by the NPC derived from C. perfringens (PDB ID: 1QM6), which possesses an additional C-terminal domain that plays a key role in the Ca 2+ -mediated recognition of membrane phospholipids (Figure S2, Supporting Information). 40 However, our structure of RePLC, the first structure derived from fungi, did not resemble any of the known bacterial phospholipase structures. Instead, it exhibited similarity to a very recently solved crystal structure of NPC4 (PDB ID: 8HAW), originating from the A. thaliana plant species.…”

The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health

“…Recently, two reports of recombinant TBV candidates produced in BEVS were published. Lee et al [ 23 ] showed that Pfs48/45 6C protein fragment expressed in BEVS does not require a fusion partner and that by preventing N-glycosylation, 6C protein is biologically active and exhibits significant transmission-reducing activity. The other report, by Qui and collaborators, showed that HAP2 from Plasmodium vivax expressed in BEVS displays transmission-blocking activity and they propose it for the first time as a novel TBV candidate [ 24 ].…”