A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia

Abstract

Clostridium perfringens epsilon toxin (ETX), one of the most potent toxins known, is a potential biological weapon; therefore, the development of an effective vaccine is important for preventing intoxication or disease by ETX. In this study, genetically detoxified epsilon toxin mutants were developed as candidate vaccines. We used site-directed mutagenesis to mutate the essential amino acid residues (His106, Ser111 and Phe199). Six site-directed mutants of ETX (mETX (H106P) , mETX (S111H) , mETX (S111Y) , mETX (F199H) , mETX (F199E) , mETX (S111YF199E) ) were generated and then expressed in Escherichia coli. Both mETX (F199E) and mETX (H106P) with low or non-cytotoxicity that retained their immunogenicity were selected to immunize mice 3 times, and the mouse survival data were recorded after challenging with recombinant wild-type ETX. mETX (F199E) induces the same protection as mETX (H106P) , which was reported previously as a promising toxin mutant for vaccine, and both of them could protect immunized mice against a 100× LD₅₀ dose of active wild-type recombinant ETX. This work showed that mETX (F199E) is another promising candidate vaccine against enterotoxemia and other diseases caused by ETX.

Keywords: Clostridium perfringens; cytotoxicity; enterotoxemia; site-directed mutagenesis; toxin mutant; vaccine; ε-toxin.

Figure 1. Affinity chromatographic profile and SDS-PAGE of mETX. (A) Purification of mETX^F199E using a Ni²⁺-cheating HP column. The mETX^F199E was eluted with buffer containing increasing concentrations of imidazole up to 500 mM (the arrow marking fraction). The purification data of other mETXs have not shown. (B) 12% SDS-PAGE analysis of purified rETX and its mutants. Lane 1-6, mETX^H106P, mETX^S111Y, mETX^S111H, mETX^F199H, mETX^F199E, mETX^S111YF199E; Lane 7, rETX; Lane M, ProteinRuler Ι protein marker (TransGen Biotech).

Figure 2. Immunoblot of purified rETX and its mutants with monoclonal mouse anti-rETX antibody detected using Super-ECL horseradish peroxidase. Lane 1-6, mETX^H106P, mETX^S111Y, mETX^S111H, mETX^F199H, mETX^F199E, mETX^S111YF199E; Lane 7, rETX; Lane 7, rETX. The pre-stained protein marker (TransGen Biotech) is indicated on the right side of the blot.

Figure 3. The three-dimensional structures of mETX^H106P (A) and mETX^F199E (B). Pro106 and Glu199 were shown in rainbow sticks and colored in red.