Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Abstract

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

“…This way, a 28-amino acid N -terminal signal peptide was identified, as well as some biochemical properties of the toxin were described. Many strategies have been employed in an attempt to obtain a non-toxic version of CPA for vaccination, the most common of which are site-directed mutagenesis, isolation of the strains that naturally produce non-toxic CPA, expression of only the N - or C -terminal domain, expression of chimeric toxins, and expression on the surface of Bacillus subtilis spores (Table 2) [32,40,41,42,43,44,45]. …”

Where does a Staphylococcus aureus vaccine stand?

“…These trials are supported by the following animal model data: AT62-IgG (rabbit anti-HLA IgG) protected mice from pneumonia and intraperitoneal challenges [47]. A fusion of Hla and Clostridium perfringens a-toxin (r-aCS) protected red blood cells from lysis [48]. Anti-Hla antibodies reduce recurrent skin infections in children, but natural infection does not provoke durable immunity [49].…”

In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

“…haemolytic property of the N and C-domain hybrid protein. Mouse erythrocyte (mRBC) suspensions used in the study were prepared according to Uppalapati et al [20]. Twenty micro moles of rN and rC-domains, individually or in combination and equal moles of native wild type alpha toxin were added to individual wells on mouse blood agar plates and incubated at 37 °C for 4h.…”