title: 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: OBJECTIVE
  To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India.
authors.forename: S R
authors.initials: SR
authors.lastname: Uppalapati
authors.forename: J J
authors.initials: JJ
authors.lastname: Kingston
authors.forename: H S
authors.initials: HS
authors.lastname: Murali
authors.forename: H V
authors.initials: HV
authors.lastname: Batra
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000187:drug effects; D005260:Female; D006460:Hemolysin Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D007106:Immune Sera / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D006460:Hemolysin Proteins; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; C021091:staphylococcal alpha-toxin; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens