title: In silico, in vitro and in vivo analysis of binding affinity between N and C-domains of Clostridium perfringens alpha toxin.
abstract: Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture.
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.
authors.forename: Siva Ramakrishna
authors.initials: SR
authors.lastname: Uppalapati
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.
authors.forename: Joseph Jeyabalaji
authors.initials: JJ
authors.lastname: Kingston
authors.affiliation: Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, India.
authors.forename: Insaf Ahmed
authors.initials: IA
authors.lastname: Qureshi
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.
authors.forename: Harishchandra Sripathy
authors.initials: HS
authors.lastname: Murali
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.
authors.forename: Harsh Vardhan
authors.initials: HV
authors.lastname: Batra
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D003016:Clostridium perfringens / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D006461:Hemolysis / Q000187:drug effects; D007273:Injections, Intramuscular; D051379:Mice; D008807:Mice, Inbred BALB C; D056004:Molecular Dynamics Simulation; D018482:Muscle, Skeletal / Q000187:drug effects / Q000473:pathology; D009336:Necrosis / Q000139:chemically induced / Q000473:pathology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D055672:Static Electricity; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens