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.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

“…This finding of higher carriage of C. perfringens, as compared to the previous studies, may be attributed to different study populations and/ or to the differences in detection sensitivities of different methods used. We targeted the α-toxin gene to make the assay specific for all the toxigenic C. perfringens strains (particularly in human feces), since α-toxin is produced by almost all the strains of C. perfringens and has been implicated in numerous gastrointestinal illnesses [ 4 , 17 , 48 ]. The counts analyzed by plc - and 16S rRNA gene-specific assays were comparable (Table 2 ), indicating that all the C. perfringens strains detected in this study harbored the α-toxin gene.…”

Perilipin 1 binds to aquaporin 7 in human adipocytes and controls its mobility via protein kinase A mediated phosphorylation

“…Far dot Western blotting was performed as previously described [16,17]. AQP7 was diluted with purification buffer B and spotted onto a nitrocellulose membrane in 1 μL drops containing 3, 10 or 12 pmol of AQP7, respectively.…”

Molecular structure and function of the carboxy‐terminus of the alpha‐toxin from Clostridium perfringens type A

“…The active site of CPA includes catalytic and binding sites by the analysis of the crystal structure. Its amino-terminus consists of nine helices and carboxyl-terminus consists of eight antiparallel sheets (Oda et al, 2012;Oda, Terao, Sakurai, & Nagahama, 2015;Uppalapati, Kingston, Qureshi, Murali, & Batra, 2013).…”