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

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 act...

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Autores principales: Uppalapati, Siva Ramakrishna, Kingston, Joseph Jeyabalaji, Qureshi, Insaf Ahmed, Murali, Harishchandra Sripathy, Batra, Harsh Vardhan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859591/
https://www.ncbi.nlm.nih.gov/pubmed/24349173
http://dx.doi.org/10.1371/journal.pone.0082024
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author Uppalapati, Siva Ramakrishna
Kingston, Joseph Jeyabalaji
Qureshi, Insaf Ahmed
Murali, Harishchandra Sripathy
Batra, Harsh Vardhan
author_facet Uppalapati, Siva Ramakrishna
Kingston, Joseph Jeyabalaji
Qureshi, Insaf Ahmed
Murali, Harishchandra Sripathy
Batra, Harsh Vardhan
author_sort Uppalapati, Siva Ramakrishna
collection PubMed
description 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.
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spelling pubmed-38595912013-12-13 In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin Uppalapati, Siva Ramakrishna Kingston, Joseph Jeyabalaji Qureshi, Insaf Ahmed Murali, Harishchandra Sripathy Batra, Harsh Vardhan PLoS One Research Article 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. Public Library of Science 2013-12-11 /pmc/articles/PMC3859591/ /pubmed/24349173 http://dx.doi.org/10.1371/journal.pone.0082024 Text en © 2013 Uppalapati et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Uppalapati, Siva Ramakrishna
Kingston, Joseph Jeyabalaji
Qureshi, Insaf Ahmed
Murali, Harishchandra Sripathy
Batra, Harsh Vardhan
In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title_full In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title_fullStr In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title_full_unstemmed In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title_short In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin
title_sort in silico, in vitro and in vivo analysis of binding affinity between n and c-domains of clostridium perfringens alpha toxin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859591/
https://www.ncbi.nlm.nih.gov/pubmed/24349173
http://dx.doi.org/10.1371/journal.pone.0082024
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