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Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel

Waraphan Toniti1, Toru Yoshida1,2, Toshiharu Tsurumura1

  • 1Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.

Plos One
|February 16, 2017
PubMed

Insights

New research reveals a novel Clostridium perfringens enterotoxin (CPILE-a) causing food poisoning. This toxin targets both alpha- and beta/gamma-actin, with distinct structural features impacting its activity.

Area of Science:

  • Microbiology
  • Structural Biology
  • Toxicology

Background:

  • Outbreaks of food poisoning in Japan were linked to Clostridium perfringens strains lacking typical enterotoxin.
  • These strains produce a novel two-component enterotoxin: C. perfringens iota-like enterotoxin-a (CPILE-a) and CPILE-b.

Purpose of the Study:

  • To elucidate the structural basis of CPILE-a's function.
  • To investigate the role of specific structural features in CPILE-a's actin-binding and ADP-ribosylation activity.
  • To compare CPILE-a's activity with the known iota toxin-a (Ia).

Main Methods:

  • X-ray crystallography was used to determine the structures of apo-CPILE-a, NAD+-CPILE-a, and NADH-CPILE-a.
  • Site-directed mutagenesis was performed on actin-binding interface regions of CPILE-a and Ia.
  • ADP-ribosylation assays were conducted using both α- and β/γ-actin.

Main Results:

  • CPILE-a possesses unique protruding loops not found in Ia.
  • CPILE-a ADP-ribosylates both α-actin and β/γ-actin, with higher sensitivity to α-actin.
  • Specific protruding loops (PT-I) and convex-concave interactions are crucial for CPILE-a's actin binding.

Conclusions:

  • CPILE-a represents a novel enterotoxin with distinct structural and functional properties compared to Ia.
  • The study provides the first detailed analysis of mutagenesis in the actin-binding regions of Ia and CPILE-a against different actin isoforms.
  • Understanding CPILE-a's mechanism is crucial for addressing foodborne illnesses caused by these strains.

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