Structure-function discrepancy in Clostridium botulinum C3 toxin for its rational prioritization as a subunit vaccine

Area of Science:

• Microbiology
• Structural Biology
• Toxicology

Background:

• Clostridium botulinum produces neurotoxins causing botulism in humans and animals.
• Physiological group III strains (type C and D) produce C2 and C3 toxins affecting cattle and avian.
• Understanding C3 toxin structure-function is crucial for developing targeted interventions.

Purpose of the Study:

• To reveal structure-function disparities between C. botulinum C3 toxins from type C (CboC) and type D (CboD) phages.
• To rationally design avirulent C3 toxin mutants for vaccine development.
• To investigate the structural basis for toxin avirulence.

Main Methods:

• Computational evaluation of C3 toxin structure-function using homology modeling.
• Analysis of sequence-structure-function relationships through covariation and point mutations.
• Generation and structural assessment of avirulent mutants.

Main Results:

• Significant differences in avirulent mutant generation were observed between CboC (8/34) and CboD (27/mutants).
• Tertiary structures remained largely unchanged, with variations in coiled and loop regions.
• Correlated mutations influenced hydrogen bonding patterns, preserving pseudocatalytic function in avirulent mutants.
• Avirulent mutants exhibited stable structures and near-native ensembles.

Conclusions:

• Site-directed mutagenesis is critical for designing avirulent toxins.
• Rational design of avirulent C3 toxins can lead to effective subunit vaccines for cattle and avian.
• Vaccine specificity is influenced by the specific C3 toxins of C. botulinum phages used.