The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

Area of Science:

• Microbiology
• Molecular Biology
• Toxicology

Background:

• Clostridioides difficile toxins TcdA and TcdB are key virulence factors in C. difficile-associated diseases.
• These toxins are large clostridial glucosyltransferases with four conserved cysteines.
• The study focuses on cysteine 2232 in TcdB, a critical residue for toxin function.

Purpose of the Study:

• To investigate the role of conserved cysteine 2232 in TcdB from C. difficile strain VPI10463 (clade I).
• To compare the function of cysteine 2232 in VPI10463 TcdB with tyrosine 2232 in hypervirulent strain R20291 TcdB (clade II).
• To elucidate the contribution of Cys-2232 to toxin-receptor binding and delivery.

Main Methods:

• Site-directed mutagenesis to replace cysteine 2232 with tyrosine in TcdB VPI10463.
• Pull-down assays to assess binding of TcdB variants to known receptors FZD2 and PVRL3.
• Competition assays using receptor-binding fragments and novel antibodies to characterize the TcdB delivery domain.

Main Results:

• Replacing Cys-2232 with tyrosine in TcdB VPI10463 significantly reduced binding to FZD2 and PVRL3.
• TcdB R20291 (with Tyr-2232) showed weak PVRL3 binding, which increased upon mutation to Cys-2232.
• TcdB R20291 did not bind FZD2, suggesting FZD2 is less critical for this toxinotype.

Conclusions:

• Conserved Cys-2232 in TcdB indirectly contributes to toxin-receptor interactions.
• The findings highlight differences in receptor usage between TcdB toxinotypes.
• Cys-2232 plays a role in modulating the binding affinity and potentially the delivery mechanism of TcdB.