1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757\u2013876)

Mary E Cook,Raquel Godoy-Ruiz,Kristen M Varney,David J Weber

doi:10.1007/s12104-020-09979-y

Abstract

Clostridioides difficile is a bacterial pathogen responsible for the majority of nosocomial infections in the developed world. C. difficile infection (CDI) is difficult to treat in many cases because hypervirulent strains have evolved that contain a third toxin, termed the C. difficile toxin (CDT), in addition to the two enterotoxins TcdA and TcdB. CDT is a binary toxin comprised of an enzymatic, ADP-ribosyltransferase (ART) toxin component, CDTa, and a pore-forming or delivery subunit, CDTb. In the absence of CDTa, CDTb assembles into two distinct di-heptameric states, a symmetric and an asymmetric form with both states having two surface-accessible host cell receptor-binding domains, termed RBD1 and RBD2. RBD1 has a unique amino acid sequence, when aligned to other well-studied binary toxins (i.e., anthrax), and it contains a novel Ca2+-binding site important for CDTb stability. The other receptor binding domain, RBD2, is critically important for CDT toxicity, and a domain such as this is missing altogether in other binary toxins and shows further that CDT is unique when compared to other binary toxins. In this study, the 1H, 13C, and 15N backbone and sidechain resonances of the 120 amino acid RBD2 domain of CDTb (residues 757–876) were assigned sequence-specifically and provide a framework for future NMR-based drug discovery studies directed towards targeting the most virulent strains of CDI.

Highlights

Clostridioides difficile infection (CDI) is caused by a sporeforming, Gram-positive bacterium, and it is the most commonly reported nosocomial infection in the world, accounting for 12% of all hospital-borne infections (Gerding 2015)
The asymmetric form, AsymCDTb, has one of its two heptameric components folding into a seven-stranded beta-barrel with an internal cavity that is reminiscent of the anthrax protective antigen (PA) (Akkaladevi et al 2013)
receptor-binding domain-2 (RBD2) was shown to provide a potent dominant negative effect for host cell toxicity when isolated indicating its important functional role within hypervirulent CDI (Xu et al 2020). With these data in hand, the sequence-specific backbone and sidechain resonance assignments of RBD2 were completed as a first step towards designing RBD2 inhibitors using NMR-based methods, which will be important for the longer-term goal of targeting toxicities associated with hypervirulent Clostridioides difficile that produces C. difficile toxin (CDT)

Summary

Biological context

Clostridioides difficile infection (CDI) is caused by a sporeforming, Gram-positive bacterium, and it is the most commonly reported nosocomial infection in the world, accounting for 12% of all hospital-borne infections (Gerding 2015). CDT has an enzymatic subunit, CDTa (47.4 kDa), with ribosyltransferase activity, and a pore-forming delivery subunit, termed CDTb (74 kDa). RBD2 was shown to provide a potent dominant negative effect for host cell toxicity when isolated (residues 757–876) indicating its important functional role within hypervirulent CDI (Xu et al 2020). With these data in hand, the sequence-specific backbone and sidechain resonance assignments of RBD2 were completed as a first step towards designing RBD2 inhibitors using NMR-based methods, which will be important for the longer-term goal of targeting toxicities associated with hypervirulent Clostridioides difficile that produces CDT

Protein expression and purification

NMR spectroscopy

Extent of assignment and data deposition

Findings

Compliance with Ethical Standards