Abstract
Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.
Highlights
Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium, which is a major cause of hospital-acquired diarrhea and pseudomembranous colitis and classified as an urgent antibiotic resistance threat by the Center for Disease Control and Prevention (CDC)
We present complementary mutagenesis, limited proteolysis, and single molecule fluorescence resonance energy transfer studies to demonstrate that the combined repetitive oligopeptides domain (CROPs) of TcdA can dynamically sample open and closed conformations relative to the rest of the toxin in a pHdependent manner, which could contribute to modulating the action of the pore-forming region in the delivery and receptor-binding domain (DRBD), and that conformational dynamics is crucial for TcdA cytotoxicity
We found that the most suitable TcdA fragment for crystallization is composed of residues L843 to T2481 including the complete DRBD and CROPs I–V (Fig 1A)
Summary
Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium, which is a major cause of hospital-acquired diarrhea and pseudomembranous colitis and classified as an urgent antibiotic resistance threat by the Center for Disease Control and Prevention (CDC). TcdA (~308 kD) and TcdB (~270 kD) belong to the large clostridial glucosylating toxin (LCGT) family, which include Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL (Aktories et al, 2017; Orrell & Melnyk, 2021). Most of these toxins are composed of four structural modules: an N-terminal glucosyltransferase domain (GTD), followed by a cysteine protease domain (CPD), a delivery and receptor-binding domain (DRBD), and a large C-terminal combined repetitive oligopeptides domain (CROPs) (Aktories et al, 2017; Orrell & Melnyk, 2021) (Fig 1A). The GTD glucosylates and inactivates the Rho and/or Ras families of small guanosine triphosphatases (GTPases) in host cells, resulting in depolymerization of the actin cytoskeleton, cell rounding, and cell death (Just et al, 1995a, 1995b; Chen et al, 2015; Liu et al, 2021)
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