Structural insights into the transition of Clostridioides difficile binary toxin from prepore to pore

Abstract

Clostridioides (formerly Clostridium) difficile is a gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in U.S. hospitals 1. The disease state is most often preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe diarrhea. C. difficile infection (CDI) is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT) 2. While TcdA and TcdB are considered the primary virulence factors, recent studies suggest that CDT increases the severity of CDI in some of the most problematic clinical strains 3. To better understand how CDT functions, we used cryo-electron microscopy (cryo-EM) to define the structure of the cell-binding component of CDT (CDTb). We obtained structures of several oligomeric forms that highlight the conformational changes that permit conversion from a prepore to a β-barrel pore. The structural analysis also reveals a glycan binding domain and residues involved in binding the host cell-receptor, lipolysis-stimulated lipoprotein receptor (LSR). Together, these results provide a framework to understand how CDT functions at the host cell interface. CDT belongs to the Iota family of binary toxins and consists of two proteins: an ADPribosyltransferase (CDTa) and a cell binding, pore-forming, and CDTa translocating protein (CDTb) 4. CDTb engages host cells through LSR 5 and undergoes proteolytic cleavage and oligomerization to facilitate CDTa binding. The complex is internalized by endocytosis, and endosome acidification leads to CDTb pore formation and CDTa translocation. Inside the cell, CDTa catalyzes ADP-ribosylation of G-actin which leads to disruption of the cytoskeleton, formation of microtubule protrusions, and increased C. difficile adherence Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

Insights into the Role of Human Gut Microbiota in Clostridioides difficile Infection

“…However, TcdB is a cytotoxin that appears to be required for the pathogenic effect of the microorganism [22]. Some C. difficile strains produce an additional toxin, a binary toxin (ADP-ribosyl-transferase, CDT) that consists of two components-CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb, which binds to host cells and translocates CDTa into the cytosol [23][24][25]. Basic predisposing factors for the C. difficile infection are the intake of antibiotics that disrupt the gut microbiota, an extended hospital stay, and advanced patient age.…”

Structural elucidation of the Clostridioides difficile transferase toxin reveals a single-site binding mode for the enzyme

“…Since CDTa was bound to CDTb in a preinsertion state, we considered the possibility that CDTa had moved from an outer site into the center following the conversion of prepore to the preinsertion state. We thus sought to confirm that CDT retained this unique binding mode throughout all stages of pore maturation by analyzing the binding of CDTa to large oligomeric CDTb particles that we have previously shown consist of CDTb in a prepore, partial β-barrel, and pore form (17). We used negative stain EM to visualize CDTa bound to the prepore, partial β-barrel, and full β-barrel forms of CDTb and noted that CDTa was bound in the center each time (Fig.…”

“…Fig. 1A) (17). These domains are responsible for preventing premature oligomerization (D1), forming the oligomerization interface and β-barrel pore (D2/D3), interacting with glycans (D3′), and binding to the host cell receptor (D4).…”

“…This structure consists of seven phenylalanines positioned in a ring at the entrance to the pore and was first characterized in the context of the related protein, anthrax toxin protective antigen (PA) (18). The φ-clamp is needed to support cargo translocation and has been shown to be important for CDT function (17,18).…”

“…The acidification of the endosome is thought to trigger CDTb pore formation and the delivery of CDTa through the pore. Previous work on oligomeric CDTb yielded structures in four distinct states that depict the transition of CDTb from soluble prepore to preinsertion state and then from a partial β-barrel to a full β-barrel pore (17). Three of these states were obtained from particles that consisted of double heptamers: a complex of a prepore and a full β-barrel pore and a complex of two heptamers in the partial β-barrel state.…”