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7 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2022

Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

Clostridioides difficile is one of the most common causes of antibiotic-associated diarrhea in developed countries. As key virulence factors of C. difficile, toxin A (TcdA) and toxin B (TcdB) act by glucosylating and inactivating Rho and Ras family small GTPases in host cells, which leads to actin cytoskeleton disruption, cell rounding, and ultimately cell death. Here we present the co-crystal structure of the glucosyltransferase domain (GTD) of TcdA in complex with its substrate human RhoA at 2.60-angstrom resolution. This structure reveals that TcdA GTD grips RhoA mainly through its switch I and switch II regions, which is complemented by interactions involving RhoA’s pre-switch I region. Comprehensive structural comparisons between the TcdA GTD–RhoA complex and the structures of TcdB GTD in complex with Cdc42 and R-Ras reveal both the conserved and divergent features of these two toxins in terms of substrate recognition. Taken together, these findings establish the structural basis for TcdA recognition of small GTPases and advance our understanding of the substrates selectivity of large clostridial toxins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling

Background & Aims: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. Methods: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA 57-80 ), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. Results: The TcdA 57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA 57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. Conclusions: TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and conformational dynamics of Clostridioides difficile toxin A

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Neutralizing epitopes on Clostridioides difficile toxin A revealed by the structures of two camelid VHH antibodies

Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural Basis for Binding of Neutralizing Antibodies to Clostridioides difficile Binary Toxin

Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb—the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol—showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗