Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdB_HV) was more cytotoxic in vitro than TcdB from historical strains (TcdB_HIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdB_HV and TcdB_HIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdB_HV were found to be substantially broader and more pronounced than those caused by TcdB_HIST. The increased toxicity of TcdB_HV was related to the toxin''s ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdB_HIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdB_HV undergoes acid-induced conformational changes at a pH much higher than that of TcdB_HIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.     

Clostridium difficile 027/BI/NAP1 Encodes a Hypertoxic and Antigenically Variable Form of TcdB

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB₀₀₃). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB₀₂₇) was at least four fold more lethal than TcdB₀₀₃. TcdB₀₂₇ caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB₀₀₃ and TcdB₀₂₇ also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB₀₂₇ and TcdB₀₀₃ identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB₀₂₇, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB₀₂₇ is a more potent toxin than TcdB₀₀₃ as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.     

Glucosyltransferase‐dependent and ‐independent effects of TcdB on the proteome of HEp‐2 cells

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase‐dependent and ‐independent effect on treated HEp‐2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase‐deficient TcdB_NXN and their proteomes were analyzed by LC‐MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdB_NXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to “GTPase mediated signaling” and the “cytoskeleton” while “chromatin” and “cell cycle” related proteins were altered by both, TcdB and TcdB_NXN. The obtained dataset of HEp‐2 cell proteome helps us to better understand glucosyltransferase‐dependent and ‐independent mechanisms of TcdB and TcdB_NXN, particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 ( https://proteomecentral.proteomexchange.org/dataset/PXD006658 ).     

Exposure of Neutralizing Epitopes in the Carboxyl-terminal Domain of TcdB Is Altered by a Proximal Hypervariable Region

The sequence, activity, and antigenicity of TcdB varies between different strains of Clostridium difficile. As a result, ribotype-specific forms of TcdB exhibit different toxicities and are not strongly cross-neutralized. Using a combination of biochemical and immunological approaches, we compared two important variants of TcdB (TcdB₀₁₂ and TcdB₀₂₇) to identify the mechanisms through which sequence differences alter epitopes and activity of the toxin. These analyses led to the discovery of a critical variation in the 1753–1851 (B2′) region of TcdB, which affects the exposure of neutralizing epitopes in the toxin. Sequence comparisons found that the B2′ region exhibits only 77% identity and is the most variable sequence between the two forms of TcdB. A combination of biochemical, analytical, and mutagenesis experiments revealed that the B2′ region promotes protein-protein interactions. These interactions appear to shield neutralizing epitopes that would otherwise be exposed in the toxin, an event found to be less prominent in TcdB₀₁₂ due to sequence differences in the 1773–1780 and 1791–1798 regions of the B2′ domain. When the carboxyl-terminal domains of TcdB₀₁₂ and TcdB₀₂₇ are swapped, neutralization experiments suggest that the amino terminus of TcdB interacts with the B2′ region and impacts the exposure of neutralizing epitopes in the carboxyl terminus. Collectively, these data suggest that variations in the B2′ region affect protein-protein interactions within TcdB and that these interactions influence the exposure of neutralizing epitopes.     

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1‐glucosylation kinetics are decisive for cell fate

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species‐mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC‐5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC‐5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.     

NOX4 inhibition protects enteric glial cells against Clostridium difficile toxin B toxicity via attenuating oxidative and Endoplasmic reticulum stresses

Enteric glial cells (EGCs), one main cell population of the enteric nervous system (ENS), play a major role in regulating intestinal barrier function. Clostridium difficile toxin B (TcdB) is the major virulence factor produced by C. difficile and estimated to be toxic to EGCs by inducing cell death, cell cycle arrest, and inflammatory cytokine production; however, the detailed mechanism for such effect is still unclear. In this study, we further evaluated the toxic effect of TcdB on EGCs and the involvement of NADPH oxidases in such process using the rat-transformed EGCs (CRL-2690). The results showed that NOX4 was activated by TcdB in EGCs and functioned as the major factor causing cytotoxicity and cell apoptosis. Mechanically, NOX4-generated H₂O₂ was the inducer of oxidative stress, Ca²⁺ homeostasis disorder, and ER stress in EGCs upon TcdB treatment, and NOX4 inhibition protected EGCs against TcdB toxicity via attenuating these dysfunctions. These findings contribute to our understanding of the mechanism by which TcdB affects EGCs and suggest the potential value of NOX4 inhibition for treatment against C. difficile infection.     

Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile – associated disease.     

Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1_401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal receptors. This structural study of TcdB in complex with frizzled-2 and chondroitin sulfate proteoglycan 4 reveals how TcdB binds to human receptors and primes itself for host entry.     

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co‐produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH‐9048 and TcsL‐9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN‐TcsH‐9048 and rN‐TcdA‐10463 glucosylated preferably Rho‐GTPases but also Ras‐GTPases to some extent. In this respect, rN‐TcsH‐9048 and rN‐TcdA‐10463 differ from the respective full‐length TcsH‐9048 and TcdA‐10463, which exclusively glucosylate Rho‐GTPases. rN‐TcsL‐9048 and full length TcsL‐9048 glucosylate both Rho‐ and Ras‐GTPases, whereas rN‐TcdB‐10463 and full length TcdB‐10463 exclusively glucosylate Rho‐GTPases. Vero cells treated with full length TcsH‐9048 or TcdA‐10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho‐GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto‐proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB‐1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro‐apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118‐paxillin and of pS144/141‐PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.     

Clostridium difficile toxin B induces autophagic cell death in colonocytes

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3⁺ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.     

MARTX effector cross kingdom activation by Golgi‐associated ADP‐ribosylation factors

Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxin consisting of conserved repeats‐containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmX_Vv). A structure‐based homology search revealed that DmX_Vv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmX_Vv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmX_Vv cytopathicity is activated by amino‐terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild‐type, but not catalytically inactive, DmX_Vv. DmX_Vv was found to localize to Golgi and to directly interact with Golgi‐associated ADP‐ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmX_Vv. These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmX_Vv effector domain of MARTX toxin.     

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.     

Cwp22, a novel peptidoglycan cross-linking enzyme,plays pleiotropic roles in Clostridioides difficile

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.     

Induced autoprocessing of the cytopathic Makes caterpillars floppy‐like effector domain of the Vibrio vulnificus MARTX toxin

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX_Vv) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the ‘Makes caterpillars floppy‐like’ effector domain (MCF_Vv) is previously unstudied. Using transient expression and protein delivery, MCF_Vv and MCF_Ah from the Aeromonas hydrophila MARTX_Ah toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C‐terminal subdomain of MCF_Vv identified an Arg‐Cys‐Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF_Vv. The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF_Vv, indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCF_Vv within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.     

Cwl0971, a novel peptidoglycan hydrolase,plays pleiotropic roles in Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.     

Detection of toxin B of Clostridium difficile based on immunomagnetic separation and aptamer-mediated colorimetric assay

Clostridium difficile can cause antibiotic-associated diarrhoea or pseudo-membranous colitis in humans and animals. Currently, the various methods such as microbiological culture, cytotoxic assay, ELISA and polymerase chain reaction have been used to detect Clostridium difficile infection (CDI). These conventional methods, however, require long detection time and professional staff. The paper is to describe a simple strategy which employs immunomagnetic separation and aptamer-mediated colorimetric assay for the detection of toxin B of C. difficile (TcdB) in the stool samples. HRP-labelled aptamer against TcdB selected by SELEX was firstly captured on the surface of magnetic beads (MB) by DNA hybridization with a complementary strand. In the presence of TcdB, aptamer specifically recognized and bound TcdB, disturbing the DNA hybridization and causing the release of HRP-aptamer from MB. This reduced the catalytic capacity of HRP and consequently the absorption intensity. As there was a relationship between the decrease in the absorption intensity and target concentration, a quantitative analysis of TcdB can be accomplished by the measurement of the absorption intensity. Under the optimal conditions, the assay system is able to detect TcdB at a concentration down to 5 ng ml⁻¹. Moreover the method had specificity of 97% and sensitivity of 66% and the system remained excellent stability within 4 weeks. The proposed method is a valuable screening procedure for CDI and can be extended readily to detection of other clinically important pathogens.     

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.     

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus

Recent studies have defined several virulence factors as vaccine candidates against Vibrio vulnificus. However, most of these factors have the potential to cause pathogenic effects in the vaccinees or induce incomplete protection. To overcome these drawbacks, a catalytically inactive form, CPD_Vv(C3725S), of the well‐conserved cysteine protease domain (CPD) of V. vulnificus multifunctional autoprocessing repeats‐in‐toxin (MARTX_Vv/RtxA1) was recombinantly generated and characterized. Notably, active and passive immunization with CPD_Vv(C3725S) conferred protective immunity against V. vulnificus strains. These results may provide a novel framework for developing safe and efficient subunit vaccines and/or therapeutics against V. vulnificus that target the CPD of MARTX toxins.     

Pyknotic cell death induced by Clostridium difficile TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity

TcdA and TcdB are the main pathogenicity factors of Clostridium difficile‐associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase‐deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor‐mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.