Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

ABSTRACTVibrio vulnificusis a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modifiedV. vulnificusMARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVvtoxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVvtoxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysisin vitrobut that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVvtoxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.IMPORTANCEUp to 95% of deaths from seafood-borne infections in the United States are due solely to one pathogen,V. vulnificus. Among its various virulence factors, the MARTXVvtoxin has been characterized as a critical exotoxin for successful pathogenesis ofV. vulnificusin mouse infection models. Similarly to MARTX toxins of other pathogens, MARTXVvtoxin is comprised of repeat-containing regions, central effector domains, and an autoprocessing cysteine protease domain. Yet how each of these regions contributes to essential activities of the toxins has not been fully identified for any of MARTX toxins. Using modified MARTXVvtoxin fused with β-lactamase as a reporter enzyme, the portion(s) responsible for toxin secretion from bacteria, effector domain translocation into host cells, rapid host cell rounding, and necrotic host cell death was identified. The results are relevant for understanding how MARTXVvtoxin serves as both a necrotic pore-forming toxin and an effector delivery platform.

Domain Organization and Evolution of Multifunctional Autoprocessing Repeats-in-Toxin (MARTX) Toxin inVibrio vulnificus

ABSTRACTThe objective of this study was to analyze multifunctional autoprocessing repeats-in-toxin (MARTX) toxin domain organization within the aquatic speciesVibrio vulnificusas well as to study the evolution of thertxA1gene. The species is subdivided into three biotypes that differ in host range and geographical distribution. We have found three different types (I, II, and III) ofV. vulnificusMARTX (MARTXVv) toxins with common domains (an autocatalytic cysteine protease domain [CPD], an α/β-hydrolase domain, and a domain resembling that of the LifA protein ofEscherichia coliO127:H6 E2348/69 [Efa/LifA]) and specific domains (a Rho-GTPase inactivation domain [RID], a domain of unknown function [DUF],a domain resembling that of thertxAprotein ofPhotorhabdus asymbiotica[rtxAPA], and an actin cross-linking domain [ACD]). Biotype 1 isolates harbor MARTXVvtoxin types I and II, biotype 2 isolates carry MARTXVvtoxin type III, and biotype 3 isolates have MARTXVvtoxin type II. The analyzed biotype 2 isolates harbor two identical copies ofrtxA1, one chromosomal and the other plasmidic. The evolutionary history of the gene demonstrates that MARTXVvtoxins are mosaics, comprising pieces with different evolutionary histories, some of which have been acquired by intra- or interspecific horizontal gene transfer. Finally, we have found evidence that the evolutionary history of thertxA1gene for biotype 2 differs totally from the gene history of biotypes 1 and 3.