scholarly journals Proinflammatory Cytokines: Possible Accomplices for the Systemic Effects of Clostridioides difficile Toxin B

2021 ◽  
Vol Volume 14 ◽  
pp. 57-62
Author(s):  
Katia Fettucciari ◽  
Alessandro Fruganti ◽  
Andrea Marchegiani ◽  
Stefano Brancorsini ◽  
Pierfrancesco Marconi ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Systemic Effects ◽  
Toxin B ◽  
Clostridioides Difficile

scholarly journals Analysis of C. difficile infection–related outcomes in European participants in the bezlotoxumab MODIFY I and II trials

2020 ◽  
Vol 39 (10) ◽  
pp. 1933-1939
Author(s):  
Emilio Bouza ◽  
Oliver A. Cornely ◽  
Antonio Ramos-Martinez ◽  
Robert Plesniak ◽  
Misoo C. Ellison ◽  
...  
Keyword(s):  
Human Monoclonal Antibody ◽  
Patient Characteristics ◽  
Toxin B ◽  
Treatment Groups ◽  
Clostridioides Difficile ◽  
Post Infusion ◽  
Baseline Characteristics ◽  
Post Hoc ◽  
Single Intravenous Infusion ◽  
Trial Participants

Abstract The MODIFY I/II trials demonstrated that bezlotoxumab, a human monoclonal antibody against Clostridioides difficile toxin B, given during antibiotic treatment for Clostridioides difficile infection (CDI) significantly reduced C. difficile recurrence (rCDI) in adults at high risk for rCDI. Efficacy of CDI-directed intervention may depend on ribotype regional epidemiology, and patient characteristics. This post hoc analysis assessed the efficacy of bezlotoxumab in the subgroup of MODIFY I/II trial participants enrolled in Europe. Data from the bezlotoxumab (10 mg/kg single intravenous infusion) and placebo (0.9% saline) groups from MODIFY I/II were compared to assess initial clinical cure (ICC), rCDI, all-cause, and CDI-associated rehospitalizations within 30 days of discharge, and mortality through 12 weeks post-infusion. Of 1554 worldwide participants, 606 were from Europe (bezlotoxumab n = 313, 51%; placebo n = 292; 48%). Baseline characteristics were generally similar across groups, although there were more immunocompromised participants in the bezlotoxumab group (27.2%) compared with placebo (20.1%). Fifty-five percent of participants were female, and 86% were hospitalized at randomization. The rate of ICC was similar between treatment groups. The rate of rCDI in the bezlotoxumab group was lower compared with placebo among European participants overall, and among those with ≥ 1 risk factor for rCDI. Bezlotoxumab reduced 30-day CDI-associated rehospitalizations compared with placebo. These results are consistent with overall results from the MODIFY trials and demonstrate that bezlotoxumab reduces rCDI and CDI-associated rehospitalizations in European patients with CDI. MODIFY I/II (NCT01241552 and NCT01513239)

scholarly journals Rab5a Promotes Cytolethal Distending Toxin B-Induced Cytotoxicity and Inflammation

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Ming-xian Chen ◽  
Yu Chen ◽  
Rui Fu ◽  
Guo-qun Mao ◽  
Sai-yue Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proinflammatory Cytokines ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Small Gtpase ◽  
Cytolethal Distending Toxin ◽  
Colonic Epithelial Cells ◽  
Toxin B ◽  
Proinflammatory Responses ◽  
Underlying Mechanisms

ABSTRACT The cytolethal distending toxin B subunit (CdtB) induces significant cytotoxicity and inflammation in many cell types that are involved in the pathogenesis of postinfectious irritable bowel syndrome (PI-IBS). However, the underlying mechanisms remain unclear. This study tested the potential role of Rab small GTPase 5a (Rab5a) in the process. We tested mRNA and protein expression of proinflammatory cytokines (interleukin-1β [IL-1β] and IL-6) in THP-1 macrophages by quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs), respectively. In the primary colonic epithelial cells, Cdt treatment induced a CdtB-Rab5a-cellugyrin association. Rab5a silencing, by target small hairpin RNAs (shRNAs), largely inhibited CdtB-induced cytotoxicity and apoptosis in colon epithelial cells. CRISPR/Cas9-mediated Rab5a knockout also attenuated CdtB-induced colon epithelial cell death. Conversely, forced overexpression of Rab5a intensified CdtB-induced cytotoxicity. In THP-1 human macrophages, Rab5a shRNA or knockout significantly inhibited CdtB-induced mRNA expression and production of proinflammatory cytokines (IL-1β and IL-6). Rab5a depletion inhibited activation of nuclear factor-κB (NF-κB) and Jun N-terminal protein kinase (JNK) signaling in CdtB-treated THP-1 macrophages. Rab5a appears essential for CdtB-induced cytotoxicity in colonic epithelial cells and proinflammatory responses in THP-1 macrophages.

scholarly journals Structural elucidation of theClostridioides difficiletransferase toxin reveals a single-site binding mode for the enzyme

2020 ◽  
Vol 117 (11) ◽  
pp. 6139-6144 ◽  
Author(s):  
Michael J. Sheedlo ◽  
David M. Anderson ◽  
Audrey K. Thomas ◽  
D. Borden Lacy
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Binding Mode ◽  
The United States ◽  
Binding Interaction ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Cryo Electron Microscopy ◽  
Hospital Acquired ◽  
Site Binding

Clostridioides difficileis a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms ofC. difficileinfection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and theC. difficiletransferase toxin (CDT). Reported here is a 3.8-Å cryo–electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to β-barrel pore, although we also observe a single bound CDTa in the prepore and β-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.

scholarly journals Epidemic Ribotypes Of Clostridium (Now Clostridioides) Difficile Are Likely To Be More Virulent Than Non-Epidemic Ribotypes In Animal Models

2019 ◽  
Author(s):  
John C. Vitucci ◽  
Mark Pulse ◽  
Leslie Tabor-Simecka ◽  
Jerry W. Simecka
Keyword(s):  
Animal Models ◽  
Toxin Production ◽  
Intestinal Epithelial ◽  
Toxin B ◽  
Golden Syrian Hamster ◽  
Clostridioides Difficile ◽  
Conflicting Evidence ◽  
Epithelial Cell Lines

Abstract Background: Clostridioides difficile infections have become more frequently diagnosed and associated with greater disease severity, which has resulted in an increase burden on the healthcare system. These increases are attributed to the increased prevalence of hypervirulent strains encompassing select ribotypes. These epidemic ribotypes were characterized as hypervirulent due to higher in vitro spore and toxin production, as well as increased incidence, severity and mortality within patients. However, it is unclear whether epidemic ribotypes are truly more virulent than non-epidemic ribotypes in vivo. Furthermore, there is conflicting evidence about the ability of a strain’s in vitro phenotype to be predictive of their in vivo virulence. The goals of the current studies were to determine if epidemic ribotypes are more virulent than other ribotypes in animal models, and whether the in vitro virulence phenotype of an isolate or ribotype predict in vivo virulence. Results. To determine if epidemic strains were truly more virulent than other non-epidemic strains, the in vivo virulence of thirteen C. difficile isolates (7 non-epidemic and 6 epidemic ribotype isolates) were determined in murine (C57BL/6 mice) and hamster (golden Syrian hamster) models of C. difficile infections. The isolates of epidemic ribotype of C. difficile were found to be more virulent in both the murine and hamster models than non-epidemic isolates. In particular, the group of epidemic ribotypes of C. difficile had lower LD 50 values in hamsters. The increased severity of disease was associated with higher levels of Toxin A and Toxin B production found in fecal samples, but not numbers of organisms recovered. The isolates were further characterized for their in vitro virulence phenotypes, e.g. toxin production, growth rates, spore formation and adherence of spores to intestinal epithelial cell lines. Although there were higher levels of toxins produced and greater adherence for the group of epidemic ribotypes, the in vitro profiles of individual isolates were not always predictive of their in vivo virulence. Conclusions. Overall, the group of epidemic ribotypes of C. difficile were more virulent in vivo despite individual isolates having similar phenotypes to the non-epidemic isolates in vitro .

scholarly journals Murine intrarectal instillation of purified recombinant C. difficile toxins enables mechanistic studies of pathogenesis

2020 ◽  
Author(s):  
Nicholas O. Markham ◽  
Sarah C. Bloch ◽  
John A. Shupe ◽  
Erin N. Laubacher ◽  
M. Kay Washington ◽  
...  
Keyword(s):  
Ex Vivo ◽  
The United States ◽  
Immunofluorescent Staining ◽  
Inflammatory Infiltration ◽  
Toxin B ◽  
Tissue Sections ◽  
Clostridioides Difficile ◽  
Hematoxylin And Eosin ◽  
Negative Controls

AbstractClostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota, et al. first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin (H&E) for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore-formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared with slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

scholarly journals Comparative Whole Genome Sequence Analysis and Biological Features of Clostridioides difficile Sequence Type 2‡

2021 ◽  
Vol 12 ◽  
Author(s):  
Xingxing Xu ◽  
Qiao Bian ◽  
Yun Luo ◽  
Xiaojun Song ◽  
Shan Lin ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Antibiotic Resistance ◽  
Sequence Type ◽  
Gene Clustering ◽  
Whole Genome ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Significant Difference ◽  
Sporulation Capacity

Clostridioides difficile sequence type 2 (ST2) has been increasingly recognized as one of the major genotypes in China, while the genomic characteristics and biological phenotypes of Chinese ST2 strains remain to be determined. We used whole-genome sequencing and phylogenetic analysis to investigate the genomic features of 182 ST2 strains, isolated between 2011 and 2017. PCR ribotyping (RT) was performed, and antibiotic resistance, toxin concentration, and sporulation capacity were measured. The core genome Maximum-likelihood phylogenetic analysis showed that ST2 strains were distinctly segregated into two genetically diverse lineages [L1 (67.0% from Northern America) and L2], while L2 further divided into two sub-lineages, SL2a and SL2b (73.5% from China). The 36 virulence-related genes were widely distributed in ST2 genomes, but in which only 11 antibiotic resistance-associated genes were dispersedly found. Among the 25 SL2b sequenced isolates, RT014 (40.0%, n = 10) and RT020 (28.0%, n = 7) were two main genotypes with no significant difference on antibiotic resistance (χ2 = 0.024–2.667, P > 0.05). A non-synonymous amino acid substitution was found in tcdB (Y1975D) which was specific to SL2b. Although there was no significant difference in sporulation capacity between the two lineages, the average toxin B concentration (5.11 ± 3.20 ng/μL) in SL2b was significantly lower in comparison to those in L1 (10.49 ± 15.82 ng/μL) and SL2a (13.92 ± 2.39 ng/μL) (χ2 = 12.30, P < 0.05). This study described the genomic characteristics of C. difficile ST2, with many virulence loci and few antibiotic resistance elements. The Chinese ST2 strains with the mutation in codon 1975 of the tcdB gene clustering in SL2b circulating in China express low toxin B, which may be associated with mild or moderate C. difficile infection.

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