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The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.

Clostridium difficile infection is the main cause of healthcareacquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

A Clostridioides difficile infection (CDI) is the most common nosocomial infection worldwide. The main virulence factors of pathogenic C. difficile are TcdA and TcdB, which inhibit small Rho-GTPases. The inhibition of small Rho-GTPases leads to the so-called cytopathic effect, a reorganization of the actin cytoskeleton, an impairment of the colon epithelium barrier function and inflammation. Additionally, TcdB induces a necrotic cell death termed pyknosis in vitro independently from its glucosyltransferases, which are characterized by chromatin condensation and ROS production. To understand the underlying mechanism of this pyknotic effect, we conducted a large-scale phosphoproteomic study. We included the analysis of alterations in the phosphoproteome after treatment with TcdA, which was investigated for the first time. TcdA exhibited no glucosyltransferase-independent necrotic effect and was, thus, a good control to elucidate the underlying mechanism of the glucosyltransferase-independent effect of TcdB. We found RAS to be a central upstream regulator of the glucosyltransferase-independent effect of TcdB. The inhibition of RAS led to a 68% reduction in necrosis. Further analysis revealed apolipoprotein C-III (APOC3) as a possible crucial factor of CDI-induced inflammation in vivo.

C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Clostridioides difficile accessory gene regulator 1 ( agr1 ) locus consists of two genes, agrB1 and agrD1 , that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1 , a grD1 , or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1 / agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action. IMPORTANCE C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile . These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr’s mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.

Clostridioides (C.) difficile is a spore-forming, toxin-producing nosocomial human gut pathogen and a causative agent of gastrointestinal infections, leading to mild to severe diarrhea. Severe C. difficile infections (CDI) can cause life-threatening conditions, such as pseudomembranous colitis, colonic perforation, or toxic megacolon. The main virulence factors of C. difficile and responsible for CDI symptoms are two AB-type protein toxins, toxin A (TcdA) and toxin B (TcdB). TcdA and TcdB are large, single-chain proteins with multiple domains and glucosyltransferase activity. After receptor-mediated endocytosis, acidification of endosomes triggers insertion and pore formation of the toxins into the endosomal membrane for the delivery of their toxic glucosyltransferase domain (GTD) into the cytosol. There, the GTD glucosylates its target proteins, small GTPases of the Rho and/or Ras family, which leads amongst others to the collapse of the actin cytoskeleton and eventually to cell death. Here, we describe in silico predicted antimicrobial peptides, denoted as Angies, since they derive from the human endogenous protein angiogenin, as inhibitors for TcdA and TcdB. The strongest inhibitory capacity provided the derivative Angie 5, consistently in HeLa and Vero cells, as well as in the physiologically more relevant colon carcinoma cell line CaCo-2. Angie 5 delayed TcdA/TcdB-mediated glucosylation of its substrate proteins and, consequently, toxin-induced cell rounding as a consequence of actin-depolymerization. Moreover, the same Angie peptides that neutralized TcdA/TcdB also prevented the growth of C. difficile in vitro. In conclusion, our study paves the way for the development of antimicrobial peptide-based anti-toxin strategies to address C. difficile -associated diseases (CDADs).

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

The gut microbiota composition of intensive care unit (ICU) patients suffering from Clostridium difficile-positive diarrhea (CDpD) is poorly understood. This prospective study aims to use 16S rDNA (and metagenome) sequencing to compare the microbiota composition of 58 (and 5) ICU patients with CDpD (CDpD group), 33 (and 4) ICU patients with C. difficile-negative diarrhea (CDnD group), and 21 (and 5) healthy control subjects (control group), as well as CDpD patients in the A+B+ (N = 34; A/B: C. difficile TcdA/B), A−B+ (N = 7), and A−B− (N = 17) subgroups. For 16S rDNA data, OTU clustering (tool: UPARSE), taxonomic assignment (tool: RDP classifier), α-diversity, and β-diversity analyses (tool: QIIME) were conducted. For metagenome data, metagenome assembly (tool: SOAPdenovo), gene calling (tools: MetaGeneMark, CD-HIT, and SoapAligner), unigene alignment (tool: DIAMOND), taxon difference analysis (tool: Metastats), and gene annotation (tool: DIAMOND) were performed. The microbial diversity of the CDpD group was lower than that of the CDnD and control groups. The abundances of 10 taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) were significantly higher in the CDpD group than in the CDnD group. The abundances of Saccharomycetes and Clostridia were significantly lower in CDpD in comparison with control. Some taxa were significantly different between the A+B+ and A−B− subgroups. CDpD might relate to a decrease in beneficial taxa (i.e., Saccharomycetes and Clostridia) and an increase in harmful taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) in gut microbiota of ICU patients. C. difficile toxin type might be slightly associated with gut microbiota composition.

Rising incidences and mortalities have drawn attention to Clostridioides difficile infections (CDIs) in recent years. The main virulence factors of this bacterium are the exotoxins TcdA and TcdB, which glucosylate Rho-GTPases and thereby inhibit Rho/actin-mediated processes in cells. This results in cell rounding, gut barrier disruption and characteristic clinical symptoms. So far, treatment of CDIs is limited and mainly restricted to some antibiotics, often leading to a vicious circle of antibiotic-induced disease recurrence. Here, we demonstrate the protective effect of the human antimicrobial peptide α-defensin-6 against TcdA, TcdB and the combination of both toxins in vitro and in vivo and unravel the underlying molecular mechanism. The defensin prevented toxin-mediated glucosylation of Rho-GTPases in cells and protected human cells, model epithelial barriers as well as zebrafish embryos from toxic effects. In vitro analyses revealed direct binding to TcdB in an SPR approach and the rapid formation of TcdB/α-defensin-6 complexes, as analyzed with fluorescent TcdB by time-lapse microscopy. In conclusion, the results imply that α-defensin-6 rapidly sequesters the toxin into complexes, which prevents its cytotoxic activity. These findings extend the understanding of how human peptides neutralize bacterial protein toxins and might be a starting point for the development of novel therapeutic options against CDIs.

Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea and pseudomembranous colitis. The major virulence factors responsible for the disease symptoms are two secreted cytotoxic proteins, TcdA and TcdB. A parenteral vaccine based on formaldehyde-inactivated TcdA and TcdB supplemented with alum adjuvant, has previously been investigated in humans but resulted in an insufficient immune response. In search for an improved response, we investigated a novel toxin inactivation method and a novel, potent adjuvant. Inactivation of toxins by metal-catalyzed oxidation (MCO) was previously shown to preserve neutralizing epitopes and to annihilate reversion to toxicity. The immunogenicity and safety of TcdA and TcdB inactivated by MCO and combined with a novel carbohydrate fatty acid monosulphate ester-based (CMS) adjuvant were investigated in rabbits. Two or three intramuscular immunizations generated high serum IgG and neutralizing antibody titers against both toxins. The CMS adjuvant increased antibody responses to both toxins while an alum adjuvant control was effective only against TcdA. Systemic safety was evaluated by monitoring body weight, body temperature, and analysis of red and white blood cell counts shortly after immunization. Local safety was assessed by histopathologic examination of the injection site at the end of the study. Body weight gain was constant in all groups. Body temperature increased up to 1 ˚C one day after the first immunization but less after the second or third immunization. White blood cell counts, and percentage of neutrophils increased one day after immunization with CMS-adjuvanted vaccines, but not with alum. Histopathology of the injection sites 42 days after the last injection did not reveal any abnormal tissue reactions. From this study, we conclude that TcdA and TcdB inactivated by MCO and combined with CMS adjuvant demonstrated promising immunogenicity and safety in rabbits and could be a candidate for a vaccine against CDI.