Search Results (43)

C. perfringens type F isolates are a leading cause of food poisoning and antibiotic-associated diarrhea. Type F isolate virulence requires production of C. perfringens enterotoxin [CPE], which acts by forming large pore complexes in host cell plasma membranes. During GI disease, CPE is produced in the intestines when type F strains undergo sporulation. The toxin is then released into the intestinal lumen when the mother cell lyses at the completion of sporulation. Once present in the lumen, CPE encounters proteases. This study examined the in vitro, ex vivo, and in vivo processing of CPE by intestinal proteases and the effects of this processing on CPE activity. Results using purified trypsin or mouse intestinal contents detected the rapid cleavage of CPE to a major band of ~32 kDa and studies with Caco-2 cells showed that this processed CPE still forms large complexes and retains cytotoxic activity. When mouse small intestinal loops were challenged with CPE, the toxin caused intestinal histologic damage, despite rapid proteolytic processing of most CPE to 32 kDa within 15 min. Intestinal large CPE complexes became more stable with longer treatment times. These results indicate that CPE processing involving trypsin occurs in the intestines and the processed toxin retains enterotoxicity. Full article

Bacillus spp. have emerged as pivotal sources of probiotic preparations, garnering considerable attention in recent years owing to their vigorous bacteriostatic activity and antimicrobial resistance. This study aimed to investigate these probiotic characteristics in depth and verify the safety of Bacillus velezensis K12, a strain isolated from broiler intestine. The K12 strain was identified as Bacillus velezensis based on its morphology and 16S rDNA sequence homology analysis. Subsequently, B. velezensis K12 was evaluated for acid resistance, bile salt resistance, gastrointestinal tolerance, drug sensitivity, and antimicrobial activity. Additionally, whole-genome sequencing technology was employed to dissect its genomic components further, aiming to explore its potential applications as a probiotic strain. B. velezensis K12 was sensitive to six antibiotics and had acid tolerance. Furthermore, it showed potent antimicrobial activity against a wide range of pathogenic bacteria, including Escherichia coli (E. coli), Staphylococcus aureus, Salmonella, Clostridium perfringens, Bacillus cereus, and Vibrio parahaemolyticus. The complete genome sequencing of B. velezensis K12 revealed a genomic length of 3,973,105 base pairs containing 4123 coding genes, among which 3973 genes were functionally annotated. The genomic analysis identified genes associated with acid and bile tolerance, adhesion, antioxidants, and secondary metabolite production, whereas no functional genes related to enterotoxins or transferable antibiotic resistance were detected, thereby confirming the probiotic properties of B. velezensis K12. B. velezensis K12 exhibits broad-spectrum bacteriostatic activity and in vitro safety, positioning it as a potential candidate strain for developing probiotic Bacillus preparations. Full article

Several recent studies have reported a significantly greater prevalence of Clostridium perfringens encoding the novel pore-forming netF toxin gene in dogs with acute hemorrhagic diarrhea syndrome. However, the presence of netF in other canine diarrheal diseases remains poorly characterized. This retrospective, cross-sectional study aimed to describe the prevalence and abundance of netF-positive C. perfringens in fecal samples from 352 dogs with acute and chronic gastrointestinal diseases. Dogs were divided into five groups: acute hemorrhagic diarrhea syndrome (AHDS), acute diarrhea (AD), chronic enteropathy (CE), exocrine pancreatic insufficiency (EPI), and healthy controls (HCs). The abundances of C. perfringens 16S rRNA, the C. perfringens enterotoxin gene and the C. perfringens netF gene in fecal samples were analyzed by quantitative polymerase chain reaction. In total, 7 of 15 (46.7%) dogs with AHDS, 10 of 75 (13.3%) dogs with AD, 2 of 120 (1.7%) dogs with CE, 1 of 12 (8.3%) dogs with EPI, and 1 of 130 (0.8%) HC dogs tested positive for netF. This study provides further evidence that NetF may be a significant contributor to the etiology of AHDS and potentially to a subset of acute nonhemorrhagic diarrhea cases, while it was only rarely detected in chronic gastrointestinal disease phenotypes. Full article

Clostridium perfringens (C. perfringens) is an anaerobic, spore-forming Gram-positive rod responsible for necrotizing gangrene, bacteremia in patients with cancer or gastrointestinal tract infection. C. perfringens virulence is due in large part to toxin production. In 2014, a new enterotoxin, BEC (binary enterotoxin of Clostridium perfringens) encoded by becA and becB genes, distinct from enterotoxin (CPE) encoded by the cpe gene, has been described. BEC-producing strains can be causative agents of acute gastroenteritis in humans. We present herein the case of a 64-year-old man who presented to the emergency department of Toulouse University Hospital with pneumonia and septic shock, without digestive symptoms. Blood cultures showed C. perfringens bacteremia and despite appropriate antibiotic treatment the patient passed away 7 h after admission. The characterization of the strain by whole genome sequencing revealed the presence of typical genes of C. perfringens: plc gene (alpha-toxin, phospholipase C) and pfoA (theta-toxin, perfringolysine). Surprisingly, this strain also harbored becA and becB genes encoding the recently described BEC toxin. Interestingly, alpha-toxin typing of our isolate and other published BEC isolates showed that they belonged to different PLC subtypes, confirming the high genetic diversity of these strains. To our knowledge, it is the first clinical case reporting bacteremia due to a BEC-producing C. perfringens isolate. Full article

Clostridium perfringens enterotoxin (Cpe)-producing strains cause gastrointestinal infections in humans and account for the second-largest number of all foodborne outbreaks caused by bacterial toxins. The Cpe toxin is only produced during sporulation; this process might be affected when C. perfringens comes into contact with host cells. The current study determined how the cpe expression levels and spore formation changed over time during co-culture with Caco-2 cells (as a model of intestinal epithelial cells). In co-culture with Caco-2 cells, total C. perfringens cell counts first decreased and then remained more or less stable, whereas spore counts were stable over the whole incubation period. The cpe mRNA level in the co-culture with Caco-2 cells increased more rapidly than in the absence of Caco-2 cells (3.9-fold higher levels in coculture than in the absence of Caco-2 cells after 8 h of incubation). Finally, we found that cpe expression is inhibited by a cue released by Caco-2 cells (8.3-fold lower levels in the presence of supernatants of Caco-2 cells than in the absence of the supernatants after 10 h of incubation); as a consequence, the increased expression in co-culture with Caco-2 cells must be caused by a factor associated with the Caco-2 cells. Full article

The objective of this study was to evaluate the occurrence of Clostridium perfringens in stool samples and swabs collected from wild mammals in the Amazon biome. Sixty-five faecal and swab samples were collected in situ and ex situ from 16 species and three genera of wild mammals, some of which were in good health and some of which had diarrhoea. After pre-enrichment, the samples were plated on selective agar for C. perfringens. Characteristic colonies were subjected to multiplex PCR for the detection of genes encoding the main C. perfringens toxins (alpha, beta, epsilon, and iota toxin and enterotoxin). Among the 65 samples, 40 (61.5%) were positive for the gene encoding the alpha toxin and were classified as type A, 36 of which were asymptomatic animals and four were diarrheal. No other toxinotypes were found. The findings of this study suggest that C. perfringens type A is commonly found in mammal species of the Amazon biome. This seems to be the first study to identify C. perfringens type A in species such as B. variegatus (common ground sloth), C. didactylus (two-toed sloth), P. flavus (Jupará), T. tetradactyla (anteater), S. collinsi (squirrel monkey), S. niger (black marmoset), and S. apella (Guyana capuchin) and in the genus Didelphis sp. (opossum). Full article

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan–Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production. Full article

Clostridium perfringens enterotoxin (CpE) is a β-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic β-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and β-pore formation. This study sheds light on trypsin’s role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics. Full article

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract. Full article

Bacteraemia brought on by Clostridium perfringens has a very low incidence but is severe and fatal in fifty per cent of cases. C. perfringens is a commensal anaerobic bacterium found in the environment and in the intestinal tracts of animals; it is known to produce six major toxins: α-toxin, β-toxin, ε-toxin, and others. C. perfringens is classified into seven types, A, B, C, D, E, F and G, according to its ability to produce α-toxin, enterotoxin, and necrotising enterotoxin. The bacterial isolates from humans include types A and F, which cause gas gangrene, hepatobiliary infection, and sepsis; massive intravascular haemolysis (MIH) occurs in 7–15% of C. perfringens bacteraemia cases, resulting in a rapid progression to death. We treated six patients with MIH at a single centre in Japan; however, unfortunately, they all passed away. From a clinical perspective, MIH patients tended to be younger and were more frequently male; however, there was no difference in the toxin type or genes of the bacterial isolates. In MIH cases, the level of θ-toxin in the culture supernatant of clinical isolates was proportional to the production of inflammatory cytokines in the peripheral blood, suggesting the occurrence of an intense cytokine storm. Severe and systemic haemolysis is considered an evolutionary maladaptation as it leads to the host’s death before the bacterium obtains the benefit of iron utilisation from erythrocytes. The disease’s extraordinarily quick progression and dismal prognosis necessitate a straightforward and expedient diagnosis and treatment. However, a reliable standard of diagnosis and treatment has yet to be put forward due to the lack of sufficient case analysis. Full article

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target. Full article

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterium capable of producing four major toxins which cause disease symptoms and pathogenesis in humans and animals. C. perfringens strains carrying enterotoxins can cause food poisoning in humans and are associated with meat consumption. An endolysin, named LysCP28, is encoded by orf28 from C. perfringens bacteriophage BG3P. This protein has an N-terminal glycosyl–hydrolase domain (lysozyme) and a C-terminal SH3 domain. Purified LysCP28 (38.8 kDa) exhibited a broad spectrum of lytic activity against C. perfringens strains (77 of 96 or 80.21%), including A, B, C, and D types, isolated from different sources. Moreover, LysCP28 (10 μg/mL) showed high antimicrobial activity and was able to lyse 2 × 10⁷ CFU/mL C. perfringens ATCC 13124 and C. perfringens J21 (animal origin) within 2 h. Necessary due to this pathogenic bacterium’s ability to form biofilms, LysCP28 (18.7 μg/mL) was successfully evaluated as an antibiofilm agent in both biofilm removal and formation inhibition. Finally, to confirm the efficacy of LysCP28 in a food matrix, duck meat was contaminated with C. perfringens and treated with endolysin (100 µg/mL and 50 µg/mL), which reduced viable bacteria by 3.2 and 3.08 units-log, respectively, in 48 h at 4 °C. Overall, the endolysin LysCP28 could potentially be used as a biopreservative to reduce C. perfringens contamination during food processing. Full article

Clostridium perfringens type F food poisoning (FP) strains produce C. perfringens enterotoxin (CPE) to cause a common bacterial food-borne illness in the United States. During FP, CPE is synthesized in the intestines when C. perfringens sporulates. Besides CPE, FP strains also produce sialidases. Most FP strains carry their cpe gene on the chromosome and all surveyed chromosomal cpe (c-cpe) FP strains produce NanH sialidase or both NanJ and NanH sialidases. NanR has been shown previously to regulate sialidase activity in non-FP strains. The current study investigated whether NanR also regulates sialidase activity or influences sporulation and CPE production for c-cpe FP strains SM101 and 01E809. In sporulation medium, the SM101 nanR null mutant showed lower sialidase activity, sporulation, and CPE production than its wild-type parent, while the 01E809 nanR null mutant showed roughly similar sialidase activity, sporulation, and CPE production as its parent. In vegetative medium, the nanR null mutants of both strains produced more spores than their parents while NanR repressed sialidase activity in SM101 but positively regulated sialidase activity in 01E809. These results demonstrate that NanR regulates important virulence functions of c-cpe strains, with this control varying depending on strain and culture conditions. Full article

Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE’s high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast–brain metastatic tumors. Full article

Clostridium perfringens is the prevalent enteric pathogen in humans and animals including chickens, and it remains largely elusive on the mechanism of C. perfringens-induced enteritis because of limited animal models available. In this study, we investigated the role of C. perfringens sporulation proteins as vaccine candidates in chickens to reduce necrotic enteritis (NE). C. perfringens soluble proteins of vegetative cells (CP-super1 and CP-super2) and spores (CP-spor-super1 and CP-spor-super2) were prepared, and cell and chicken experiments were conducted. We found that deoxycholic acid reduced C. perfringens invasion and sporulation using the Eimeria maxima and C. perfringens co-infection necrotic enteritis (NE) model. C. perfringens enterotoxin (CPE) was detected in the CP-spor-super1&2. CP-spor-super1 or 2 induced cell death in mouse epithelial CMT-93 and macrophage Raw 264.7 cells. CP-spor-super1 or 2 also induced inflammatory gene expression and necrosis in the Raw cells. Birds immunized with CP-spor-super1 or 2 were resistant to C. perfringens-induced severe clinical NE on histopathology and body weight gain loss. CP-spor-super1 vaccine reduced NE-induced proinflammatory Ifnγ gene expression as well as C. perfringens luminal colonization and tissue invasion in the small intestine. Together, this study showed that CP-spor-super vaccines reduced NE histopathology and productivity loss. Full article