Clostridium perfringens beta-toxin targets endothelial cells in necrotizing enteritis in piglets

ABSTRACT Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C β-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

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Platelet Endothelial Cell Adhesion Molecule 1 (CD31) Is Essential for Clostridium perfringens Beta-Toxin Mediated Cytotoxicity in Human Endothelial and Monocytic Cells

Toxins ◽

10.3390/toxins13120893 ◽

2021 ◽

Vol 13 (12) ◽

pp. 893

Author(s):

Basma Tarek ◽

Julia Bruggisser ◽

Filippo Cattalani ◽

Horst Posthaus

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Gene Knockout ◽

Specific Interaction ◽

Cell Type Specificity ◽

Monocytic Cells ◽

Beta Toxin

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals.

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Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells

Microbial Pathogenesis ◽

10.1006/mpat.1999.0323 ◽

2000 ◽

Vol 28 (1) ◽

pp. 45-50 ◽

Cited By ~ 59

Author(s):

Valgerdur Steinthorsdottir ◽

Haraldur Halldórsson ◽

OÂlafur S Andrésson

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Human Endothelial Cells ◽

Transmembrane Pores ◽

Beta Toxin

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Clostridium perfringens Beta-Toxin Induces Necrostatin-Inhibitable, Calpain-Dependent Necrosis in Primary Porcine Endothelial Cells

PLoS ONE ◽

10.1371/journal.pone.0064644 ◽

2013 ◽

Vol 8 (5) ◽

pp. e64644 ◽

Cited By ~ 36

Author(s):

Delphine Autheman ◽

Marianne Wyder ◽

Michel Popoff ◽

Katharina D’Herde ◽

Stephan Christen ◽

...

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Beta Toxin ◽

Porcine Endothelial Cells

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Application of an Endothelial Cell Culture Assay for the Detection of Neutralizing Anti-Clostridium Perfringens Beta-Toxin Antibodies in a Porcine Vaccination Trial

Toxins ◽

10.3390/toxins11040225 ◽

2019 ◽

Vol 11 (4) ◽

pp. 225 ◽

Cited By ~ 2

Author(s):

Olivia K. Richard ◽

Sven Springer ◽

Jacqueline Finzel ◽

Tobias Theuß ◽

Marianne Wyder ◽

...

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Neutralizing Antibody ◽

Antibody Activity ◽

Antibody Titers ◽

Type C ◽

Cell Culture Assay ◽

Beta Toxin

Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development.

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Effects of Clostridium perfringens Beta-Toxin on the Rabbit Small Intestine and Colon

Infection and Immunity ◽

10.1128/iai.00547-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4396-4404 ◽

Cited By ~ 54

Author(s):

Jorge E. Vidal ◽

Bruce A. McClane ◽

Juliann Saputo ◽

Jaquelyn Parker ◽

Francisco A. Uzal

Keyword(s):

Small Intestine ◽

Clostridium Perfringens ◽

Time Course ◽

Defense Mechanism ◽

Intestinal Damage ◽

Loop Model ◽

Type B ◽

Ileal Loop ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

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The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

Veterinary Research ◽

10.1186/1297-9716-44-45 ◽

2013 ◽

Vol 44 (1) ◽

pp. 45 ◽

Cited By ~ 26

Author(s):

Stefanie Verherstraeten ◽

Evy Goossens ◽

Bonnie Valgaeren ◽

Bart Pardon ◽

Leen Timbermont ◽

...

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Alpha Toxin ◽

Bovine Endothelial Cells

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Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein

FEMS Microbiology Letters ◽

10.1016/0378-1097(95)00218-t ◽

1995 ◽

Vol 130 (2-3) ◽

pp. 273-278 ◽

Cited By ~ 13

Author(s):

V Steinpórsdótti

Keyword(s):

Fusion Protein ◽

Clostridium Perfringens ◽

Expression And Purification ◽

Glutathione S Transferase ◽

Beta Toxin

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Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

Infection and Immunity ◽

10.1128/iai.00818-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4354-4363 ◽

Cited By ~ 25

Author(s):

Menglin Ma ◽

Jihong Li ◽

Bruce A. McClane

Keyword(s):

World War Ii ◽

Heat Resistance ◽

Clostridium Perfringens ◽

Type A ◽

World War ◽

Content Type ◽

Post World War Ii ◽

Heat Resistant ◽

Type C ◽

Beta Toxin

ABSTRACTClostridium perfringenstype C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates arecpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores withD100values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomalcpegene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both thecpeandcpbgenes can be mobilized in Darmbrand isolates. These results suggest thatC. perfringenstype A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomalcpegene.

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