Endothelial Binding of Beta Toxin to Small Intestinal Mucosal Endothelial Cells in Early Stages of Experimentally Induced Clostridium Perfringens Type C Enteritis in Pigs

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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Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

Infection and Immunity ◽

10.1128/iai.01848-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 2958-2970 ◽

Cited By ~ 26

Author(s):

Menglin Ma ◽

Abhijit Gurjar ◽

James R. Theoret ◽

Jorge P. Garcia ◽

Juliann Beingesser ◽

...

Keyword(s):

Clostridium Perfringens ◽

Synergistic Effects ◽

Fluid Accumulation ◽

Content Type ◽

Positive Type ◽

Intestinal Infections ◽

Type C ◽

Small Intestinal ◽

Beta Toxin ◽

Enteritis Necroticans

ABSTRACTThe ability ofClostridium perfringenstype C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also expressC. perfringensenterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and sinceC. perfringenscan sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogeniccpbandcpenull mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of thecpbandcpemutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing thecpemutant or reversing thecpbmutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions duringC. perfringensintestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.

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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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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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Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias

Infection and Immunity ◽

10.1128/iai.00825-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5291-5299 ◽

Cited By ~ 40

Author(s):

Francisco A. Uzal ◽

Juliann Saputo ◽

Sameera Sayeed ◽

Jorge E. Vidal ◽

Derek J. Fisher ◽

...

Keyword(s):

Mouse Models ◽

Clostridium Perfringens ◽

Clinical Signs ◽

Passive Immunization ◽

Small Animal ◽

Abdominal Distension ◽

Virulent Type ◽

Genes Encoding ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

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The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽

10.1128/mbio.00338-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 24

Author(s):

Menglin Ma ◽

Jorge Vidal ◽

Juliann Saputo ◽

Bruce A. McClane ◽

Francisco Uzal

Keyword(s):

Clostridium Perfringens ◽

Regulatory System ◽

Gas Gangrene ◽

Necrotic Enteritis ◽

Vegetative Cells ◽

Content Type ◽

Two Component ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

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