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Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium.

Roos S, Wyder M, Candi A, Regenscheit N, Nathues C, van Immerseel F, Posthaus H - Toxins (Basel) (2015)

Bottom Line: Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach.This, however, was not inhibited by CPB neutralization.Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Pathobiology, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern 3012, Switzerland. simone.roos@vetsuisse.unibe.ch.

ABSTRACT
Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

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Reduction of TEER upon basolateral exposure of IPEC-J2 cells to C. perfringens type C culture supernatants but not rCPB. IPEC-J2 cells were incubated in the basolateral compartment of the Transwell® with the same toxins and supernatants (diluted 1:10 and 1:5) as in Figure 3. Aerolysin and the C. perfringens type C supernatant NCTC 3180 (1:10) caused a rapid and sustained drop in TEER. This effect could not be inhibited by neutralization of CPB using mAb-CPB. Supernatants of JF 3721 induced a drop in TEER within 12 h and supernatants of JF 3693 within 48 h. rCPB (32 µg/mL), or TGY (1:5) resulted in a small drop of TEER values, which however never dropped below 2 kΩ cm2 and subsequently increased towards the end of the experiment. Values represent means of three independent experiments with a total of eight separate Transwells®. Error bars represent two-fold standard deviation. Asterisks indicate values which differ significantly from the TGY control group.
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toxins-07-01235-f004: Reduction of TEER upon basolateral exposure of IPEC-J2 cells to C. perfringens type C culture supernatants but not rCPB. IPEC-J2 cells were incubated in the basolateral compartment of the Transwell® with the same toxins and supernatants (diluted 1:10 and 1:5) as in Figure 3. Aerolysin and the C. perfringens type C supernatant NCTC 3180 (1:10) caused a rapid and sustained drop in TEER. This effect could not be inhibited by neutralization of CPB using mAb-CPB. Supernatants of JF 3721 induced a drop in TEER within 12 h and supernatants of JF 3693 within 48 h. rCPB (32 µg/mL), or TGY (1:5) resulted in a small drop of TEER values, which however never dropped below 2 kΩ cm2 and subsequently increased towards the end of the experiment. Values represent means of three independent experiments with a total of eight separate Transwells®. Error bars represent two-fold standard deviation. Asterisks indicate values which differ significantly from the TGY control group.

Mentions: Basolateral exposure of IPEC-J2 cell layers to aerolysin as a positive control resulted in a rapid and complete loss of the TEER of polarized IPEC-J2 layers (Figure 4). TGY did not have an effect on the TEER up to a dilution of 1:5 and the control C. perfringens type A supernatant also did not induce a drop in TEER below 2 kΩ cm2 at this concentration. Basolateral incubation of polarized IPEC-J2 cells with the supernatant of NCTC 3180 diluted 1:10 in cell culture medium resulted in a rapid decline of TEER. After 1 h, TEER values dropped below 2 kΩ cm2 and subsequently declined (Figure 4). Pre-incubation of the supernatants with neutralizing mAb-CPB did not reduce this effect. Basolateral incubation with supernatant of JF 3721 diluted 1:10 in cell culture medium did not induce a drop of TEER values below 2 kΩ cm2 (Figure S4), however increasing the concentration to a 1:5 dilution resulted in a significant drop at 24 h with a subsequent further decline (Figure 4). Pre-incubation with mAb-CPB had no effect on this drop in TEER. Recombinant CPB at a concentration of 32 µg/mL did not induce a significant drop in TEER when applied basolaterally. Dunn’s Z multiple comparison tests revealed a significant difference between TEER values of the TGY control group and TEER values of IPEC-J2 grown on Transwells® which had been incubated with aerolysin and the supernatant of NCTC 3180 (1:10) neutralized with mAb-CPB from 1 h–48 h. TEER values of non-neutralized NCTC 3180 differed significantly from 2 h to 48 h from the TGY control group. TEER values of Transwells® incubated with supernatant of JF 3721 neutralized or non-neutralized differed significantly from 24 h to 48 h from the TGY control group. Similar to apical exposure no significant differences were present between Transwells® incubated with type C supernatants (NCTC 3180, JF 3721) and the corresponding neutralized supernatants. Similar to apical exposure experiments, 32 µg/mL CPB in the basolateral chamber of the Transwells® did not reduce the TEER below 2 kΩ cm2.


Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium.

Roos S, Wyder M, Candi A, Regenscheit N, Nathues C, van Immerseel F, Posthaus H - Toxins (Basel) (2015)

Reduction of TEER upon basolateral exposure of IPEC-J2 cells to C. perfringens type C culture supernatants but not rCPB. IPEC-J2 cells were incubated in the basolateral compartment of the Transwell® with the same toxins and supernatants (diluted 1:10 and 1:5) as in Figure 3. Aerolysin and the C. perfringens type C supernatant NCTC 3180 (1:10) caused a rapid and sustained drop in TEER. This effect could not be inhibited by neutralization of CPB using mAb-CPB. Supernatants of JF 3721 induced a drop in TEER within 12 h and supernatants of JF 3693 within 48 h. rCPB (32 µg/mL), or TGY (1:5) resulted in a small drop of TEER values, which however never dropped below 2 kΩ cm2 and subsequently increased towards the end of the experiment. Values represent means of three independent experiments with a total of eight separate Transwells®. Error bars represent two-fold standard deviation. Asterisks indicate values which differ significantly from the TGY control group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4417965&req=5

toxins-07-01235-f004: Reduction of TEER upon basolateral exposure of IPEC-J2 cells to C. perfringens type C culture supernatants but not rCPB. IPEC-J2 cells were incubated in the basolateral compartment of the Transwell® with the same toxins and supernatants (diluted 1:10 and 1:5) as in Figure 3. Aerolysin and the C. perfringens type C supernatant NCTC 3180 (1:10) caused a rapid and sustained drop in TEER. This effect could not be inhibited by neutralization of CPB using mAb-CPB. Supernatants of JF 3721 induced a drop in TEER within 12 h and supernatants of JF 3693 within 48 h. rCPB (32 µg/mL), or TGY (1:5) resulted in a small drop of TEER values, which however never dropped below 2 kΩ cm2 and subsequently increased towards the end of the experiment. Values represent means of three independent experiments with a total of eight separate Transwells®. Error bars represent two-fold standard deviation. Asterisks indicate values which differ significantly from the TGY control group.
Mentions: Basolateral exposure of IPEC-J2 cell layers to aerolysin as a positive control resulted in a rapid and complete loss of the TEER of polarized IPEC-J2 layers (Figure 4). TGY did not have an effect on the TEER up to a dilution of 1:5 and the control C. perfringens type A supernatant also did not induce a drop in TEER below 2 kΩ cm2 at this concentration. Basolateral incubation of polarized IPEC-J2 cells with the supernatant of NCTC 3180 diluted 1:10 in cell culture medium resulted in a rapid decline of TEER. After 1 h, TEER values dropped below 2 kΩ cm2 and subsequently declined (Figure 4). Pre-incubation of the supernatants with neutralizing mAb-CPB did not reduce this effect. Basolateral incubation with supernatant of JF 3721 diluted 1:10 in cell culture medium did not induce a drop of TEER values below 2 kΩ cm2 (Figure S4), however increasing the concentration to a 1:5 dilution resulted in a significant drop at 24 h with a subsequent further decline (Figure 4). Pre-incubation with mAb-CPB had no effect on this drop in TEER. Recombinant CPB at a concentration of 32 µg/mL did not induce a significant drop in TEER when applied basolaterally. Dunn’s Z multiple comparison tests revealed a significant difference between TEER values of the TGY control group and TEER values of IPEC-J2 grown on Transwells® which had been incubated with aerolysin and the supernatant of NCTC 3180 (1:10) neutralized with mAb-CPB from 1 h–48 h. TEER values of non-neutralized NCTC 3180 differed significantly from 2 h to 48 h from the TGY control group. TEER values of Transwells® incubated with supernatant of JF 3721 neutralized or non-neutralized differed significantly from 24 h to 48 h from the TGY control group. Similar to apical exposure no significant differences were present between Transwells® incubated with type C supernatants (NCTC 3180, JF 3721) and the corresponding neutralized supernatants. Similar to apical exposure experiments, 32 µg/mL CPB in the basolateral chamber of the Transwells® did not reduce the TEER below 2 kΩ cm2.

Bottom Line: Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach.This, however, was not inhibited by CPB neutralization.Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Pathobiology, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern 3012, Switzerland. simone.roos@vetsuisse.unibe.ch.

ABSTRACT
Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

Show MeSH
Related in: MedlinePlus