Characterizing the Contributions of Various Clostridium perfringens Enterotoxin Properties to <i>In Vivo</i> and <i>In Vitro</i> Permeability Effects.

Clostridium perfringens enterotoxin (CPE) is thought to cause lethal enterotoxemia when absorbed from the intestinal lumen into the circulation. CPE action sequentially involves receptor-binding, oligomerization into a prepore, and pore formation. To explore the mechanistic basis by which CPE alters permeability, this study tested the permeability effects of several recombinant CPE (rCPE) species: rCPE and rCPE_C186A (which form pores), rC-CPE and rCPE_D48A (which bind to receptors but cannot oligomerize), rCPE_C186A/F91C (which binds and oligomerizes without pore formation), and rCPE_Y306A/L315A (which has poor receptor-binding ability). On Caco-2 cells, i) only rCPE and rCPE_C186A were cytotoxic; ii) rCPE and rCPE_C186A affected transepithelial resistance (TEER) and 4 kDa fluorescent dextran (FD4) transit more quickly than binding-capable, but noncytotoxic, rCPE variants; whereas iii) rCPE_Y306A/L315A did not affect TEER or FD4 transit. Using mouse intestinal loops, rCPE (but not noncytotoxic rC-CPE, rCPE_D48A or rCPE_Y306A/L315A) was lethal and caused intestinal histologic damage within 4 h. After 2 h of treatment, rCPE was more strongly absorbed into the serum than those noncytotoxic rCPE species but by 4 h rC-CPE and rCPE_D48A became absorbed similarly as rCPE, while rCPE_Y306A/L315A absorption remained low. This increased rC-CPE and rCPE_D48A absorption from 2 to 4 h did not involve a general intestinal permeability increase because Evans Blue absorption from the intestines did not increase between 2 and 4 h of treatment with rC-CPE or rCPE_D48A. Collectively, these results indicate that CPE receptor binding is sufficient to slowly affect permeability, but CPE-induced cytotoxicity is necessary for rapid permeability changes and lethality. <b>IMPORTANCE</b> Clostridium perfringens enterotoxin (CPE) causes lethal enterotoxemia when absorbed from the intestines into the bloodstream. Testing recombinant CPE (rCPE) or rCPE variants impaired for various specific steps in CPE action showed that full CPE-induced cytotoxicity causes rapid Caco-2 monolayer permeability alterations, as well as enterotoxemic lethality and rapid CPE absorption in mouse small intestinal loops. However, receptor binding-capable, but noncytotoxic, rCPE variants did cause slow-developing <i>in vitro</i> and <i>in vivo</i> permeability effects. Absorption of binding-capable, noncytotoxic rCPE variants from the intestines did not correlate with general intestinal permeability alterations, suggesting that CPE binding can induce its own uptake. These findings highlight the importance of binding and, especially, cytotoxicity for CPE absorption during enterotoxemia and may assist development of permeability-altering rCPE variants for translational purposes.