Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections

Abstract

Clostridium perfringens causes enteritis and enterotoxemia in humans and livestock due to prolific toxin production. In broth culture, C. perfringens uses the Agr-like quorum sensing (QS) system to regulate production of toxins important for enteritis/enterotoxemia, including beta toxin (CPB), enterotoxin, and epsilon toxin (ETX). The VirS/VirR two-component regulatory system (TCRS) also controls CPB production in broth cultures. Both the Agr-like QS and VirS/VirR systems are important when C. perfringens senses enterocyte-like Caco-2 cells and responds by upregulating CPB production; however, only the Agr-like QS system is needed for host cell-induced ETX production. These in vitro observations have pathophysiologic relevance since both the VirS/VirR and Agr-like QS signaling systems are required for C. perfringens strain CN3685 to produce CPB in vivo and to cause enteritis or enterotoxemia. Thus, apparently upon sensing its presence in the intestines, C. perfringens utilizes QS and TCRS signaling to produce toxins necessary for intestinal virulence.

Keywords: Agr; Clostridium perfringens; VirS/VirR; beta toxin; enterotoxin; epsilon toxin; intestinal infection; quorum sensing; two component regulatory system.

Figure 1. Regulation of C. perfringens toxin production by the Agr-like QS system. (A) western blot analysis of CPE production by 16 h sporulating cultures of CPE-positive type A strain F5603 (F5603WT), an isogenic agrB null mutant (F5603::agrB) or an agr locus complementing strain (F5603AgrBcomp). (B) western blot analysis of ETX production by 4 or 8 h vegetative cultures of type D strain CN3718 (WT), the isogenic agrB null mutant (Δ) or the agr locus complementing strain (P3). (C) western blot of CPB production by overnight vegetative cultures of type C strain CN3685 (WT), an isogenic agrB null mutant (agrBKO) or the agr locus complementing strain (P3). Reproduced with permission from refs. , , and .

Figure 2. Agr-like QS system regulation of C. perfringens toxin production involves a diffusible signal. (A) Inoculation of Transwell chambers with, as indicated, the type C parent strain CN3685, the isogenic cpb null mutant (CN3685::cpb) or an isogenic agrB null mutant (CN3685::agrB) in two chambers, or CN3685::cpb in one chamber and CN3685::agrB in the other chamber. After 5 h, CPB production in each culture was examined by CPB western blot. (B) Inoculation of Transwell chambers with, as indicated, type D parent strain CN3718, an isogenic etx null mutant (CN3718::etx) or an agrB null mutant (CN3718::agrB) in two chambers, or CN3718::etx in one chamber and CN3718::agrB in the other chamber. After 5 h, ETX production in each culture was examined by ETX western blot. Reproduced with permission from refs. and .

Figure 3. Regulation of C. perfringens toxin production by the VirS/VirR TCRS. (A) western blot analysis of CPB production by overnight vegetative cultures of type C strain CN3685, an isogenic virR null mutant (VirR knockout) or the virS/virR complementing strain (VirR/pTS405). For comparison, the migration of purified CPB is also shown in the left lane of the blot. (B) western blot analyses of ETX production by 4 h vegetative broth cultures of type D strain CN3718, the isogenic virR null mutant (VirR knockout) or the complementing strain (VirR/pTS405). Reproduced with permission from refs. and .

Figure 4. Contact with host Caco-2 cells induces an upregulation of toxin production by C. perfringens. (A) western blot analysis of ETX production by type D strain CN3718 (WT), the isogenic ΔAgrB null mutant (Δ) or the P3 agr locus complementing strain (P3). Shown is ETX production by these strains after 1‒2 h in the presence (Caco-2 cells) or absence (MEM) of enterocyte-like Caco-2 cells. (B) western blot analysis of CPB production by type C strain CN3685 (WT), the isogenic ΔAgrB null mutant (Δ) or the P3 complementing strain (P3) after 2 h in the presence of Caco-2 cells. No CPB production was detected after 2 h growth of these strains in the absence of Caco-2 cells. (C) western blot analysis of CPB production by CN3685, the isogenic virR null mutant (VirR ko) or the VirR/pTS405 complementing strain after 2 h in the presence of Caco-2 cells. Reproduced with permission from refs. and .

Figure 5. The Agr-like QS system controls the intestinal virulence of type C strain CN3685. (A) Histologic damage in rabbit small intestinal loops treated for 6 h with control (sterile) broth or washed cells of wild-type CN3685, an isogenic luxS null mutant, an isogenic agrB null mutant or the an agr locus complementing strain. Tissues were processed routinely for production of hematoxylin and eosin-stained sections. Sections were photographed at 200 × final magnification. (B) Fluid accumulation in small intestinal loops after challenge with the indicated strains. The asterisk indicates statistically significant (p < 0.05) differences from CN3685 and BMJV13. (C) western blot analysis of in vivo CPB production in luminal fluids recovered from small intestinal loops challenged for 6 h with washed cells of each indicated strain. The migration of purified CPB is indicated to the left of the blot. Similar analyses of luminal fluids collected from loops challenged with washed cells of the luxS null mutant detected wild-type levels of CPB production (not shown). Reproduced with permission from ref. .

Figure 6. The VirS/VirR TCRS controls the intestinal virulence of type C strain CN3685. (A) Histologic damage in rabbit small intestinal loops challenged for 6 h with sterile control broth (top left), washed cells of wild-type CN3685 (top right), an isogenic virR null mutant (bottom left) or a virS/virR complementing strain (bottom right). Tissues were processed by histology and stained using hematoxylin and eosin. Sections were photographed at 200 × final magnification. (B) Fluid accumulation in small intestinal loops after challenge with the indicated strains. The asterisk indicates statistically significant (p < 0.05) differences from CN3685. (C) western blot analysis of in vivo CPB production in luminal fluids recovered from small intestinal loops challenged for 6 h with washed cells of each indicated strain. The 35 kDa size of purified CPB is indicated to the left of the blot. Reproduced with permission from ref. .

Figure 7. Models for C. perfringens toxin gene regulation by the Agr-like QS system and VirS/VirR TCRS. (A) Regulation of toxin production in type C and D strains. The Agr-like QS system generates an autoinducing peptide (AIP) that interacts with one or more TCRS, possibly including VirS/VirR and/or for ETX regulation in type D strain CN3718, another still unidentified TCRS (sensor = membrane sensor; RR = response regulator). This signaling results in direct or indirect increased production of CPA and PFO in both type C and D strains, CPB in type C strain CN3685 and ETX in type D strain CN3718 (but not in type B strains). (B) Regulation of sporulation and CPE production in CPE-positive type A strains. The Agr-like QS system generates AIP, which (possibly via a TCRS) increases Spo0A production and, under sporulation-inducing conditions, alternative sigma factor expression levels to promote sporulation and CPE production.