Lethal toxin is a critical determinant of rapid mortality in rodent models of Clostridium sordellii endometritis

Abstract

The toxigenic anaerobe Clostridium sordellii is an uncommon but highly lethal cause of human infection and toxic shock syndrome, yet few studies have addressed its pathogenetic mechanisms. To better characterize the microbial determinants of rapid death from infection both in vitro and in vivo studies were performed to compare a clinical strain of C. sordellii (DA-108), isolated from a patient who survived a disseminated infection unaccompanied by toxic shock syndrome, to a virulent reference strain (ATCC9714). Rodent models of endometrial and peritoneal infection with C. sordellii ATCC9714 were rapidly lethal, while infections with DA-108 were not. Extensive genetic and functional comparisons of virulence factor and toxin expression between these two bacterial strains yielded many similarities, with the noted exception that strain DA-108 lacked the tcsL gene, which encodes the large clostridial glucosyltransferase enzyme lethal toxin (TcsL). The targeted removal by immunoprecipitation of TcsL protected animals from death following injection of crude culture supernatants from strain ATCC9714. Injections of a monoclonal anti-TcsL IgG protected animals from death during C. sordellii ATCC9714 infection, suggesting that such an approach might improve the treatment of patients with C. sordellii-induced toxic shock syndrome.

Figure 1

The clinical C. sordellii strain DA-108 does not cause acute death in (A) a rat model of endometritis or (B) mouse peritonitis. (A) Rats were inoculated intrauterine with 1 × 10¹⁰ CFU C. sordellii clinical strain DA-108 or the reference strain ATCC9714. (B) SvEV129 mice were infected intraperitoneally with 1 × 10⁸ CFU C. sordellii DA-108 or 10-fold less (1 × 10⁷ CFU) C. sordellii ATCC9714. CFU = colony forming units; n = 5 animals per group. (C) Cell-free toxins prepared as described in Materials and Methods from strain ATCC9714 caused rapid death in SvEV129 mice following i.p. injection while toxins similarly prepared from the DA-108 strain did not cause death. n = 5 animals per group. (D) Rats were infected with 1 × 10¹⁰ CFU C. sordellii ATCC9714 admixed with a goat polyclonal anti-toxin and then received 300 µl once daily i.p. injections of this IgG on days 1, 2, and 3 post-infection as detailed in Materials and Methods. n = 5 rats

Figure 2

The clinical C. sordellii strain DA-108 lacks the gene for lethal toxin (tcsL). (A and B) Genetic comparison of virulence factor genes between the DA-108 and ATCC9714 strains. Genomic DNA was isolated and subjected to PCR for the genes encoding the cholesterol-dependent cytolysin (sordelliilysin); lecithinase (phospholipase C); neuraminidase; and lethal toxin. Four different primer pairs were used to amplify the lethal toxin gene. (C) Western immunoblot detection of TcsL in crude toxin preparations from ATCC9714 and DA-108 strains. The TcsL appears at ∼250 kDa. (D) Phospholipase C activity was confirmed for both strains using an egg yolk-phospholipid hydrolysis activity assay. Results are the mean ± half-range for two independent determinations. A.U. = arbitrary units. (E) Neuraminidase activities for each strain and a C. perfringens neuraminidase (positive control) were determined as described in Materials and Methods and data expressed as the mean ± half-range for two determinations (absorbance units at 560 nm).

Figure 3

C. sordellii TcsL is important for causing rapidly lethal death during infection. (A) Rats (n = 5 per group) or C57BL/6J mice (n = 10 per group) were infected i.u. with 1 × 10¹⁰ or 1 × 10⁶ CFU C. sordellii ATCC9714, respectively, admixed with the mouse monoclonal anti-TcsL IgG. Animals then received twice daily i.p. injections of this IgG on days 0, 1, 2, and 3 post-infection as detailed in Materials and Methods. Survival at 72 hr (after the last IgG injection) was determined (*P<0.05, **P<0.01 compared to untreated animals by Mantel-Cox log-rank test). (B) Mice (n = 5 per group) were injected i.p. with a 1:5 dilution of crude toxins prepared from strain ATCC9714 after immunoprecipitation (IP) was performed with anti-TcsL or anti-C. difficile toxin A IgG (control). (C) Mice (n = 5 per group) were infected i.u. with 1 × 10⁷ CFU of C. sordellii ATCC9714 or DA-108 (or were left uninfected) and pleural fluid volumes were measured as described in Materials and Methods. ***P < 0.0001 vs. uninfected animals.