TcsL is an essential virulence factor in Clostridium sordellii ATCC 9714

Abstract

Clostridium sordellii is an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis of C. sordellii infections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids into C. sordellii using RP4-mediated conjugation from Escherichia coli and have successfully used these techniques to insertionally inactivate the tcsL gene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections by C. sordellii ATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor in C. sordellii and opens the way for in-depth studies of this important human pathogen at the molecular level.

FIG. 1.

Southern hybridization analysis of tcsL mutants. Genomic DNA was purified and digested with HaeIII and EcoRI prior to electrophoresis on 0.8% agarose gels. The Southern blots were probed with probes specific for (A) tcsL, (B) ermB, and (C) catP. WT, wild-type ATCC 9714; DLL5001 to DLL5003, tcsL mutants; V, vector control pDLL2; M, DIG-labeled λ-HindIII molecular size markers (kb).

FIG. 2.

Comparative analysis of TcsL production. The wild type and isogenic mutant derivatives were examined by Western immunoblotting using TcsL-specific antibodies and precipitated supernatant proteins from the strains indicated. WT, wild-type ATCC 9714; DA108, tcsL-nonexpressing negative-control strain; DLL5001 to DLL5003, tcsL mutants; M, molecular mass size markers (kDa).

FIG. 3.

Quantitative Vero cell cytotoxicity assays. Serial doubling dilutions of C. sordellii culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in cytotoxicity assays. (A) The morphological changes of the Vero cells were observed and scored by microscopy after 24 h. The endpoint was taken as the last dilution at which CPE was observed. The toxin titer is the reciprocal of the endpoint dilution. WT, wild-type ATCC 9714; DA108, tcsL-nonexpressing negative-control strain; DLL5001 to DLL5003, tcsL mutants. (B) Cell viability following incubation of Vero cells with C. sordellii supernatants for 24 h was determined by using MTT reagent. The percent death was calculated by comparison to untreated wells, which were assumed to have 100% viability. •, wild-type ATCC 9714; ▪, tcsL mutant DLL5001; ▴, tcsL mutant DLL5002; ▾, tcsL mutant DLL5003; ♦, tcsL-nonexpressing negative-control strain DA108; ✖, TY medium control. All assays were performed in duplicate on three independent culture supernatants, and the mean values of these assays are shown together with the standard errors of the means.

FIG. 4.

Kaplan-Meier survival curve using the mouse infection model. The graph shows days from inoculation with C. sordellii to death. Two groups of 5 female 129/SvJ mice were injected intraperitoneally with each of the indicated strains, and mouse survival was monitored for 6 days. •, wild-type ATCC 9714; ○, tcsL mutant DLL5001; ×, tcsL mutant DLL5003.

FIG. 5.

Representative histological findings in mouse uterine horns 48 h after unilateral inoculation with the wild-type C. sordellii strain ATCC 9714 (A and C) or the tcsL mutant DLL5003 (B and D). Panels E and F are representative images taken from the uninfected, contralateral uterine horns of mice infected with the wild-type strain ATCC 9714 or the mutant strain DLL5003, respectively. Sections were stained with hematoxylin and eosin. Magnification: A and B, ×100; C, D, E, and F, ×400.