Dissecting the Contributions of Clostridium perfringens Type C Toxins to Lethality in the Mouse Intravenous Injection Model

Abstract

The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/ toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.

ANÁLISIS In vitro e in vivo DE SOBREDONANTES CRUDOS DE Clostridium perfringens AISLADOS DE CASOS DE ENTEROTOXEMIA EN ALPACAS.

“…Calculada en forma triplicada para cada aislado, obteniéndose la media y la desviación estándar de las medias (SD) 4 Capacidad de producción de Cp-PLC y clasificada por actividad lecitinasa: baja (<1 u.a./mL), mediana (1-3 u.a./mL) y alta (>3 u.a./mL) 5 Actividad hemolítica siguiendo lo descrito por Titball et al (1989), expresada en unidades arbitrarias hemolíticas (u.a.h.) por mL y calculada en forma duplicada (A y B) para cada aislado, obteniéndose la media y la desviación estándar de las medias (SD) 6 Actividad perfringolisina, según la metodología descrita por Fisher et al (2006), expresada en unidades arbitrarias hemolíticas (u.a.h.) por mL y calculada en forma duplicada (A y B) para cada aislado, obteniéndose la media y la desviación estándar de las medias (SD) 7 Actividad citotóxica evaluada en línea celular HEp-2.…”

“…Posee gen plc [Cp-PLC]) pero carecen de gen cpb2 (toxina β2) y gen cpe (CPE) 6 Genotipo A β2 +vo . Poseen genes plc [Cp-PLC]) y cpb2 (toxina β2) pero carecen del gen cpe (CPE) la escasa producción de Cp-PLC o la ausencia de hemolisinas tipo Cp-PLC de manera similar a reportes en aislados C. perfringens genotipo C cpb2 +vo (Fisher et al, 2006).…”

“…Se determinó siguiendo la metodología descrita por Fisher et al (2006), para la cual el sobrenadante de cada aislado fue seriadamente diluida (1/2 hasta 1/128; [Log 2 ]) en BBI suplementado con 1 mM EDTA (inhibidor de la Cp-PLC) hasta alcanzar volúmenes finales de 150 ì L y ser distribuidas en una microplaca de 96 pocillos. Se agregó a cada pocillo 150 ì L de solución de eritrocitos de caballo [resuspendidos en BBI suplementado con 1 mM EDTA al 1% (vol/vol)].…”

Clostridium perfringenstype C isolates rapidly upregulate their toxin production upon contact with host cells

“…Several other mammalian cells lines, in addition to Caco-2 cells, were able to induce the quick increase in virulent in rabbit ileal loops. This study 7 further determined that, in the presence of TI, purified β-toxin induces hemorrhagic necrosis in rabbit ileal loops. Collectively, these results indicated that β-toxin is necessary and sufficient for the enteric virulence of type C isolate CN3685 in rabbit ileal loops.…”

“…7 However, since type C disease involves toxin production in the intestines, we recently examined 11 whether contact with human enterocytelike Caco-2 cells might affect the kinetics of toxin secretion or production by type C isolates.…”