RESUMEN
We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.
Asunto(s)
Bacillus anthracis/crecimiento & desarrollo , Mediciones Luminiscentes , Esporas Bacterianas/aislamiento & purificación , Animales , Recuento de Colonia Microbiana , Femenino , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/microbiología , Macrófagos Alveolares/microbiología , Ratones , Neutrófilos/microbiología , Factores de Tiempo , Imagen de Cuerpo EnteroRESUMEN
Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the DeltabclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of DeltabclA and wild-type spores. The DeltabclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.
Asunto(s)
Bacillus anthracis/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Esporas/química , Esporas/crecimiento & desarrollo , Animales , Bacillus anthracis/patogenicidad , Eliminación de Gen , Dosificación Letal Mediana , Ratones , Conejos , Análisis de Supervivencia , VirulenciaRESUMEN
Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Inmunización Secundaria , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/uso terapéutico , Colágeno/inmunología , Reacciones Cruzadas , Femenino , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Ratones , Análisis de Supervivencia , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Ferrets were evaluated as a possible small animal model for the development of colitis and/or signs of the hemolytic uremic syndrome after oral infection with Escherichia coli O157:H7 or other Shiga toxin--producing E. coli (STEC). Ferrets treated with streptomycin (Stm) had higher counts of E. coli O157:H7 strain 86-24 Stm-resistant (Stm(r)) or O91:H21 strain B2F1 Stm(r) in their stools than non--Stm-treated animals. None of the animals displayed evidence of colitis, but Stm-treated animals fed strain 86-24 Stm(r) exhibited weight loss significantly greater than that exhibited by ferrets fed an isogenic mutant negative for the adhesin intimin. Moreover, 11 (23%) of the 47 Stm-treated ferrets inoculated with 86-24 Stm(r) or B2F1 Stm(r) developed hematuria and/or histological damage to glomeruli or thrombocytopenia, compared with 0 of 14 uninfected control animals receiving Stm in water. Thus, the ferret may serve as a model for renal disease secondary to intestinal infection with STEC.