Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model.
The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10(6)CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100microg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.
Department of Medical Microbiology and Immunology, University of Göteborg, S-413 46, Göteborg, Sweden.
Catharina
C
Wising
Liselott A
LA
Svensson
Hinda J
HJ
Ahmed
Vivianne
V
Sundaeus
Karin
K
Ahlman
Ing-Marie
IM
Jonsson
Lena
L
Mölne
Teresa
T
Lagergård
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000633:toxicity*; D002460:Cell Line; D002602:Chancroid / Q000382:microbiology / Q000503:physiopathology / Q000517:prevention & control*; D003601:Cytotoxicity Tests, Immunologic; D004195:Disease Models, Animal; D018073:Haemophilus Vaccines / Q000008:administration & dosage / Q000276:immunology; D006191:Haemophilus ducreyi / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011817:Rabbits; D012867:Skin / Q000473:pathology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; C468508:CdtA protein, Haemophilus ducreyi; C468509:CdtC protein, Haemophilus ducreyi; D018073:Haemophilus Vaccines; C067044:cytolethal distending toxin