Effect of formalin toxoiding on Pseudomonas aeruginosa toxin A: biological, chemical, and immunochemical studies

Abstract

We investigated the effect of Formalin toxoiding on the biological, chemical, and immunological activities of Pseudomonas aeruginosa toxin A. Formalin treatment alone resulted in a 1,000-fold decrease in toxin-induced cell cytotoxicity and altered the antigenicity of the toxin A molecule without adversely affecting enzymatic activity. Competitive blocking experiments indicated that Formalinmediated detoxification proceeded via alterations in a region of the toxin molecule other than the active site of the enzyme. The addition of lysine to Formalin-toxin mixtures not only increased the rate and extent of detoxification and antigenic alteration, but also completely destroyed enzymatic activity. The immunogenicities of different toxoids varied substantially. Upon dialysis and storage, Formalinderived toxoids underwent partial toxic reversion, whereas a Formalin-lysinederived toxoid did not. The sodium dodecyl sulfate-polyacrylamide gel patterns of Formalin-and Formalin-lysine-treated toxins indicated that these treatments caused the formation of a heterogenous group of high-molecular-weight species and produced substantial changes in the electrophoretic mobility of toxin A.

Protection against Pseudomonas aeruginosa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, antielastase, and antilipopolysaccharide

“…DISCUSSION In the present study, we used a murine bum wound sepsis model to evaluate the ability of passively transferred AT-IgG, AE-IgG, and ALPS-IgG to protect against fatal P. aeruginosa infection. Passive, rather than active immunization, was selected for comparing the efficacy of these antibodies due to the following: (i) differences in the immunogenicity of these three antigens (native elastase and stable toxin A toxoids) are poor immunogens in mice unless administered with potent adjuvants, whereas LPS is a good immunogen (8,32,37); (ii) adjuvants, such as muramyl dipeptide, have been shown to evoke nonspecific immunity to P. aeruginosa (10); and (iii) passive immunity has been shown to be an effective means of conferring protection against P. aeruginosa (9,33,46). We were unable to demonstrate an appreciable degree of protection against lethal infections by two virulent strains of P. aeruginosa by the transfer of AT-IgG or AE-IgG.…”

“…soy broth dialysate (TSBD) medium described by Bjorn et al (3). P. aeruginosa PA220, M2, SBI-C, and SBI-N produced elastase, as detected by elastin degradation (31), and toxin A, determined in an adenosinediphosphate ribosyl transferase assay (8). The challenge inoculum for the burn wound sepsis model was prepared by incubating 10 ml of TSBD medium with 0.1 ml of an overnight culture.…”

Immunization of mice against various strains of Pseudomonas aeruginosa by using Pseudomonas lectin vaccine

“…used as potential P. aeruginosa vaccines. These include LPS [4], endotoxin protein [5], exotoxin [6] 'slime' polysaccharides [7] 'slime' glycolipoprotein [8], cell wall extracts [9], membrane proteins [10], proteoids (protease-elastase) [11], flagella [12], pill [13] and ribosomal [3] as well as other preparations [1][2][3]. Most of these preparations were found to significantly reduce the incidence of animal mortality due to Pseudomonas infection, some of them without and others with visible deleterious side-effects [1][2][3] as well as varying degrees of success in coping with the variability of the serotypes involved in the Pseudomonas infections [1-31.…”

Immunological cross-reactivity in the absence of DNA homology between Pseudomonas toxin A and diphtheria toxin

“…Enzyme assay and neutralization by antibody. Enzyme neutralization assays were performed by incubating toxin A (100 ng; treated with urea and DTT) or fragment A of diphtheria toxin in 10 ,ul of 5% normal rabbit serum with an equal volume of immune serum for 10 min at 37°C as previously described (9,13). The ADP-ribose transferase activities of toxin A and fragment A of diphtheria toxin were then measured as described previously (13,15).…”