Protective Properties of Nontoxic Recombinant Exotoxin A (Domain I-II) Against Pseudomonas aeruginosa Infection

Abstract

Background: Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. Objectives: In this study, recombinant exotoxin A (domains I and II), (ExoA I-II) protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. Materials and Methods: The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A (Domains I, II). Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 (7.5 x 10(7) CFU) of clinical strain of mucoid P. aeruginosa. Results: Sequencing of the cloned gene showed that the sequence of ExoA I-II gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA I-II increased survival times against intra-peritoneal challenge with an approximate 7.5 x 107 CFU (2 x LD50) of clinical strain of P. aeruginosa. Conclusions: Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa.