Dual-Function Vaccine for Pseudomonas aeruginosa : Characterization of Chimeric Exotoxin A-Pilin Protein

Abstract

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64⌬553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64⌬553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseudomonas colonization in susceptible individuals.Colonization of cystic fibrosis (CF) individuals with Pseudomonas aeruginosa represents a significant negative milestone in the progression of this disease. Once colonized, patients are subject to the damaging effects of various secreted virulence factors and to the inflammatory response of the host immune system. A key component of colonization is the adhesion of type IV pili to asialo-GM1 receptors on the surface of epithelial cells (26,45,48; for a review, see reference 21). Type IV pili are composed of pilin polymers arranged in a helical structure with five subunits per turn (19,41). The portion of the pilin protein responsible for cell binding is located near the C terminus (amino acids 129 to 142) in a ␤-turn-␤-turn loop subtended from a disulfide bond (5,6,23,36). A 12-or 17-aminoacid sequence (depending on the specific strain) in this loop interacts with receptors on epithelial cells. For CF individuals, the overproduction of the R domain of the mutant CF transmembrane conductance regulator can lead to an increased level of asialo-GM1 and, accordingly, an increased binding of P. aeruginosa (3,26,45). Functional studies of pilin have indicated that only the last pilin subunit (the tip) of a pilus interacts with epithelial cell receptors (31). To interfere with bacterial adhesion, anti-pilin antibodies will need to recognize residues that are normally located at the C-terminal loop of pilin (32). Structural studies have indicated that this loop is dominated by main chain residues; and this may explain why pilins from distinct strains bind the same receptor despite sequence variation and the presence of both 12-and 17-aminoacid loops. Generating antibodies to the C-terminal pilin loop may be useful in reducing or eliminating colonization (15,22,47). Table 1 lists the pilin loop sequences from several strains of P. aeruginosa.Pseudomonas exotoxin A (here called PE), a prominent virulence factor secreted by P. aeruginosa, is cytotoxic for mammalian cells by virtue of its ability to enter cel...

Genetic Characterization Indicates that a Specific Subpopulation of Pseudomonas aeruginosa Is Associated with Keratitis Infections

“…The predicted type IV pilin (encoded by pilA) of the keratitis isolate 39016 shares 99 to 100% amino acid identity with 3 of the 44 PilA sequences in the database. Two are represented by accession numbers AAK68044 and AK68042 and are from strains 82932 and 82935, which are clinical isolates of unknown source (20). There was also a third match to strain PA5235 (rectal swab; AAM44066) (29).…”

Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients

“…The importance of TFP in the biology and virulence of P. aeruginosa has made them attractive targets for vaccine development (Hertle et al, 2001;Sheth et al, 1995). Saiman et al (1989) raised cross-reactive monoclonal antibodies that recognized some pilin variants and reduced binding to eukaryotic cells.…”

Intranasal Immunization Strategy To Impede Pilin-Mediated Binding ofPseudomonas aeruginosato Airway Epithelial Cells

“…Neutralizing antibodies to PE have been shown to not only promote uptake and killing of P. aeruginosa by neutrophils (11) but also be beneficial in clearing P. aeruginosa infections (18,59). Antibodies to PE can also reduce the binding of both piliated and nonpiliated P. aeruginosa to tracheal cells in vitro (42), and several studies have described strategies to generate neutralizing immune responses to PE as a method of reducing the clinical impact of this virulence factor (8,20,25,50).…”