The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice

Abstract

We expressed three Actinobacillus pleuropneumoniae ApxI deletion derivatives to map the domain that could induce protective immunity. Antiserum to ApxI N-terminal covered by residues 40 to 380 was found to neutralize ApxI hemolytic activity but not ApxIII cytotoxicity. When used as a subunit vaccine in mice, this recombinant N-terminal fragment elicited protection against lethal infection with heterologous A. pleuropneumoniae serovars.

FIG. 1.

(A) Structural organization of ApxI and the relative positions of each truncated ApxI fragment. Fragment X1F1 covered the N-terminal and putative transmembrane domains. Fragment X1F2 covered the gene C-mediated activation domain, and fragment X1F3 covered the calcium-binding domain of the glycine-rich repeats. (B and C) Expression and immunoblotting of ApxI deletion derivatives. Panel B shows Coomassie blue staining of partially purified ApxI recombinant proteins indicated by filled arrows. Lanes: 1, X1F1; 2, X1F2; 3, X1F3. Panel C shows that A. pleuropneumoniae-infected swine serum was reactive to all recombinant proteins. Molecular mass markers are shown on the left (in kilodaltons).

FIG. 2.

Neutralization of the ApxI hemolytic activity. Hemolytic neutralization assays were performed with native ApxI toxin obtained from A. pleuropneumoniae 3906 preincubated with guinea pig preimmune serum (▪), X1F1 antiserum (⧫), X1F2 antiserum (▵), X1F3 antiserum (▴), and field serum (□). The results show the arithmetic means of the hemolytic neutralization findings from three experiments performed in duplicate ± the standard deviations.

FIG. 3.

Neutrophil protection assay with anti-ApxI antisera. Cytotoxic neutralization assays were performed with native ApxIII toxin obtained from A. pleuropneumoniae serotype 8 preincubated with guinea pig preimmune serum (▪), X1F1 antiserum (⧫), X1F2 antiserum (▵), X1F3 antiserum (▴), and field serum from A. pleuropneumoniae serotype 8-infected pigs (□). The results are the arithmetic means of the cytotoxic neutralization findings from three experiments performed in duplicate ± the standard deviations.