Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5

Abstract

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A. A. Potter, and P. J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate. Actinobacillus pleuropneumoniae is a highly infectious porcine respiratory tract pathogen (22). The disease caused by this bacterium is encountered worldwide and frequently causes severe losses in infected herds (21). Infected animals develop disease symptoms ranging from acute fibrinous pneumonia and pleuritis with high mortality to chronic lung lesions resulting in reduced growth rates (22). Pigs which survive an infection develop a protective immune response but can still be carriers of the pathogen (22). The rapid onset and severity of disease mean that losses may occur before antibiotic therapy has time to become effective. Attempts to immunize against A. pleuropneumoniae disease have been hampered by the occurrence of two biotypes and more than 14 serotypes (2, 6, 10-14, 16) and the lack of reliable cross-protection (9). It has been hypothesized that a cross-protective immune response might be achievable by immunization with a mixture of serotype-specific antigens that are recognized by convalescent sera (3). Recently, we were able to show that two recombinant proteins could protect pigs from death in an A. pleuropneumoniae serotype 7 challenge (17). One of these antigens was a truncated RTX toxin, and the other was an iron-regulated transferrin-binding protein. However, no cross-protection against A. pleuropneumoniae serotype 1 infection was observed (17). In order to achieve protection against A. pleuropneumoniae serotype 1 (3), we were successful in cloning and expressing a protective outer membrane lipoprotein (OmlA) of A. pleuropneumoniae serotype 1. In an A. pleuropneumoniae serotype 1 challenge experiment, all animals immunized with the recombinant OmlA were protected from death (3). However, antiserum raised against the transferrin-binding proteins of A. pleuropneumoniae serotypes

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

“…The antiserum also reacted very weakly with 43 kDa proteins of serotypes 5a, 5b, and 10 ( Fig. 3) although the antiserum against OmlA S has not reacted with OmlA U [9,10].…”

“…The ¢rst 134 amino acids of OmlA U were identical to those of OmlA S . However, the proteins have been reported to be antigeni-cally distinct, based on the fact that antiserum against OmlA S did not react with OmlA U in Western blot analyses [9,10]. These data indicate that the major epitopes a¡ecting antigenic variations are located in the amino acids downstream of the positions at 135 of OmlA S and OmlA U .…”

“…These data indicate that the major epitopes a¡ecting antigenic variations are located in the amino acids downstream of the positions at 135 of OmlA S and OmlA U . Previous studies on the omlA genes of serotypes 1 and 5 using the cloned genes as a probe in a Southern blot analysis have shown that the genes highly homologous to the omlA I were present in serotypes 1, 2, 8, 9, 11, and 12 [8], and that the genes homologous to the omlA S were present in serotypes 5a, 5b, and 10 [9,10]. In the present study, a 0.7-kb fragment derived from the cloned omlA U gene was used as a probe in a Southern blot.…”

Genetic and Immunologic Analyses of PlpE, a Lipoprotein Important in Complement-Mediated Killing ofPasteurella haemolyticaSerotype 1

“…A search of GenBank sequences and subsequent sequence alignments revealed that the deduced amino acid sequence of PlpE has 18% identity and 32% similarity to an outer membrane lipoprotein (OmlA) produced by Actinobacillus pleuro- pneumoniae serotype 1 and 20% identity and 35% similarity to the OmlA protein from A. pleuropneumoniae serotype 5 (data not shown). Although both of those proteins lack the hexapeptide repeat mentioned above, they contain regularly spaced PK and PQ repeats in the same region (4,17,23).…”

Infección por Actinobacillus pleuropneumoniae en un modelo murino = Actinobacillus pleuropneumoniae infection in a murine model

“…La inmunización de cerdos con estas proteínas confiere cierta protección frente a la infección (Chiang et al, 1991;Rossi-Campos et al, 1992;Gerlach et al, 1993;Bunka et al, 1995).…”