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

Abstract

Pasteurella haemolytica serotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies against P. haemolytica outer membrane proteins (OMPs) correlates statistically with resistance to experimental P. haemolytica challenge in cattle. Until now, specific P. haemolytica OMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to an Actinobacillus pleuropneumoniae lipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes of P. haemolytica except serotype 11. We found that intact P. haemolytica and recombinant E. coli expressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed in P. haemolytica and assumes a similar surface-exposed conformation in E. coli. In complement-mediated killing assays, we observed a significant reduction in killing of P. haemolytica when bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense against P. haemolytica.on July 4, 2020 by guest http://iai.asm.org/ Downloaded from bution of antibodies against this protein to complement-mediated killing of P. haemolytica. We found that the 45-kDa protein is a lipoprotein, designated PlpE, and that antibodies against PlpE, present in bovine immune sera, contribute to complement-mediated killing of P. haemolytica. MATERIALS AND METHODSBacteria, bacteriophage, culture media, and genomic library. P. haemolytica (89010807N) S1 was grown in BHI broth or on BHI agar (Difco Laboratories, Detroit, Mich.) as previously described (32). Escherichia coli BB4 and XL1-Blue and bacteriophages ZAPII and R408 were supplied with a P. haemolytica genomic DNA library (Clontech Laboratories, Palo Alto, Calif.) (37) and were grown according to the manufacturer's instructions. Recombinant E. coli strains were grown in the presence of ampicillin (50 g/ml).Bovine immune sera and purification of antibodies. Two bovine immune sera were used, one from a calf hyperimmunized with live P. haemolytica (25) and one from a calf that was vaccinated with P. haemolytica OMPs and was resistant to experimental P. haemolytica challenge (7). Briefly, the OMP-vaccinated calf was vaccinated subcutaneously on day 0 and day 21 with P. haemolytica S1 OMPs (2 mg in 1 ml of phosphate buffered saline [PBS] and 1 ml of an aluminum hydroxide-DDA-bromide adjuvant which has been described elsewhere in more detail [8]). On day 36, the calf was experimentally challenged transthoracically with 5 ml of a mixture containing 10 9 CFU of P. haemolytica S1/ml in each caudal lung lobe. Lung damage was evaluated upon necropsy 4 days after challenge, by using a previous...

Mannheimia haemolyticaand bovine respiratory disease

“…A surface-exposed 45-kDa OMP, designated PlpE, was sequenced and cloned by Pandher et al (1999) . Mosier et al (1989) reported this protein to be immunogenic in cattle and Pandher et al (1998) found that antibodies to PlpE were associated with complement-mediated killing of M. haemolytica . A recombinant PlpE was highly immunogenic when injected subcutaneously in vaccination studies (Confer et al , 2003).…”

Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica

“…Outer membrane proteins and lipoproteins of M. haemolytica may be involved in serum sensitivity (111,112) and they are believed to be important protective antigens. Antibodies directed against some of these antigens are capable of inducing phagocytosis and complement-mediated killing (112). As a result, these surface-exposed proteins are of interest as potential vaccine candidates.…”

“…Though the M. haemolytica pomA gene has been cloned, attempts to inactivate it have been unsuccessful (G. Murphy, personal communication), suggesting that PomA may be involved in transport of nutrients or that it may be critical to the integrity of the outer membrane. M. haemolytica also produces at least five distinct lipoproteins with apparent molecular weights that range from 19 to 45 kD (112,(114)(115)(116). A mutant lacking three of these lipoproteins (PlpA, PlpB and PlpD) was created by allelic exchange using the ROB-1 beta-lactamase gene from M. haemolytica (117).…”

Molecular Cloning of thePasteurella haemolytica pomAGene and Identification of Bovine Antibodies against PomA Surface Domains

“…Therefore, our work has focused on identifying and characterizing P. haemolytica OMPs with surface domains that are targets of antibodies present in sera from immune cattle (30). We found that the 45-kDa lipoprotein (PlpE) elicits bovine antibodies that effect complement-medi-ated killing of P. haemolytica (28). Our previous studies with PomA revealed that it is recognized by antibodies from cattle immune to P. haemolytica challenge and that it possesses surface-exposed regions (21).…”

“…The sera were from cattle that were resistant to experimental P. haemolytica challenge after natural exposure to the bacterium or after vaccination with live P. haemolytica or with P. haemolytica OMPs (30). Sera were absorbed with intact P. haemolytica cells as previously described (28). Absorbed and unabsorbed sera, diluted 1:100 in Tris-saline-nonfat dry milk (TSM) (10mM Tris [pH 7.4], 0.9% [wt/vol] NaCl, 1% nonfat dry milk), were used to probe Western immunoblots of purified rPomA and P. haemolytica whole-cell lysates (Fig.…”