Adhesion protein ApfA of Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development

Abstract

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes of A. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation of apfA dramatically reduced the ability of A. pleuropneumoniae to colonize mouse lung, suggesting that apfA is a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges with A. pleuropneumoniae serovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection with A. pleuropneumoniae.

Fig 1

The expression of type IV fimbriae is induced upon contact with host cells, and ApfA mediates the adhesion of A. pleuropneumoniae to host cells. (A) The relative mRNA levels of the apfA, apfB, and apfC genes were upregulated after A. pleuropneumoniae 4074 was cocultured with SJPL cells (SJPLC), compared to those of bacteria without cell coculture. The mRNA level was determined by qRT-PCR. (B) Adhesion capabilities of wt strain 4074, mutant strain 4074^ΔapfA, and complementary strain C4074^ΔapfA to SJPL and PIEC cells. (C) Blockage of A. pleuropneumoniae adhesion to SJPL and PIEC cells by rApfA. SJPL and PIEC cells were pretreated with rApfA (rApfA-treated) or BSA (Control) before being subjected to contact with bacteria. The data shown are means ± SD of data determined with triplicate samples of one experiment representative of three independent experiments. The asterisks show significant differences (*, P < 0.05; **, P < 0.01). Confocal microscopy showed the adhesion of wt strain 4074 (D), mutant strain 4074^ΔapfA (E), and complementary strain C4074^ΔapfA (F) to SJPL cells. A. pleuropneumoniae cells were labeled with CFDA (green fluorescence). SJPL cell nuclei were stained with DAPI (blue fluorescence) (scale bars = 50 μm).

Fig 2

ApfA mediates the colonization of lung by A. pleuropneumoniae. The bacterial burdens in lungs of mice infected with wt strain 4074 or mutant strain 4074^ΔapfA were evaluated at 24 h postinfection. The data shown are representative of the results of one of three independent experiments. The asterisks show significant differences (**, P < 0.01).

Fig 3

Naturally infected piglets show high anti-ApfA immune responses. (A) SDS-PAGE analysis of rApfA purified from E. coli cells. Left lane, protein marker; right lane, protein rApfA (2 μg). (B) Piglets infected with A. pleuropneumoniae show an elevated anti-ApfA immune response. The convalescent-phase sera are serum samples from pigs infected with A. pleuropneumoniae, and the negative sera are samples from healthy pigs. The anti-rApfA IgG level was determined using ELISA. The asterisks show significant differences (**, P < 0.01).

Fig 4

Immunization with rApfA protein protects mice against lethal challenges with strain 4074 (serovar 1) and strain WF83 (serovar 7). (A) A high level of specific anti-rApfA IgG antibody was induced in mice by immunization with rApfA. (B and C) Survival of mice immunized with rApfA, bacterin (positive control), and adjuvant (negative control) following i.p. infection with (B) strain 4074 (serovar 1) and (C) strain WF83 (serovar 7). The data shown are representative of the results of one of three independent experiments; the survival data were analyzed using the log rank (Mantel-Cox) test. The asterisks show significant differences (**, P < 0.01).

Fig 5

Serum from mice immunized with rApfA afforded passive protection against lethal challenges with strain 4074 (serovar 1) and strain WF83 (serovar 7). Serum against rApfA or bacterin significantly increased the survival rates after a challenge with (A) strain 4074 (serovar 1) or (B) strain WF83 (serovar 7), while control mice immunized with serum from adjuvant-immunized mice all succumbed to infection. The data shown shown are representative of the results of one of three independent experiments; the survival data were analyzed using the log rank (Mantel-Cox) test. The asterisks show significant differences (*, P < 0.05; **, P < 0.01).