Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE

Abstract

Background: There is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens.

Principal findings: A high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested, only one, PlpE in denatured insoluble form, protected chickens against fowl cholera challenge. PlpE also elicited comparable levels of protection in mice. PlpE was localized by immunofluorescence to the bacterial cell surface, consistent with its ability to elicit a protective immune response. To explore the role of PlpE during infection and immunity, a plpE mutant was generated. The plpE mutant strain retained full virulence for mice.

Conclusion: These studies show that PlpE is a surface exposed protein and was the only protein of 71 tested that was able to elicit a protective immune response. However, PlpE is not an essential virulence factor. This is the first report of a denatured recombinant protein stimulating protection against fowl cholera.

Figure 1. Western immunoblot analysis of PlpE expression in P. multocida whole cell lysates probed with chicken antiserum against recombinant PlpE.

Lanes: wild type strain (lane 1); plpE mutant (lane 2); complemented mutant (lane 3); mutant transformed with empty vector (lane 4). Numbers on the left indicate the positions of molecular mass standards (in kDa). Arrow indicates the position of the 39 kDa PlpE. Pre-bleed serum showed no reactivity (data not shown).

Figure 2. Surface localisation of P. multocida PlpE by immunofluorescence assay.

Bacteria were fixed with paraformaldehyde, incubated with chicken antiserum against PlpE, stained with Alexa Fluor 488 goat anti-chicken IgG, and visualized by fluorescence microscopy. Fluorescence (panels: a, c, e) and DIC (Differential Interference Contrast) (panels: b, d, f) selected images of the bacterial Z-stack cross-sections. Control staining with antiserum raised against an unrelated protein showed no surface fluorescence (data not shown). Scale bar  = 1 µm.