Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein

Abstract

Background: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated.

Results: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 μmol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum.

Conclusion: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Figure 1

Analysis of rMsEno expression and purification using SDS-PAGE followed by Coomassie blue staining. M: PageRuler™ Prestained Protein Ladder (SM0671, Fermentas). Lane 1: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-28a (+). Lane 2: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 3: Supernatant of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 4: Sediment of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 5: Purified recombinant protein.

Figure 2

rMsEno enzymatic activity and its influence factors. (A) The enzymatic activity of rMsEno was determined by measuring the conversion of 2-PGA to PEP. (B) The effect of substrate (2-PGA) concentration on the enzymatic activity of rMsEno. (C) Km and Vmax for rMsEno were determined as 1.1 × 10⁻³ M and 0.793 μmol/L/min respectively, based on the Lineweaver–Burk plot (double-reciprocal plot).

Figure 3

Determination of the localization of M. synoviae enolase. (A) Western blot analysis. Lane 1: Total cellular proteins of M. synoviae. Lane 2: Purified rMsEno was used as a positive control. Lane 3: The cytosolic proteins of M. synoviae. Lane 4: The membrane proteins of M. synoviae. Lane 5: BSA was used as a negative control. (B) Immunoelectron microscopy examination. Arrows pointed the M. synoviae enolase to be stained with goat anti-rabbit IgG labeled with 12 nm diametral colloidal gold particles. (C) Non-immunized rabbit serum showed no any binding activity.

Figure 4

Binding assays. (A) Western blot analysis of the binding ability of rMsEno to chicken Plg. Lane 1: rMsEno; Lane 2: BSA. (B) Western blot analysis of the binding ability of rMsEno to human Fn. Lane 1: rMsEno; Lane 2: BSA. (C) The binding ability of rMsEno to chicken plg. (D) The binding ability of rMsEno to human Fn.

Figure 5

Adherence and inhibition assays. (A) DF-1 cells pre-treated with Plg were infected with M. synoviae WVU 1853 strain. (B) The adherence was inhibited by rabbit anti-rMsEno serum. (C) Treatment with non-immune rabbit serum showed no influence on the bacterial adherence. (D) Negative control of uninfected DF-1 cells showed no bacterial attachment when incubated with the goat anti-rabbit IgG (whole molecule)-FITC.