Glaesserella parasuis autotransporters EspP1 and EspP2 are novel IgA-specific proteases

Abstract

Background: Glaesserella parasuis causes Glässer's disease, which is associated with severe polyarthritis, fibrinous polyserositis and meningitis, and leads to significant economic losses to the swine industry worldwide. IgA is one of the most important humoral immune factors present on mucosal surfaces, and it plays a crucial role in neutralizing and removing pathogens. G. parasuis is able to colonize the mucosal membrane of respiratory tract without being eliminated. Nevertheless, the immune evasion mechanism of G. parasuis in thwarting IgA remains unclear.

Aims: The object of this study is to characterize the IgA degradation activity of Mac-1-containing autotransporter EspP1 and EspP2 from G. parasuis.

Methods: The swine IgA was purified and incubated with EspP1 and EspP2 respectively. Western blotting was used to detect the cleavage of swine IgA. Generation of EspP1 and EspP2 mutant protein were used to explore the putative active sites of EspPs. LC-MS/MS based N/C-terminal sequencing was performed to measure the cleavage sites in swine IgA.

Result: Our results show that G. parasuis EspP1 and EspP2 cleave swine IgA in a dose- and time- dependent manner. G. parasuis lose the IgA protease activity after simultaneously delete espP1 and espP2 indicating that EspP1 and EspP2 are the only two IgA proteases in G. parasuis. The IgA protease activity of EspP1 and EspP2 is affected by the putative active sites which contain Cys47, His172 and Asp194/195. Swine IgA is cleaved within Cα1 and Cα3 domains upon incubation with EspPs. Moreover, EspPs can degrade neither IgG nor IgM while G. parasuis possess the ability to degrade IgM unexpectedly. It suggests that G. parasuis can secrete other proteases to cleave IgM which have never been reported.

Conclusion: We report for the first time that both EspP1 and EspP2 are novel IgA-specific proteases and cleave swine IgA within the Cα1 and Cα3 domains. These findings provide a theoretical basis for the EspPs-induced immune evasion.

Keywords: EspP1; EspP2; Glaesserella parasuis; IgA-protease; immune evasion.

Figure 1

Glaesserella parasuis exhibits the ability to cleave swine IgA. (A) Culture supernatants of 2% heat-inactivated swine serum alone (−) and G. parasuis strain CF7066 cultured in the presence of 2% heat-inactivated swine serum to stationary phase were analysed by Western blot. IgA was detected with HRP conjugated Goat anti-Pig IgA antibody. The cleavage products are indicated with asterisk (*). SDS-PAGE (B) and Western blot (C) were used to assess the purification of swine IgA. (D) Culture supernatants and lysates were incubated with purified IgA at 37°C for different periods of time and analysed by Western blot.*p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA.

Figure 2

Glaesserella parasuis EspP1- and EspP2-mediated cleavage of swine IgA heavy chain. Cleavage of purified swine IgA by recombinant EspP1 and EspP2 was detected by SDS-PAGE (A) and Western blot (B,C). The cleavage products are indicated with asterisk (*). And the IgA was cleaved in a time- (B) and dose- (C) dependent manner. (D) Lysates and culture supernatants of CF 7066 wild type (WT) and EspP1 and EspP2 defivient strain (AespP1, AespP2, and AespP1AespP2) were incubated with purified swine IgA, and the degradation of IgA was detected by Western blot. *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA.

Figure 3

Protease activity of EspP1 and EspP2 are affected by the putative active sites which consisted of cysteine, histidine and aspartate. (A) The multiple-sequence alignment of G. parasuis EspP1 (aa 1-250) and EspP2 (aa 1-249) amino acids with the published sequence of S. pyogenes IdeS (aa 1-341), S. suis IdeS (aa 1-454) and S. equi IdeE (aa 1-349). The putative active sites were marked with yellow stars, and the D144 was acted as control, which was marked with a green star. (B) Schematic of EspP1 and EspP2. Positions of C47, H172, D194/D195, and D144 were highlighted and these amino acid residues were mutated to alanine separately. Protease activity of EspP1 (C) and EspP2 (D) disappeared when Cys-47, His-172, or Asp-194/Asp-195 were mutated to alanine (Ala). And the degradation activity of EspP1^D144A and EpspP2^D144A were not affected by the mutation.

Figure 4

Swine lgA is cleaved within the Cd1 and Cu3 domain. (A,B) EspP1 and EspP2 have three different cleavage sites in swine IgA, which led to two different cleavage products with the molecular weight of ~33 and ~27 kDa under reducing condition. The cleavage sites are indicated with scissors.

Figure 5

Glaesserella parasuis EspP1 and EspP2 are IgA-specific protease. (A,B) CF7066 and its derivatives EspPI and EspP2 deficient strains were incubated in the presence of 2% heat-inactiveted swine serum to stationary phase and detected by Western blot. The cleavage product is indicated with asterisk (*). (C,D) Recombinant EspP1 and EspP2 were incubated with 2% swine serum overnight at 37°C, and then subjected to Western blot.