FIG 1.

Identification of Glaesserella parasuis sialidase (NanH)-deficient mutant. (A) Schematic diagram showing wild-type (WT) G. parasuis, ΔnanH::kan mutant, and ΔnanH::kan+pNanH complementary strain. G. parasuis was identified using the primers 16SrRNA-F/R (822 bp). The primers P7/P8 were used to amplify nanH in the WT and the fragment including ΔnanH::kan in the ΔnanH::kan and ΔnanH::kan+pNanH strains (2,462 bp). The primers P5/P6 were used to amplify the kanamycin cassette in the ΔnanH::kan and ΔnanH::kan+pNanH strains (909 bp). The primers P13/P14 were used to amplify the inner fragment of nanH in WT and ΔnanH::kan+pNanH strains (594 bp). ORF, open reading frame. (B) PCR amplification without primers served as a PCR negative control. M, molecular size marker. (C) NanH consisted of a signal peptide (amino acids [aa] 1 to 29), transmembrane domain (aa 13 to 32), and functional domain (aa 33 to 801) according to the Signal P4.1 server. (D) Recombinant sialidase (rNanH) was expressed by plasmid pET-nanH after induction with isopropyl-β-d-1-thiogalactopyranoside. The vector pET-25b and noninduced plasmid pET-nanH did not express rNanH. rNanH was primarily included in the pellets but not in supernatants of the sonicated bacterial cells. The purified rNanH was obtained using the His tag and Ni Sepharose 6 Fast Flow. (E) Western blots using the mouse anti-sialidase antibodies revealed that WT and ΔnanH::kan+pNanH strains express NanH but the ΔnanH::kan strain does not. (E) Equivalent quantity of proteins from sonicated bacterial cells was used for sialidase activity detection. (F) The WT had the highest sialidase activity (P < 0.001), and the ΔnanH::kan+pNanH strain exhibited restoration of the activity to a level higher than the ΔnanH::kan strain (P < 0.01). (G) The sialidase activity of rNanH was dose dependent. For G. parasuis and rNanH, sialidase activity was detected in three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.