Abstract
Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.
Highlights
Haemophilus (Glaesserella) parasuis, a member of the Pasteurellaceae family, is a common commensal extracellular bacterium that colonizes the upper respiratory tract in swine
The results showed that the purified protein was the H. parasuis Outer membrane protein P2 (OmpP2) protein
The effects of loops L1–L8 of H. parasuis OmpP2 on proinflammatory cytokine expression in porcine alveolar macrophages (PAMs) First, we examined whether OmpP2 and the synthesized peptides corresponding to loops L1–L8 affected the viability of PAMs when stimulated by different concentrations and at various durations
Summary
Haemophilus (Glaesserella) parasuis, a member of the Pasteurellaceae family, is a common commensal extracellular bacterium that colonizes the upper respiratory tract in swine. Virulent strains of H. parasuis can cause Glässer’s disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis [1]. H. parasuis infection causes a significant increase in mortality and morbidity in swine, leading to major economic losses in the pig industry. 15 H. parasuis serovars with apparent differences in virulence. Outer membrane protein P2 (OmpP2), a member of the porin family, is the most abundant protein in the outer membrane of H. parasuis [7]. H. parasuis OmpP2 induces inflammatory responses and upregulates
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