Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4

Abstract

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL–1β, IL–6, and TNF–α using qPCR and ELISA. Blocking signal-regulated JNK–MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK–MAPK and RelA(p65) pathways.

Deletion of Polyamine Transport Protein PotD Exacerbates Virulence in Glaesserella (Haemophilus) parasuis in the Form of Non-biofilm-generated Bacteria in a Murine Acute Infection Model

“…In previous studies, we and others have found that PotD is a stress-responsive protein and one of the differentially expressed virulence-related proteins in G. parasuis [Han et al, 2007;Zhang et al, 2014a]. We also found that recombinant PotD protein in G. parasuis could trigger an effectively immunoprotective effect against severe pathological lesions in a mouse model by triggering both cellular and humoral immunity [Dai et al, 2019b]. However, little evidence of the exact biological function of PotD, especially whether it is related to bacterial virulence in vivo, was unveiled in G. parasuis.…”

“…The anti-rPotD antisera were prepared as previously described [Dai et al, 2019b]. Each mouse was immunized subcutaneously on days 0, 14, and 21 with 0.1 mg of rPotD (0.2 ml) containing a one-tenth volume of water adjuvant MONTANIDE™ GEL 01 (SEPPIC, Paris, France) [Xu et al, 2019].…”

“…Overnight-grown suspended bacterial culture was spun down and pellets were resuspended in electron microscope fixing solution (2.5% glutaraldehyde; Goodbio technology CO. LTD, Wuhan, China) for 2 h in the dark. Ultrathin sections were prepared as previously described [Dai et al, 2019b], the samples were washed, blocked, and incubated with primary antibody (mice-sourced PotD antisera). Secondary antibody (Sigma), which was chelated with colloidal gold particles, was added, and the sections were incubated for 1 h at 37°C.…”

“…For suspended bacterium culture, samples were spun down; pellets added to the electron microscope fixing solution and fixed as above. After sections were prepared as previously described [Dai et al, 2019b], the samples were visualized using SEM (HITACHI, SU8100, Japan) and TEM (HITACHI, HT7700, Japan), respectively.…”

The effects of baicalin on piglets challenged with Glaesserella parasuis

“…The expression levels of IL-10 and TNF-α in the spleen were upregulated after colostrum-deprived pigs were infected with G. parasuis [ 35 ]. Stimulation of Raw 264.7 macrophages with G. parasuis PotD could induce the production of IL1β, IL6, and TNF-α [ 36 ]. Therefore, these cytokines play important roles in the inflammatory damage caused by G. parasuis infection.…”