Functional Characterization Of Porcine Coronin1A During Haemophilus Parasuis Infection

Haemophilus parasuis (H.parasuis), the causative agent of Glasser’s disease, has generated a worldwide threaten to the swine herds, and leads to a huge economic loss to the pig industry, and the disease is characterized by fibrinous polyserositis, meningitis and polyarthritis. As we all know, H.parasuis has to survive host pulmonary defences in order to produce systemic disease, and the first line of defence is composed of porcine alveolar macrophages (PAM), whose main role is the elimintion of airborne pathogens and other environmental particles. Interestingly, high virulent strains of H.parasuis could prevent phagocytosis, whereas low virulent strains of H.parasuis are susceptible in phagocytosis. A recent study demonstrated that H.parasuis infection in PK-15cells could induce the NF-κB activation via IκB degradation and the phosphorylation of p65, which allows NF-κB to stimulate expression of target genes associated with variuos inflammations. However, the pathogenesis of H.parasuis infection is still unknown.Coronin1A, a member of mammalian coronin family, also known as Tryptophan Aspartate containing coat protein (TACO) or p57, is mainly expressed in hematopoietic cells and equally distributes in the cytosol. It has been demonstrated that Coronin1A plays a crucial role in T lymphocyte activation, migration, survival, and calcium signal transduction. Previous study has clarified the association between human coronin1A and the intracellular survival of pathogenic mycobacterium, suggesting that Coronin1A specially participate in modulating phagosome-lysosome fusion upon mycobacterial infection. In addition, human Coronin1A is mentioned as a novel inhibitor of TLR-mediated NF-κB activation in Mycobacterium leprae infection. Hence, Coronin1A is a crucial protein that plays important roles in immune response and phagocytosis against bacterial infection.In our previous study, we firstly identified the porcine Coronin1A in Haemophilus parasuis(H.parasuis) infected porcine alveolar macrophages (PAMs), and the relative expression of Coronin1A was up-regulated in PAM following H.parasuis infection at the transcriptional level. To clarify the molecular mechanisms of porcine Coronin1A during H.parasuis infection, we studied the interactions between porcine Coronin1A and formation of phagolysosome as well as the NF-κB signal pathway in innate immune responses during H.parasuis infection. The main research works were as following: 1. Porcine Coronin1A may be involved in preventing the formation of phagolysosome during H.parasuis infection.Using immunohistochemical and STRING analysis, we demonstrated that the expression of Coronin1A was up-regulated in the lymph node, spleen and lung of pigs infected with H.parasuis, implying that porcine Coronin1A was probably involved in the infection process of H.parasuis. Besides,7DE genes related with Coronin1A were identified as follows:Slc9a3r1、Ltb、Il-1β、Fcgr2β、Ly86、Cd14、Cc14, and all of which were up-regulated. The related DE genes may contribute to the phagocytosis against H.parasuis or immune response during H.parasuis infection. Furthermore, the Subcellular localization showed that porcine Coronin1A was mainly distributed in the cytosol, partially overlapped with tubulin, which is involved in formation of phagolysosome. Above all, the localization of Coronin1A in PAM infected with H.parasuis identified that Coronin1A is actively retained on the phagosomal membrane containing live bacteria. In contrast, during phagocytosis of heat-killed bacteria, Coronin1A initially associates with phagosomes but is rapidly released concomitant with lysosomal delivery of the H.parasuis.2. Porcine Coronin1A contributes to inhibit NF-κB activation during Haemophilus parasuis infectionUsing quantitative RT-PCR and stimulation with lipopolysaccharide (LPS) and polyinosinic acid-polycytidylic acid [Poly (I:C)], we investigated that porcine coronin1A was mainly expressed in cells of lymphoid/myeloid lineage, and the immunostimulation assay indicated that both LPS and Poly (I:C) can induce the expression of porcine coronin1A in vitro. In addition, we identified porcine coronin1A could suppress NF-κB activation and investigated the potential mechanism by which it acts, revealing that porcine Coronin1A significantly suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65. Overexpression of porcine Coronin1A inhibited the transcription of NF-κB-mediated downstream genes (IL-6, IL-8and COX-2) through down-regulation of NF-κB. Furthermore, we found porcine Coronin1A is a key molecular that inhibit NF-κB activation during H.parasuis infection.