RfaD和RfaF在副猪嗜血杆菌LPS致病性中的作用及机制

Haemophilus parasuis (HPS) is an important respiratory-tract pathogen in swine. It is well known that the lipopolysaccharide (LPS) of Gram-negative bacteria plays an important role in pathogenesis, and core oligosaccharide is an essential component of the LPS. The ADP-L-glycro-D-manno -heptose-6-epimerase (RfaD) and heptosyltransferase II (RfaF), coding rfaD and rfaF gene, were directly involved in the synthesis of core oligosaccharide and the bacterial pathogenesis. Nevertheless, the pathogenic roles of both RfaD and RfaF were obviously different during the bacteria infection. In previous study, we first confirmed that loss of the rfaD or rfaF gene expression in H. parasuis resulted in decreased cell-adherence and invasion properties. Therefore, we speculated that loss of the rfaD or rfaF gene expression in H. parasuis affected the synthesis of core oligosaccharide of LPS that impaired the pathogenesis of bacteria. In this study, the purified LPS of the HPS rfaD and rfaF mutants were used as the study objection to observe the pathogenesis in mice, to detect the expression of proinfammatory cytokine and to analyze the signaling pathways of NF-κB and MAPK in porcine alveolar macrophages cells. Hence, this study further confirmed the precise role and mechanism of RfaD and RfaF in the pathogenesis of the LPS and played a significant role to further study the basic biology and pathogenic mechanism of H. parasuis.

副猪嗜血杆菌(HPS)是一种重要的猪病原菌。细菌脂多糖(LPS)在致病过程中发挥重要作用，核心多糖是LPS的组成成分。rfaD和rfaF基因分别编码的ADP-L-甘油-D-甘露庚糖-6-异构体(RfaD)和庚糖转移酶II (RfaF)直接参与核心多糖的形成，并影响细菌的致病性，但不同细菌RfaD和RfaF发挥的作用有所差别。申请人前期首次证实HPS LPS的RfaD和RfaF基因缺失株均降低了对宿主细胞的黏附入侵能力，推测缺失HPS的RfaD和RfaF基因后影响了LPS核心多糖的形成，从而降低了菌株的致病性。本课题拟利用HPS rfaD 和rfaF缺失株的LPS为研究对象，研究其对小鼠致病力和对宿主细胞炎性因子表达的影响，分析其对NF-κB和MAPK信号通路影响，确定RfaD 和RfaF在HPS LPS致病中的作用和机制。本项目对HPS基础生物学和致病机制的研究有重要的意义。

一、项目的背景.副猪嗜血杆菌(Haemophilus parasuis，HPS)是危害全球养猪业一个重要的细菌性病原，已造成巨大的经济损失。LPS是HPS一个毒力因子，RfaD和RfaF是HPS核心多糖合成过程中关键的成分，也参与了HPS的致病过程。本项目以HPS rfaD和rfaF缺失株的LPS为对象，研究了RfaD和RfaF对HPS LPS致病性中的作用及其机制，对HPS基础生物学和致病机制的研究具有重要意义。.二、主要研究内容.（1）HPS亲本株SC096、rfaD和rfaF缺失株LPS提取纯化；.（2）HPS rfaD和rfaF缺失株LPS在猪肺泡巨噬细胞（PAM）中诱导炎性因子的表达；.（3）HPS rfaD和rfaF缺失株LPS诱导炎性因子的表达机制。.三、重要结果.（1）RfaD和RfaF参与了HPS LPS诱导炎性因子转录应答和表达的作用；.（2）rfaF和rfaD基因通过调节NF-κB和MAPKp38信号通路来影响HPS-LPS诱导猪PAM炎症反应。.四、关键数据及其科学意义.（1）ΔrfaF-LPS和ΔrfaD-LPS刺激小鼠后，血清中IL-1α，IL-6，IL-8和TNF-α的蛋白表达量显著降低，证实了rfaF和rfaD基因在HPS LPS诱导小鼠炎性因子表达中起着重要的作用。.（2）ΔrfaF-LPS和ΔrfaD-LPS刺激猪PAM后，IL-1α、IL-1β、IL-6、IL-8及TNF-α的mRNA转录水平显著降低，IL-6和IL-8蛋白水平明显降低，说明了RfaD和RfaF参与了HPS LPS诱导炎性因子转录应答和表达的作用。.（3）ΔrfaF-LPS和ΔrfaD-LPS刺激猪PAM后，phospho-NF-κB p65和phospho-p38的蛋白表达量显著降低，而IκBα的蛋白表达量显著升高，说明了rfaF和rfaD基因通过调节NF-κB和MAPK p38信号通路来影响HPS-LPS诱导PAM炎症反应，对HPS基础生物学和致病机制的研究有重要意义。