PhoP/Q对APEC关键毒力因子及β-防御素抗感染的调控研究

The PhoP/Q is one of the key regulatory factor in pathogenic bacterial virulence and resistance to antimicrobial peptides(AMPs).Little was known about PhoP/Q dependent molecular mechanism by which APEC survives.It is significant to study different regulation of APEC crucial virulence and avianβ-defensins(AVBDs)by PhoP/Q. Firstly,the purpose of this investigation is to test the hypothesis that the PhoP/Q TCS is critical to the virulence of APEC and anti-infection of AvBDs.The double knockout mutants of △PhoP/Q will be constructed using λ-Red recombinant system in APEC(CVCC1565), these mutants will be tested for various attributes of virulence, including in chicken vivo assays and various in vitro assays. And cloning of PhoP/Q gene and efficient expression in escherichia coli,the recombinant gene of PhoP/Q will be translocated into BL21(DE3)respectively,And then generate the recombinant strains to express the relevant proteins, under induction with IPTG，the fusion proteins will be expressed and purified under denaturing condition using Ni-NTA, respectly． Next，we compare the different expressed genes between △PhoP/Q strain and the parent strain grown to middle-exponential phase by DNA microarray ,the microarray data will be confirmed by RT- PCR independently.Bioinformatics methods will be used to analyze the transcriptional regulation of virulence and promoter of defensins gene family in APEC, Screening the correlate target genes of outer-membrane protease T,lipopolysaccharide ,high pathogenicity island, the EMSA will be carried out to analyze the in vitro accosiation between regulatory proteins and their targeting promoter DNA regions.In addition,virulence and pathogenicity of △PhoP/Q mutation in the APEC will be evaluated by chicken LD50 test and cell invasion assays. Finally，in order to compare the difference of mRNA expression, chicken models infected with APEC strains will be established. AVBDs（1-14）genes expression will be determined using RT- PCR, Screening the correlated genes of AVBDs,and the protein content of crucial AVBDs will be measured by immunohistochemistry methods. In order to study the intervention of AVBDs & PhoP/Q , chicken trachea epithelium cells（CTECs）models will be established and divided into control group and experimental groups．The CTECs will be cultured with PhoP/Q proteins & APEC strains in different concentrations and different time conditions.The mRNA and proteins expression leve of crucial AVBDs will be tested by RT-PCR and western blot,respectively.

PhoP/Q是调控细菌毒力和内源性抗菌肽的重要途径之一。针对禽致病性大肠杆菌（APEC）难于防制问题，依据靶向细菌毒力的抗感染新思路，探讨PhoP/Q对APEC毒力因子致病性及鸡β-防御素（AVBDs）抗感染的调控研究具有重要意义。 本项目基于Red系统构建APEC△PhoP/Q双基因缺失突变株,表达纯化PhoP和PhoQ蛋白。在此基础上，利用APEC基因芯片、RT-PCR、凝胶阻滞及致病性试验，开展PhoP/Q对APEC毒力调控研究，通过生物信息学分析，筛选 PhoP/Q 调控APEC关键毒力靶基因。分别建立鸡APEC感染和气管上皮细胞模型，针对PhoP/Q与AVBDs靶向拮抗效应,选择PhoP/Q蛋白及APEC菌株进行体内外干预试验，开展PhoP/Q对鸡AVBDs抗APEC感染的调控研究,发现鸡抗APEC感染更有效AVBDs,最终为研发抗菌肽制剂和有效防治APEC提供支撑。

phoP/Q二元调控系统广泛存在于革兰氏阴性菌中，是调控细菌毒力及内源性抗菌肽的重要途径之一。针对禽致病性大肠杆菌（APEC）难于防制问题，依据靶向细菌毒力的抗感染新思路，探讨PhoP/Q对APEC毒力因子致病性及鸡β-防御素（AVBDs）抗感染的调控研究具有重要意义。本课题对APEC的phoP/Q二元调控系统开展研究，获得如下成果：(1)成功构建phoP、phoQ、phoP/Q的缺失株及回复株，生物学特性评价发现phoP/Q基因的缺失导致APEC的运动能力、毒力基因转录水平、APEC黏附和入侵CEF细胞的能力、LD50出现了显著性的降低，为进一步研究phoP/Q对APEC毒力机制的调控奠定基础。(2)基因芯片筛选受phoP/Q调控的差异表达基因，获得显著性差异基因292个，其中fliA、ompT、rstA、rstB、crcA、yrbL、ybjX可能是二元调控系统PhoP/Q直接调控的毒力相关基因，为进一步筛选phoP/Q调控的靶基因提供参考。(3)原核表达载体构建PhoP和PhoQ蛋白的工程菌，EMSA验证受PhoP蛋白调控的毒力相关基因，分别为iss、mgtA、chuA、uspA、slyB、hlyF，为进一步探究PhoP/Q对APEC的调控作用提供研究依据。(4) 建立APEC及其 PhoP/Q双基因缺失株感染罗曼雏鸡动物模型，RNA-Seq筛选感染雏鸡小肠后的应答基因，获得CALB1、TMIGD1、B-G、CCL19、DDX3X、SUSD2、CCLI10、CD274、MT4、LEAP2等肠道免疫应答相关基因； RT-PCR检测AvBD6基因在感染过程中肠段的表达，免疫组化检测AvBD6在肠道中的蛋白表达情况。结果显示AvBD6基因在小肠各段的绒毛上皮细胞和十二指肠、空肠及回肠的肠腺部分有不同程度的表达；AvBD6蛋白在小肠各段中的含量以回肠最高。本课题针对PhoP/Q与AVBDs靶向拮抗效应，开展PhoP/Q对鸡AVBDs抗APEC感染的调控研究，最终为研发抗菌肽制剂和有效防治APEC提供支撑。