禽致病性大肠杆菌（Avian pathogenic E.coli, APEC）属于肠道外致病性大肠杆菌（ExPEC）的一种致病亚型，对大肠杆菌进行系统演化分群，主要可分为八个系统群--A、B1、B2、C、D、E、F和Clade I，研究指出多数B2群禽源大肠杆菌属于致病性APEC，而非共生性、弱毒性大肠杆菌，B2群APEC对禽类具有较强致病力，还具有人畜共患潜力。本研究第一部分是探究禽致病性大肠杆菌的分子流行病学特征，主要研究F群禽源大肠杆菌。对F群禽源大肠杆菌进行毒力基因型和致病表型，研究发现多数F群禽源大肠杆菌具有致病力，类似于B2群高致病力APEC，甚至具有人畜共患潜力。第二部分探究了APEC分子致病机理，DksA是一种重要的转录因子。我们构建了转录因子DksA基因缺失株和回补株，研究DksA基因缺失对APEC强毒株致病表型的影响，缺失DksA导致APEC菌株致病力显著下降，生物信息分析发现DksA缺失株与野生株的转录组、蛋白组存在明显差异，进而深入探究DksA分子调节机制，研究发现DksA是APEC关键的毒力调控因子，DksA通过small RNA (sRNA)转录后调控途径介导APEC感染巨噬细胞。第三部分则对FY26ΔDksA作为弱毒疫苗候选毒株的免疫保护效果进行了评价，研究发现缺失株FY26ΔDksA作为弱毒疫苗候选毒株，对APEC感染具有较好的保护效果，同时足够致弱，具备较高的生物安全性。

1.禽源大肠杆菌F群分离株致病力和人畜共患潜力研究

通过MLST对F群大肠杆菌进行分型，这些F群禽源大肠杆菌属于29个STs，将含有8个以上菌株的ST类群，称作优势ST，总共有13个优势ST (ST59、ST62、ST115、ST117、ST135、ST354、ST362、ST393、ST405、ST457、ST501、ST648和ST1158)。对F系统群大肠杆菌进行基因型和表型分析，结果表明，多数F群禽源大肠杆菌具有与APEC强毒株相似的基因型和毒力特征。F群大肠杆菌能产生生物被膜，普遍具有运动性；75.4%的菌株表现出补体耐受。相比之下，含ColV/BM大质粒菌株的补体耐受性显著高于阴性菌株，毒力基因评分显示毒力因子（Virulence factor, VF）分数≥15的菌株补体耐受比例高于VF <15的菌株。F群禽大肠杆菌导致禽患大肠杆菌病的能力较强，并且ColV/BM质粒阳性菌株引起雏鸡感染死亡率高于ColV/BM阴性菌株。筛选38株F群优势ST类群菌株，用败血症、脑膜炎和尿路感染动物模型评估这些F群禽大肠杆菌的人畜共患病风险，这38株菌在感染3天后（3 dpi）平均疾病评分，及小鼠和雏鸡死亡率，均接近阳性对照APEC B2群强毒株DE205B。所有38株菌均能引起大鼠的血流感染，并在24小时内诱发败血症。F群强毒株在小鼠尿道膀胱中的增殖水平明显高于阴性对照弱毒株MG1655。这些结果表明F群禽源性大肠杆菌与人源ExPEC致病性相似，并具有人畜共患潜力。综上所述，F群禽源大肠杆菌对家禽普遍具有致病力，是典型的禽致病性大肠杆菌(APEC)，部分F群菌株具有人畜共患潜力。

2.DksA基因缺失株的致病表型变化和分子调节机制

转录因子可以通过与启动子或启动子附近区域的结合来调控RNA聚合酶(RNAP)的转录功能，DksA是大肠杆菌重要的转录因子。构建DksA基因缺失株FY26ΔDksA，动物试验表明，缺失株FY26ΔDksA体内竞争能力显著低于强毒野生株FY26，与野生株FY26相比，FY26ΔDksA感染雏鸡的肺和血液中细菌载量大幅下降。在雏鸡感染模型中，缺失株FY26ΔDksA和野生株FY26的半数致死量(LD50)分别为2.5×107 CFU/只和2.0×105 CFU/只；而在小鼠模型中，缺失株FY26ΔDksA和野生株FY26的半数致死量(LD50)分别为6.3×106 CFU/只和3.2×105 CFU/只，结果表明缺失株FY26ΔDksA的毒力较野生株明显下降。在LB液体培养基中37°C过夜培养野生株、缺失株和回补株，测量OD600值绘制其生长曲线，结果显示各菌株间无明显差异。细胞黏附和胞内存活试验结果表明，与野生株FY26相比，缺失株FY26ΔDksA的黏附能力和胞内存活能力显著下降。通过RNA-seq对野生株FY26与缺失株FY26ΔDksA进行转录组测序，鉴定发现野生株FY26与缺失株FY26ΔDksA转录表达谱差异较明显。筛选转录水平受DksA调控较为明显的sRNA，如sRNA057、sRNA091、sRNA051、sRNA167、sRNA029和DsrA，通过Northern blot进行验证，结果与转录组测序结果一致。与转录组结果相类似，Label-free蛋白组测序发现野生株FY26与缺失株FY26ΔDksA蛋白表达谱差异也较明显。

3. DksA基因缺失株作为弱毒疫苗候选毒株的免疫效果评价

对野生株FY26、缺失株FY26ΔDksA与回补株FY26CΔC-DksA进行荚膜染色与透射电镜观察，结果发现缺失株荚膜较野生株和回补株明显增厚，这与转录组数据结果一致。缺失株FY26ΔDksA致病力较弱，而荚膜增厚，该特性表明其具有弱毒疫苗应用潜力。对FY26ΔDksA的免疫效果及血清效价进行评定，结果显示无论免疫剂量1×106 CFU/只或是5×105 CFU/只，一免后的免疫保护率均大于90%，而二免后免疫保护率则为100%，弱毒疫苗候选株FY26ΔDksA能产生高水平的血清效价。体内定殖清除试验的结果表明，FY26ΔDksA在体内随着时间的推移而逐渐被清除。综上所述，FY26ΔDksA弱毒疫苗候选毒株对同源菌株感染，具有较好的保护效果，同时具备较高的安全性，有待评估该弱毒疫苗候选毒株对不同血清型菌株感染的保护力。

      Avian pathogenic E. coli (APEC) belongs to a pathogenic subtype of extra-intestinal pathogenic E. coli (ExPEC). E. coli can be divided into eight system groups (A, B1, B2, C, D, E, F, and Clade I) by phylogenetically grouping. Several reports have pointed out that most of the phylogroup B2 poultry-derived E coli isolates are considered as pathogenic APECs, rather than symbiotic and non-virulent E. coli. The B2 pholygroup APEC has strong pathogenicity to poultry, and also has the potential for zoonotic infection. The first part of this study is to explore the molecular epidemiological characteristics of APEC, mainly to focus on F phylogroup avian-source E. coli isolates.We analyze the virulence genotype and pathogenic phenotype of F group of poultry E. coli. Most of phylogroup F poultry-derived E. coli isolates hold virulence, similar to the high virulent phylogroup B2 APEC, and even have zoonotic potential. The second part focuses on the molecular pathogenesis of APEC. We construct the transcription factor DksA gene deletion strain FY26ΔDksA and complementary strain FY26ΔC-DksA to investigate the effect of DksA gene deletion on the APEC pathogenic phenotype. The lack of DksA gene leads to a significant decrease in the APEC virulence, and there are also significant differences in the transcriptome and proteome for the deletion strain, compared to the wild strain. We have thoroughly explored the molecular regulation mechanism of DksA, and find that DksA is a key virulence regulator of APEC. DksA mediates APEC to infect macrophages through small RNA (sRNA) post-transcriptional regulatory pathways. We mainly explore the molecular epidemiological characteristics and infection mechanisms of APEC from these two aspects. The third part evaluates the immune protection effect of the mutant strain FY26ΔDksA as a candidate strain of attenuated vaccine.

1. Study on pathogenicity and zoonotic potential of phylogroup F avian-source E. coli

MLST is used to identify STs of the phylogroup F E. coli. These phylogroup F avian E. coli belong to 29 STs. The ST group containing more than 8 strains is called the dominant ST. There are 13 dominant STs, including ST59, ST62, ST115, ST117, ST135, ST354, ST362, ST393, ST405, ST457, ST501, ST648, and ST1158. The genotype and phenotype analysis show that most phylogroup F poultry-derived E. coli share similar genotype and virulence characteristics to virulent APEC strains. Phylogroup F E. coli can produce biofilms and generally hold motility; 75.4% of phylogroup F strains hold the complement tolerance. In contrast, strains containing ColV/BM large plasmids have significantly higher complement tolerance than the negative strains, and strains with virulence factor (VF) score ≥ 15 hold higher complement tolerance ratio than strains with VF < 15. Pholygroup F avian E. coli isolates lead to severe avian colibacillosis, and the mortality rate of ColV/BM plasmid-positive strains in the chick model is higher than that of ColV/BM-negative strains. We screen 38 dominant ST strains, and animal models of sepsis, meningitis and urinary tract infection are used to assess the zoonotic risk of these 38 F E. coli strains. The average disease score and mortality within 3 day post-infection (dpi) for mice and chicks infected with these 38 strainsare are close to the positive control virulent DE205B. All 38 strains can cause bloodstream infections in rats and induce sepsis within 24 hours. The proliferation level of virulent phylogroup F strains in the urethra and bladder of mice are significantly higher than that of the negative control MG1655. These results indicate that phylogroup F poultry-derived E. coli is similar to human ExPEC in pathogenicity and has zoonotic potential. In summary, phylogroup F poultry-derived E. coli is generally pathogenic and considered as a typical avian pathogenic E. coli (APEC). Several phylogroup F strains have zoonotic potential.

2. Pathogenic phenotype changes and molecular regulation mechanism of DksA gene deletion strain

DksA is an important transcription factor. Most transcription factors can regulate the transcription function of RNA polymerase (RNAP) by binding to the promoter or the region near the promoter. Animal experiments show that the in vivo competitiveness of the deletion strain FY26ΔDksA is significantly lower than that of the wild-type strain FY26. Compared with the wild-type FY26, the bacterial load of FY26ΔDksA in the lung and blood of infected chicks is obviously reduced. In the chick model, the LD50 of the mutant strain FY26ΔDksA and the wild-type FY26 are 2.5×107 CFU/chick and 2.0×105 CFU/chick, respectively. In the mouse model, the LD50 of the mutant FY26ΔDksA and the wild-type FY26 are 6.3×106 CFU/mouse and 3.2×105 CFU/mouse, respectively. The results show that the virulence of the mutant FY26ΔDksA is significantly lower than that of the wild-type strain. The wild-type strain, mutant, and complementary strains are cultured overnight in LB liquid medium at 37°C, and the OD600 values are measured to analyse their growth curves. The results show that there are no significant differences among these strains. The results for cell adhesion and intracellular survival test show that the adhesion and survival ability of the deletion strain FY26ΔDksA are significantly reduced relative to the wild strain FY26. Transcriptome sequencing of the wild strain FY26 and the mutant strain FY26ΔDksA reveal that the transcriptional expression profiles of the wild strain FY26 and the deletion strain FY26ΔDksA are significantly different. We screen sRNAs, whose transcription levels are more obviously regulated by DksA, such as sRNA057, sRNA091, sRNA051, sRNA167, sRNA029 and DsrA.These sRNAs are verified by Northern blot, and the results are consistent with the transcriptome sequencing. Similar to transcriptome results, label free proteome sequencing shows that there is also significantly different for the protein expression profile between the wild-type strain FY26 and mutant FY26ΔDksA.

3. Evaluation of immune protection for DksA gene mutant strain as the candidate strain of attenuated vaccine

Capsular staining and transmission electron microscopic (TEM) observation of the wild-type FY26, the mutant strain FY26ΔDksA, and the complementary strain FY26CΔC-DksA show that the capsule of FY26ΔDksA is significantly thicker than that of the wild-type and the complementary strains, which is consistent with the transcriptome data. Because the mutant FY26ΔDksA has weaker pathogenicity and the obvious thick of the K1 capsule, the characteristics suggest that it has the potential of attenuated vaccine application. Therefore, we perform the immune tests to evaluate the immune protection and serum titer of FY26ΔDksA. The results show that regardless of the vaccine dose of 1.0 × 106 CFU/chick or 5.0 × 105 CFU/chick, the immune protection after the first immunization is greater than 90%. After the second immunization, the immune protection rate is about 100%. FY26ΔDksA can induce a high level of serum titer. The results of the colonization clearance test in vivo show that the attenuated strain FY26ΔDksA can be gradually cleared in the body over time. In summary, the attenuated vaccine candidate strain FY26ΔDksA has a good protective effect against infections of homologous strains, and at the same time FY26ΔDksA has high safety. The protection of the attenuated vaccine candidate strain against infection of different serotype APEC strains needs to be evaluated.