副鸡禽杆菌（Avibacterium paragallinarum, Apg）为革兰氏阴性短杆菌，属于巴氏杆菌科禽杆菌属，可引起鸡传染性鼻炎（infectious coryza, IC）。该病可导致育成鸡生长不良和产蛋鸡产蛋率下降（10%～40%），给养禽业带来巨大的经济损失。疫苗防控和药物防治是鸡传染性鼻炎主要的防控手段，但是由于副鸡禽杆菌各血清型之间交叉保护性较差，以及耐药问题的日益严重给副鸡禽杆菌的防控带来困难。生物被膜是许多细菌病原体的重要毒力因子，与细菌的毒力、黏附、宿主定植等密切相关，因此本研究从生物被膜的角度筛选与副鸡禽杆菌生物被膜形成相关的基因，并研究其生物学特性，以期为副鸡禽杆菌的防控提供新的研究角度。结果如下：

1 副鸡禽杆菌转座子突变文库的构建

本研究以副鸡禽杆菌JZIC-005株为受体菌，大肠杆菌S17-1λpir（pEVS170）为供体菌，大肠杆菌DH5α（pRK2013）为辅助菌，采用三亲本结合转导法将携带mini-Tn5转座子的质粒pEVS170导入JZIC-005菌株，实现转座子随机突变。通过转座子上的红霉素抗性和菌株自身的四环素抗性筛选阳性结合子，成功构建了包含3106个突变株的转座子随机突变文库，为后续功能基因研究奠定了基础。

2 副鸡禽杆菌生物被膜形成相关基因的筛选及鉴定

基于构建的转座子突变文库，本研究筛选了生物被膜形成能力发生显著变化的突变株，并鉴定了相关基因。从3106株突变株中，共筛选出188株生物被膜表型改变的突变株，其中172株形成能力增强，16株减弱。通过热不对称PCR定位转座子插入位点，剔除重复位点后，共鉴定出105个与生物被膜形成相关的基因。功能分析（GO、KEGG和KOG）表明，这些基因主要参与氨基酸代谢、纤毛组装、生物合成、群体感应系统、跨膜运输及多种代谢途径，揭示了生物被膜形成的复杂调控网络。

3 转座子插入突变株的生物学特性分析

为进一步探究生物被膜形成机制，本研究选取8个关键基因突变株（Tn-441、Tn-483、Tn-1216、Tn-1504、Tn-1706、Tn-2206、Tn-2428和Tn-2859）进行深入分析。生长曲线显示，这些突变株与野生株无显著差异。然而，部分突变株表现出显著的致病性变化：Tn-483（插入假设蛋白基因）、Tn-1504（插入TonB能量传感器基因）、Tn-2428（插入GntP家族通透酶基因）和Tn-2859（插入假设蛋白基因）对鸡胚成纤维细胞（DF-1细胞）的黏附、入侵及毒性均减弱；Tn-2206（插入自身诱导剂2激酶基因）和Tn-1216（插入菌毛外膜引来蛋白基因）黏附能力增强，但入侵和毒性减弱；Tn-441（插入假设蛋白基因）黏附能力不变，入侵和毒性减弱；Tn-1706（插入GDP-L-岩藻糖合酶基因）黏附和入侵能力增强，但毒性未变。

综上，本研究成功构建了副鸡禽杆菌mini-Tn5转座子突变文库，鉴定出105个生物被膜形成相关基因，并揭示了8个关键突变株的生物学特性。其中，Tn-1504、Tn-2428和Tn-2859在保持正常生长的同时，生物被膜形成能力和致病性显著减弱，符合“抗毒力策略”中削弱病原体毒力而不影响其生存的理念，有望成为减毒候选株。这些发现为副鸡禽杆菌的防控提供了新的理论依据和潜在靶点。

Avibacterium paragallinarum (Av. Paragallinarum, Apg), a Gram-negative short bacillus belonging to the genus Avibacterium within the family Pasteurellaceae, is the causative agent of infectious coryza (IC) in chickens. This disease leads to growth retardation in growing chickens and a 10%~40% decline in egg production of laying hens, resulting in significant economic losses to the poultry industry. While vaccination and antimicrobial therapy remain the primary strategies for IC control, challenges persist due to limited cross-protection among different serotypes of Av. Paragallinarum and the escalating issue of antibiotic resistance. Biofilm formation, a critical virulence factor for numerous bacterial pathogens, is closely associated with bacterial pathogenicity, adhesion, and host colonization. To address these challenges, this study aimed to screen biofilm-associated genes in Av. Paragallinarum and investigate their biological characteristics, thereby providing novel insights for developing effective control measures. The results are as follows:

1 Construction of a transposon mutant library in Avibacterium paragallinarum

In this study, the Av. Paragallinarum strain JZIC-005 was utilized as the recipient strain, with Escherichia coli S17-1λpir (pEVS170) serving as the donor strain and E. coli DH5α (pRK2013) as the helper strain. A tri-parental conjugation approach was employed to introduce the mini-Tn5 transposon-carrying plasmid pEVS170 into the JZIC-005 strain, achieving random transposon mutagenesis. Positive conjugants were selected through erythromycin resistance conferred by the transposon and tetracycline resistance inherent to the recipient strain. A random transposon mutant library comprising 3106 mutant strains was successfully constructed, laying the foundation for subsequent functional gene research.

2 Screening and identification of biofilm-associated genes in Avibacterium paragallinarum

Based on the constructed transposon mutant library, this study screened mutants with significant alterations in biofilm-forming capacity and identified associated genes. Among 3106 mutants, 188 strains exhibited phenotypic variations in biofilm formation, including 172 strains with enhanced capacity and 16 strains with reduced capacity. Through thermal asymmetric interlaced PCR (TAIL-PCR) to localize transposon insertion sites and subsequent elimination of redundant loci, 105 biofilm-associated genes were ultimately identified. Functional analyses (GO, KEGG, and KOG) revealed that these genes predominantly participate in amino acid metabolism, cilia assembly, biosynthetic processes, quorum-sensing systems, transmembrane transport, and diverse metabolic pathways, collectively elucidating the intricate regulatory network underlying biofilm formation.

3 Biological characterization of transposon insertion mutants

To further investigate the mechanisms of biofilm formation, eight key mutant strains (Tn-441, Tn-483, Tn-1216, Tn-1504, Tn-1706, Tn-2206, Tn-2428, and Tn-2859) were selected for in-depth analysis. Growth curve analysis showed no significant differences between these mutant strains and the wild-type strain. However, significant pathogenic variations were observed in some mutants: Tn-483 (inserted into a hypothetical protein gene), Tn-1504 (inserted into the energy transducer TonB gene), Tn-2428 (inserted into the GntP family permease gene), and Tn-2859 (inserted into a hypothetical protein gene) exhibited reduced adhesion, invasion, and cytotoxicity toward chicken embryo fibroblast (DF-1) cells; Tn-2206 (inserted into the autoinducer-2 kinase gene) and Tn-1216 (inserted into the fimbrial biogenesis outer membrane usher protein gene) showed enhanced adhesion but weakened invasion and cytotoxicity; Tn-441 (inserted into a hypothetical protein gene) maintained normal adhesion capacity but displayed reduced invasion and cytotoxicity; Tn-1706 (inserted into the GDP-L-fucose synthase gene) demonstrated increased adhesion and invasion capacities without changes in cytotoxicity.

In summary, this study successfully constructed a mini-Tn5 transposon mutant library for Av. Paragallinarum, identifying 105 biofilm formation-associated genes and elucidating the biological characteristics of eight key mutant strains. Among these, Tn-1504, Tn-2428, and Tn-2859 exhibited significantly attenuated biofilm-forming capacity and pathogenicity while maintaining normal growth, aligning with the "anti-virulence strategy" that aims to weaken pathogen virulence without compromising survival. These mutants are expected to become candidate attenuated strains. These findings provide new theoretical foundations and potential targets for the prevention and control of Av. Paragallinarum.

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