肌醇代谢参与猪肺炎支原体致病的机制解析

The specific metabolic capacity of pathogenic microorganisms has been considered as “nutritional virulence”, which is an important factor affecting its pathogenicity. Currently, studies on the pathogenesis of Mycoplasma hyopneumoniae (Mhp) is mainly focused on the classical virulence factors, however, the relationship between the metabolism and pathogenicity of Mhp has not been reported yet. Our previous study found that only Mhp contained myo-inositol metabolic pathways in swine mycoplasmas, and highly virulent strains were able to grow with inositol at lower concentrations more efficiently compared with that of less virulent strains, suggesting that virulent strains are more likely to use myo-inositol from the host as carbon source to survive. In addition, the single gene insertion mutant of myo-inositol metabolism, the iolC mutant, the cytotoxicity of which on porcine bronchial epithelial cells was significantly lower than that of the wild strain, indicating that inositol metabolism is related to the virulence of Mhp. In this research, the first step is to construct its complementary strain based on iolC mutant strain. To study the role of myo-inositol metabolism in the pathogenicity of Mycoplasma hyopneumoniae, the biological characteristics, the pathogenicity to cells and animal models will be assessed after myo-inositol metabolism is blocked. In addition, through transcriptomics analysis, it will be determined whether myo-inositol metabolism participates in the pathogenicity of Mhp by affecting the expression of other virulence-related factors, ultimately elucidating the mechanism of inositol metabolism involved in the pathogenesis of Mhp. This study will reveal the pathogenesis of Mhp from the prospective of metabolic adaptation. In addition, this study will provide ideas and methods which may be utilized for the study of other pathogens.

病原微生物的特定代谢能力已被视为“营养毒力”，是影响其致病力的重要因素。目前对猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）致病机制的研究主要集中于经典毒力因子，关于其代谢能力与致病力的联系尚无报道。申请人发现猪源支原体中仅Mhp含有肌醇代谢途径，且强毒株较弱毒株能更高效地利用低水平的肌醇，提示强毒株在宿主体内更易于以肌醇为碳源生存。此外，肌醇代谢单基因突变株—iolC突变株对猪支气管上皮细胞的毒性显著低于野生株，表明肌醇代谢与其毒力相关。鉴于此，本项目拟构建iolC突变株的互补株，明确肌醇代谢被阻断后对Mhp生物学特性的影响，分析肌醇代谢在其感染细胞和动物机体中的作用，通过转录组分析肌醇代谢是否影响其他毒力相关基因的表达，最终阐明肌醇代谢参与Mhp致病的机理，从代谢适应性的角度揭示该病原的致病机制，同时也为其他病原致病机制的研究提供可借鉴的思路和方法。

病原微生物的特定代谢能力已被视为“营养毒力”，是影响其致病力的重要因素。猪肺炎支原体（Mhp）致病机制的研究主要集中于经典毒力因子，关于其代谢能力与致病力的联系尚无报道。研究发现猪源支原体中仅Mhp含有肌醇代谢途径，且强毒株较弱毒株能更高效地利用低水平的肌醇，提示强毒株更易于以肌醇为碳源生存。本研究从代谢适应性角度揭示肌醇代谢参与Mhp感染的作用机制，开展以下研究：明确肌醇对Mhp生长的影响及肌醇代谢的关键基因，构建Mhp强毒株iolC突变株及其互补株；明确肌醇代谢被阻断后对Mhp生物学特性的影响；明确肌醇代谢在Mhp感染宿主细胞和引发动物致病中的作用；明确肌醇代谢对Mhp毒力相关基因表达的影响。得出以下结论：添加一定浓度肌醇可促进Mhp菌株的生长，iolC为Mhp肌醇代谢的关键基因；（2）随机插入突变体库获得肌醇代谢的双基因突变株（iolC及rbsC），对山羊丝状支原体的CRISPRi质粒和在Mhp 瞬转的PMD-TO-GFP质粒进行改造，构建了iolC的敲除和表达抑制质粒，获得iolC的大幅表达抑制株。iolC被抑制后，其RNA转录水平显著下降；（3）iolC抑制株与野生株、回补株相比，生物学特性固体克隆形态明显变化，生物被膜形成能力显著下降；（4）肌醇代谢途径能够增强Mhp对永生化猪呼吸道上皮细胞的黏附能力、氧化应激损伤和对猪肺泡巨噬细胞的抗吞噬能力；（5）iolC抑制株与野生株及其回补株相比，对BALB/c小鼠的致病力，以及在小鼠肺脏、肝脏、脾脏的定植及扩散能力显著降低，结合猪体免疫攻毒实验，初步表明 iolC 抑制株具有作为 Mhp 的减毒活疫苗应用的候选疫苗株的潜力；（6）iolC抑制株、野生株和回补株的转录组测序结果表明iolC 基因的抑制对Mhp毒力相关基因的表达有影响，对两个转录显著上调的基因NADH氧化酶和NADH黄素氧化还原酶进行了功能解析，证实二者是Mhp的新型毒力因子，为Mhp亚单位疫苗研发提供理论依据。