He Xianjing, Jiang Kai, Xiao Jiawei, Lian Shuai, Chen Yaping, Wu Rui, Wang Lina, Sun Dongbo, Guo Donghua
Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, No. 5 Xinyang Road, Sartu District, Daqing, 163319, Heilongjiang Province, China.
Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, No. 5 Xinyang Road, Sartu District, Daqing, 163319, Heilongjiang Province, China.
Vet Microbiol. 2022 Mar;266:109335. doi: 10.1016/j.vetmic.2022.109335. Epub 2022 Jan 10.
Fusobacterium necrophorum, a Gram-negative anaerobe, is an important bovine pathogen that causes hepatic abscesses, foot rot, mastitis and endometritis. We have previously shown that the 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in bacterial infections; however, the molecular mechanisms by which this protein mediates adhesion remain unclear. In this study, we investigated the role of 43 K OMP in F. necrophorum adhesion to bovine epithelial cells using 43 K OMP-deficient mutants, and identified the protein that interacts with 43 K OMP by immunoprecipitation-mass spectrometry. Our results indicated that the native 43 K OMP and recombinant 43 K OMP could bind to the cell membrane of MAC-T or bovine endometrial epithelial cells (BEECs). When F. necrophorum was preincubated with antibodies against the recombinant 43 K OMP or bovine epithelial cells were preincubated with 43 K OMP, the adhesion of F. necrophorum to MAC-T or BEECs decreased significantly (P<0.01). We successfully constructed a 43 K OMP-deficient strain (A25Δ43 K OMP) and bacterial attachment to MAC-T or BEECs was significantly higher with the F. necrophorum A25 strain than with mutant strain A25Δ43 K OMP (P<0.01). The deficiency of 43 K OMP reduced the binding of F. necrophorum to bovine epithelial cells by 90.5 %-94.9 %. Among the 39 potential differential proteins, fibronectin, collagen and myosin were selected as the target proteins, and direct interaction between 43 K OMP of F. necrophorum and fibronectin was demonstrated. Taken together, these results suggest that 43 K OMP plays a key role in adhesion of F. necrophorum to bovine epithelial cells through its interaction with fibronectin. These findings provide a theoretical basis for the pathogenic mechanism of F. necrophorum.
坏死梭杆菌是一种革兰氏阴性厌氧菌，是引起牛肝脏脓肿、腐蹄病、乳腺炎和子宫内膜炎的重要病原体。我们之前已经表明，坏死梭杆菌的43 kDa外膜蛋白（43K OMP）是一种孔蛋白，在细菌感染中起重要作用；然而，该蛋白介导黏附的分子机制仍不清楚。在本研究中，我们使用43K OMP缺陷型突变体研究了43K OMP在坏死梭杆菌黏附牛上皮细胞中的作用，并通过免疫沉淀-质谱法鉴定了与43K OMP相互作用的蛋白。我们的结果表明，天然43K OMP和重组43K OMP均可与MAC-T或牛子宫内膜上皮细胞（BEECs）的细胞膜结合。当坏死梭杆菌与抗重组43K OMP抗体预孵育或牛上皮细胞与43K OMP预孵育时，坏死梭杆菌对MAC-T或BEECs的黏附显著降低（P<0.01）。我们成功构建了一株43K OMP缺陷菌株（A25Δ43K OMP），坏死梭杆菌A25菌株对MAC-T或BEECs的细菌附着明显高于突变菌株A25Δ43K OMP（P<0.01）。43K OMP的缺陷使坏死梭杆菌与牛上皮细胞的结合减少了90.5%-94.9%。在39种潜在的差异蛋白中，选择纤连蛋白、胶原蛋白和肌球蛋白作为靶蛋白，并证明了坏死梭杆菌的43K OMP与纤连蛋白之间存在直接相互作用。综上所述，这些结果表明43K OMP通过与纤连蛋白相互作用在坏死梭杆菌黏附牛上皮细胞中起关键作用。这些发现为坏死梭杆菌的致病机制提供了理论依据。
