Department of Veterinary Disease Biology, Faculty of Health Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.
Vet Microbiol. 2013 Dec 27;167(3-4):565-72. doi: 10.1016/j.vetmic.2013.09.005. Epub 2013 Sep 12.
The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg-production worldwide. Increased knowledge of the pathogenesis and virulence factors is important to better understand and prevent the negative effects of G. anatis. To this end outer membrane vesicles (OMVs) are natural secretion products of Gram-negative bacteria, displaying an enormous functional diversity and promising results as vaccine candidates. This is the first study to report that G. anatis secretes OMVs during in vitro growth. By use of transmission electron microscopy (TEM) and SDS-PAGE, we showed that changes in in vitro growth conditions, including incubation time, media composition and temperature, affected the OMV production and protein composition. A large protein band was increased in its concentration after prolonged growth. Analysis by LC-MS/MS indicated that the band contained two proteins; the 320.1 kDa FHA precursor, FhaB, and a 407.8 kDa protein containing a von Willebrand factor type A (vWA) domain. Additional two major outer-membrane (OM) proteins could be identified in all samples; the OmpH-homolog, OmpC, and OmpA. To understand the OMV formation better, a tolR deletion mutation (ΔtolR) was generated in G. anatis. This resulted in a constantly high and growth-phase independent production of OMVs, suggesting that depletion of peptidoglycan linkages plays a role in the OMV formation in G. anatis. In conclusion, our results show that G. anatis produce OMVs in vitro and the OMV protein profile suggests that the production is an important and well-regulated ability employed by the bacteria, which may be used for vaccine production purposes.
鸡败血支原体是一种革兰氏阴性细菌，是导致产蛋鸡输卵管炎和腹膜炎的主要原因，在全球范围内导致产蛋量下降。增加对发病机制和毒力因子的了解对于更好地理解和预防鸡败血支原体的负面影响非常重要。为此，外膜囊泡（OMV）是革兰氏阴性菌的天然分泌产物，具有巨大的功能多样性，并有望成为疫苗候选物。这是第一项报道鸡败血支原体在体外生长过程中分泌 OMV 的研究。通过使用透射电子显微镜（TEM）和 SDS-PAGE，我们表明，体外生长条件（包括孵育时间、培养基组成和温度）的变化会影响 OMV 的产生和蛋白质组成。长时间生长后，大蛋白带的浓度增加。通过 LC-MS/MS 分析表明，该带包含两种蛋白质；320.1 kDa FHA 前体 FhaB 和含有血管性血友病因子 A （vWA）结构域的 407.8 kDa 蛋白。在所有样品中还可以鉴定出另外两种主要的外膜（OM）蛋白；OmpH 同源物 OmpC 和 OmpA。为了更好地理解 OMV 的形成，在鸡败血支原体中生成了 tolR 缺失突变（ΔtolR）。这导致 OMV 的产生始终很高且与生长阶段无关，表明肽聚糖键的耗尽在鸡败血支原体的 OMV 形成中起作用。总之，我们的结果表明，鸡败血支原体在体外产生 OMV，并且 OMV 蛋白谱表明这种产生是细菌的一种重要且受调控的能力，这可能用于疫苗生产目的。
