Costa-Ramos Carolina, Rowley Andrew F
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom.
Appl Environ Microbiol. 2004 Feb;70(2):729-35. doi: 10.1128/AEM.70.2.729-735.2004.
Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100 degrees C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 microg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 microg g of body weight(-1)) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.
先前的研究表明，将从感染壳病的食用蟹（黄道蟹）中分离出的大西洋假交替单胞菌的细胞外产物（ECP）注射到健康蟹体内会导致其迅速死亡。在本研究中，我们检测了ECP中活性致死因子的性质。将ECP注射到蟹体内会导致循环血细胞（血细胞）总数迅速下降，蟹在60至90分钟内死亡。死亡的个体表现出眼柄回缩、肢体麻痹和触角敏感性丧失，这表明活性因子作用于神经系统。组织病理学研究表明，受影响的蟹鳃中有大量血细胞聚集，肝胰腺中的小管遭到破坏。ECP中的活性因子对热处理（100℃30分钟）和蛋白酶K消化不敏感。由于脂多糖（LPS）是致死因子的一个潜在候选物，它从完整的大西洋假交替单胞菌或ECP中纯化出来，随后注射到蟹体内。这些蟹出现了先前用ECP观察到的所有外部症状，如肢体麻痹和眼柄回缩，并且在受到攻击后90分钟内死亡，尽管未观察到循环血细胞数量有显著下降。同样，用从大西洋假交替单胞菌中纯化的LPS（1至20微克）对血细胞进行体外培养，未导致观察到的与ECP相关的凝集反应，但确实导致了脱粒反应并最终导致细胞裂解。用大肠杆菌或铜绿假单胞菌LPS（1微克/克体重）注射蟹，未引起接触大西洋假交替单胞菌LPS后观察到的任何特征性症状。注射大肠杆菌LPS后蟹未死亡，但铜绿假单胞菌LPS在注射后2小时导致约80%的死亡率。总体而言，这些结果表明，大西洋假交替单胞菌对食用蟹的主要毒力因子是单独的LPS或与其他热稳定因子结合的LPS。
