Ward C K, Lawrence M L, Veit H P, Inzana T J
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Infect Immun. 1998 Jul;66(7):3326-36. doi: 10.1128/IAI.66.7.3326-3336.1998.
A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.
利用针对参与荚膜多糖（CP）输出的cpxD基因的特异性探针，鉴定并表征了胸膜肺炎放线杆菌5型荚膜多糖生物合成相关的DNA区域。从菌株J45基因组DNA的一个5.8 kb BamHI片段和一个8.0 kb EcoRI片段中克隆了相邻的5型特异性CP生物合成区域。DNA序列分析表明，该区域包含四个完整的开放阅读框，即cps5A、cps5B、cps5C和cps5D。Cps5A、Cps5B和Cps5C与几种参与脂多糖或CP生物合成的细菌糖基转移酶具有较低的同源性。然而，Cps5D与其他革兰氏阴性菌的KdsA蛋白（3-脱氧-D-甘露糖-2-辛酮酸8-磷酸合成酶）具有高度同源性。cps5ABC的G+C含量（28%）显著低于cps5D和胸膜肺炎放线杆菌染色体其余部分（42%）。构建了一个跨越克隆的cps5ABC开放阅读框的2.1 kb缺失片段，并通过与卡那霉素抗性盒的同源重组将其转入J45染色体，产生突变体J45-100。多重PCR证实了J45-100 DNA该区域的缺失。J45-100不产生细胞内或细胞外CP，表明cps5A、cps5B和/或cps5C参与CP生物合成。然而，Apx毒素、脂多糖和膜蛋白的生物合成不受该突变影响。除了缺乏CP生物合成外，与J45相比，J45-100生长更快，对初乳前犊牛血清中的杀伤敏感，并且在气管内攻毒剂量为菌株J45的50%致死剂量（LD50）的三倍时对猪无毒力。在J45 LD50的六倍剂量下，J45-100引起轻度至中度肺部病变，但未导致死亡。将cps5ABC电穿孔导入胸膜肺炎放线杆菌1型菌株4074，产生菌株4074(pJMLCPS5)，其表达1型和5型CP。然而，与4074相比，4074(pJMLCPS5)中1型荚膜表达减少。重组菌株在对数期产生的总CP（1型和5型CP合并）显著减少（P = 0.0012），但在稳定后期产生的总CP比4074显著更多（P < 0.0001）。此外，与菌株4074相比，菌株4074(pJMLCPS5)在呼吸道攻毒后在猪和小鼠中引起的死亡率和菌血症更低，表明毒力受荚膜产生减少的影响。这些结果强调了CP在胸膜肺炎放线杆菌血清抗性和毒力中的重要性。
