Bandara Aloka B, Lawrence Mark L, Veit Hugo P, Inzana Thomas J
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061, USA.
Infect Immun. 2003 Jun;71(6):3320-8. doi: 10.1128/IAI.71.6.3320-3328.2003.
The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.
胸膜肺炎放线杆菌的荚膜多糖（CP）是该细菌在猪体内致病所必需的。然而，关于CP的类型或数量是否影响胸膜肺炎放线杆菌毒力的分子研究尚未见报道。为开展此项研究，对胸膜肺炎放线杆菌1型中CP输出所需保守基因下游的一个DNA区域进行了克隆和测序。鉴定出三个开放阅读框，分别命名为cps1A、cps1B和cps1C，它们与细菌碳水化合物生物合成基因具有氨基酸同源性。将卡那霉素抗性盒（Kan(r)）插入跨越cps1AB的750 bp缺失区域或仅插入cps1B中的512 bp缺失区域，并将构建体克隆到自杀载体中。然后通过同源重组将Kan(r)基因转移到4074菌株的染色体上，分别产生4074Deltacps1N菌株和4074Deltacps1B菌株。通过酶联免疫吸附测定和游离CP沉淀测定，4074Deltacps1N菌株未产生可检测到的CP，但4074Deltacps1B菌株产生的1型CP量为亲本菌株4074的15%。将4074菌株的cps1ABC基因以及5a型菌株J45的cps5ABC和cps5ABCDE基因克隆到穿梭载体pLS88中，并电穿孔导入4074Deltacps1N，分别产生4074Deltacps1N(pABcps101)、4074Deltacps1N(pJMLcps53)和4074Deltacps1N(pABcps55)。4074Deltacps1N(pABcps101)产生的1型CP约为4074菌株的33%。4074Deltacps1N(pJMLcps53)和4074Deltacps1N(pABcps55)产生的5a型CP量分别与4074菌株产生的相似或多出四倍。以相似剂量对猪进行气管内攻毒时，产生1型CP的菌株（通过死亡率、肺实变、出血和纤维蛋白性胸膜炎评估）的毒力顺序如下：4074菌株>4074Deltacps1N(pABcps101)菌株≥4074Deltacps1N菌株>4074Deltacps1B菌株。4074Deltacps1N(pJMLcps53)菌株的毒力低于4074Deltacps1N(pABcps55)菌株。然而，这两种菌株产生的5a型CP量与亲本菌株4074产生的1型CP量相似或更多，但毒力低于亲本菌株。因此，胸膜肺炎放线杆菌同基因菌株产生的1型或5a型CP量与该细菌在猪体内的毒力相关。然而，毒力也受所产生CP的类型或其表达机制的影响。
