Stevenson Andrew, Macdonald Julie, Roberts Mark
Molecular Bacteriology Group, Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, Scotland, UK.
Vet Microbiol. 2003 Mar 20;92(1-2):121-34. doi: 10.1016/s0378-1135(02)00351-6.
Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.
胸膜肺炎放线杆菌是猪胸膜肺炎的病原体。关于胸膜肺炎放线杆菌定殖于呼吸道的机制，人们了解甚少。菌毛是细菌黏附于黏膜上皮的常见介质，在胸膜肺炎放线杆菌细胞表面已观察到菌毛。在此，我们报告了胸膜肺炎放线杆菌4型菌毛结构基因（apfA）的鉴定与特性。此外，在胸膜肺炎放线杆菌中还鉴定出了一些开放阅读框，它们与其他物种的4型菌毛生物合成基因具有显著同源性，包括一个假定的前导特异性肽酶（apfD）。胸膜肺炎放线杆菌apfA编码一个预测分子量约为16kDa的多肽，在预测的切割位点去除前导序列后将产生一个14.5kDa的多肽。成熟多肽的前30个残基与A组4型菌毛家族的其他成员高度保守。ApfA的信号序列长度为13个氨基酸，不同寻常的是，切割位点之前的残基是丙氨酸而非大多数其他4型菌毛中发现的甘氨酸。ApfA的C末端拥有在许多物种的4型菌毛中保守的半胱氨酸残基。在其他4型菌毛中，远端C末端半胱氨酸形成一个二硫键，产生一个环，这对菌毛功能很重要，并且也是一个主要的抗原决定簇。在ApfA预测环内的一个基序在其他嗜血性放线杆菌属（嗜血杆菌属、放线杆菌属、巴斯德菌属）生物的4型菌毛中高度保守。胸膜肺炎放线杆菌4型菌毛生物合成基因与杜克雷嗜血杆菌假定的4型菌毛基因显示出最强的同源性。胸膜肺炎放线杆菌apfA基因在胸膜肺炎放线杆菌的不同血清型中均存在且高度保守。制备了重组ApfA并用于制备抗ApfA抗血清。
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