Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Canada.
BMC Genomics. 2010 Feb 8;11:98. doi: 10.1186/1471-2164-11-98.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length.
Transcriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879) were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene APL_0920). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections.
These genes that we have identified as up-regulated in vivo, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.
胸膜肺炎放线杆菌是猪传染性胸膜肺炎的病原体，这种呼吸道疾病在全球范围内造成了巨大的经济损失。许多毒力因子参与了疾病的发病机制，包括荚膜多糖、RTX 毒素、LPS 和许多铁摄取系统。为了鉴定在自然感染过程中体内表达的基因，我们使用胸膜肺炎放线杆菌 DNA 微阵列进行了转录谱分析实验，从 5b 型血清感染的猪肺中回收细菌 mRNA 后进行实验。AppChip2 包含基于 App 5b 血清型菌株 L20 基因组序列的 2033 个 PCR 扩增子，代表长度大于 160bp 的 ORF 超过 95%。
对从患有自然感染的猪肺中回收的胸膜肺炎放线杆菌或在 BHI 培养基中生长的细菌分离株进行了转录谱分析。为了从肺部病变中提取总 RNA 时最大限度地提高细菌 mRNA 的产量和质量，我们开发了一种结合珠磨和热酸酚提取的 RNA 提取方案。我们的微阵列几乎可以检测到所有的胸膜肺炎放线杆菌转录本，并且在感染的急性阶段有 150 个基因被认为是体内差异表达的。我们的结果表明，例如，来自编码 RTX 毒素 ApxIV 的操纵子的基因 apxIVA 在体内高度上调，并且编码 IV 型菌毛（APL_0878 和 APL_0879）的操纵子中的两个基因也上调。这些转录谱分析数据与我们小组之前进行的比较基因组杂交相结合，表明 72 个上调基因中有 66 个在所有血清型中保守，其中 3 个基因编码预测的外膜蛋白（基因 irp 和 APL_0959，预测分别编码 TonB 依赖性受体和丝状血凝素/粘附素）或脂蛋白（基因 APL_0920）。在对照性实验感染中，之前仅鉴定出 72 个上调基因中的 4 个。
我们鉴定出的这些在体内上调、血清型间保守且编码潜在外膜蛋白的基因是开发针对猪传染性胸膜肺炎的交叉保护性疫苗的潜在候选基因。
