Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria, Australia.
PLoS Pathog. 2010 Feb 5;6(2):e1000750. doi: 10.1371/journal.ppat.1000750.
P. multocida is the causative agent of a wide range of diseases of animals, including fowl cholera in poultry and wild birds. Fowl cholera isolates of P. multocida generally express a capsular polysaccharide composed of hyaluronic acid. There have been reports of spontaneous capsule loss in P. multocida, but the mechanism by which this occurs has not been determined. In this study, we identified three independent strains that had spontaneously lost the ability to produce capsular polysaccharide. Quantitative RT-PCR showed that these strains had significantly reduced transcription of the capsule biosynthetic genes, but DNA sequence analysis identified no mutations within the capsule biosynthetic locus. However, whole-genome sequencing of paired capsulated and acapsular strains identified a single point mutation within the fis gene in the acapsular strain. Sequencing of fis from two independently derived spontaneous acapsular strains showed that each contained a mutation within fis. Complementation of these strains with an intact copy of fis, predicted to encode a transcriptional regulator, returned capsule expression to all strains. Therefore, expression of a functional Fis protein is essential for capsule expression in P. multocida. DNA microarray analysis of one of the spontaneous fis mutants identified approximately 30 genes as down-regulated in the mutant, including pfhB_2, which encodes a filamentous hemagglutinin, a known P. multocida virulence factor, and plpE, which encodes the cross protective surface antigen PlpE. Therefore these experiments define for the first time a mechanism for spontaneous capsule loss in P. multocida and identify Fis as a critical regulator of capsule expression. Furthermore, Fis is involved in the regulation of a range of other P. multocida genes including important virulence factors.
多杀性巴氏杆菌是多种动物疾病的病原体，包括家禽霍乱和野生鸟类。多杀性巴氏杆菌的禽霍乱分离株通常表达由透明质酸组成的荚膜多糖。已经有报道称多杀性巴氏杆菌会自发失去荚膜，但发生这种情况的机制尚未确定。在这项研究中，我们鉴定了三个自发丧失产生荚膜多糖能力的独立菌株。定量 RT-PCR 显示，这些菌株荚膜生物合成基因的转录显著减少，但 DNA 序列分析未发现荚膜生物合成基因座内的突变。然而，配对有荚膜和无荚膜菌株的全基因组测序鉴定出无荚膜菌株中 fis 基因内的单个点突变。来自两个独立衍生的自发无荚膜菌株 fis 的测序表明，每个菌株都含有 fis 内的突变。用 fis 的完整拷贝对这些菌株进行互补，预测编码转录调节剂，使所有菌株恢复荚膜表达。因此，功能性 Fis 蛋白的表达对于多杀性巴氏杆菌荚膜表达是必需的。对其中一个自发 fis 突变株的 DNA 微阵列分析鉴定出大约 30 个基因在突变体中下调，包括 pfhB_2，它编码一种已知的多杀性巴氏杆菌毒力因子丝状血凝素，和 plpE，它编码交叉保护表面抗原 PlpE。因此，这些实验首次定义了多杀性巴氏杆菌自发荚膜缺失的机制，并将 Fis 鉴定为荚膜表达的关键调节剂。此外，Fis 参与调节一系列其他多杀性巴氏杆菌基因，包括重要的毒力因子。
