Polissi A, Pontiggia A, Feger G, Altieri M, Mottl H, Ferrari L, Simon D
Department of Microbiology, Medicine Research Centre, Glaxo Wellcome S.p.A., 37100 Verona, Italy.
Infect Immun. 1998 Dec;66(12):5620-9. doi: 10.1128/IAI.66.12.5620-5629.1998.
Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.
肺炎链球菌是细菌性肺炎的主要病因，也是儿童中耳炎和脑膜炎的病原体。除荚膜外，该病原体的毒力因子尚未完全明确。细菌致病机制领域（体内表达技术和标记突变技术[STM]）的最新技术进展使得大规模鉴定毒力基因成为可能。我们将最初用于发现沙门氏菌致病相关基因的STM技术应用于肺炎链球菌。在携带独特DNA序列标签和氯霉素抗性标记的自杀质粒载体中构建了肺炎链球菌染色体片段文库（400至600 bp）。用该文库转化近期临床分离株G54。通过同源重组获得氯霉素抗性突变体，导致携带独特标签的自杀载体插入使基因失活。在小鼠肺炎模型中，筛选了1250个候选克隆；其中200个在肺部未被回收，因此被认为是毒力减弱的突变体。通过反向PCR扩增减毒突变体中氯霉素基因两侧的区域并进行测序。序列分析表明，200个突变体在126个不同基因中存在插入，这些基因可分为六类：（i）已知的肺炎链球菌毒力基因；（ii）参与代谢途径的基因；（iii）编码蛋白酶的基因；（iv）编码ATP结合盒转运蛋白的基因；（v）编码参与DNA重组/修复的蛋白质的基因；（vi）与功能未知的假设基因具有相似性的DNA序列。为了评估每个突变体的毒力减弱情况，在小鼠败血症模型中对所有126个克隆进行了单独分析。并非所有在肺炎模型中筛选出的突变体在败血症模型中都得到证实，这表明存在肺炎特异性毒力因子。
