de Lima Pimenta Andréa, Di Martino Patrick, Le Bouder Emmanuel, Hulen Christian, Blight Mark A
Laboratoire ERRMECe, Groupe Interactions Cellulaires, Université de Cergy-Pontoise, 2, avenue A. Chauvin, BP222, 95302 Cergy-Pontoise cedex, France.
Microbes Infect. 2003 Nov;5(13):1177-87. doi: 10.1016/j.micinf.2003.09.002.
By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants. Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster. Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants. A search in the databanks identified two loci in the Pseudomonas aeruginosa PAO1 genome with significant homology to the genes interrupted by the transposon insertions. Mutant IVC6 shows homology to gmd, coding for the enzyme GDP-mannose dehydratase, involved in the synthesis of A-band- O-antigen-containing lipopolysaccharide (LPS). Mutant IVG7 is significantly similar to a probable outer membrane protein of strain PAO1, with no specific function attributed thus far, yet with significant homology to Escherichia coli FadL, involved in long-chain fatty acid transport. We propose that this protein, together with LPS, is involved in the first steps of P. fluorescens adherence leading to host colonisation. Results presented here also demonstrate the pathogenic potential of P. fluorescens, assessed in an in vivo Drosophila model system, correlated with its ability to adhere to the human extracellular matrix protein, fibronectin. Correlation between the mutant phenotypes with identified virulence factors and their actual role in the virulence of P. fluorescens is discussed.
通过富集荧光假单胞菌的随机转座子插入文库，筛选出对人细胞外基质蛋白纤连蛋白黏附能力有缺陷的突变体，我们分离得到了23个黏附缺陷型突变体。这些突变体在非生物表面形成生物膜的能力存在缺陷，并且在果蝇体内毒力模型中测试时毒力受损。对这些突变体的分子特征分析表明，转座子插入定位在两个不同的染色体位置，随后从两个突变体中克隆并鉴定了这两个位置。在数据库中搜索发现，铜绿假单胞菌PAO1基因组中有两个位点与被转座子插入中断的基因具有显著同源性。突变体IVC6与编码GDP - 甘露糖脱水酶的gmd基因同源，该酶参与含A带O抗原的脂多糖（LPS）的合成。突变体IVG7与PAO1菌株的一种可能的外膜蛋白显著相似，目前尚未赋予其特定功能，但与参与长链脂肪酸运输的大肠杆菌FadL具有显著同源性。我们认为这种蛋白与LPS一起，参与了荧光假单胞菌黏附宿主并导致宿主定植的第一步。本文给出的结果还表明，在果蝇体内模型系统中评估的荧光假单胞菌的致病潜力与其黏附人细胞外基质蛋白纤连蛋白的能力相关。文中讨论了突变体表型与已鉴定的毒力因子之间的相关性以及它们在荧光假单胞菌毒力中的实际作用。
