Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis
Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis
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DOI：
10.1099/jmm.0.2008/002071-0
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发表时间：
2008-09-01
影响因子：
3
通讯作者：
Mobley, Harry L. T.
中科院分区：
文献类型：
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作者：
Himpsl, Stephanie D.;Lockatell, C. Virginia;Mobley, Harry L. T.
革兰氏阴性菌奇异变形杆菌可导致长期留置导尿管或尿路功能或结构异常的个体发生尿路感染（UTI）。已知的毒力因子包括尿素酶、溶血素、菌毛、鞭毛、DsbA、磷酸盐转运蛋白和参与细胞壁合成和代谢的基因，其中许多已经使用特征标记诱变（STM）技术鉴定。为了鉴定其他毒力决定因子并增加基因组的理论覆盖率，本研究使用该方法产生并评估了1880个奇异变形杆菌菌株HI 4320突变体。在约315只CBA小鼠中进行初次和二次体内筛选，在Luria肉汤和基本A培养基中进行初次和二次体外筛选以消除具有轻微生长缺陷的突变体，以及在约500只CBA小鼠中进行共激发竞争实验后，鉴定出对上行性UTI CBA小鼠模型定殖至关重要的基因中断的突变体。完成体内筛选后，在上行性UTI的CBA小鼠模型中总共减毒了217个转座子突变体。体外筛选后，这一数字减少到196个转座子突变体，可能在毒力中发挥作用。共攻击竞争实验证实了所测试的93个转座子突变体中的37个的显著衰减，被野生型HI 4320击败。对37个突变体进行序列分析后，在包括肽基脯氨酰异构酶surA和ppiA、糖基转移酶cpSF、生物聚合物转运蛋白exbD、转录调节因子nhaR、一种推定的菌毛蛋白、鞭毛缠绕蛋白fliF和钩蛋白flgE以及多个代谢基因的基因中鉴定出转座子插入。
The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar Wring protein fliF and hook protein flgE, and multiple metabolic genes.