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首页> 外文期刊>Infection and immunity >Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection
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Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

机译：在实验性升尿路感染过程中使用绿色荧光蛋白评估尿路致病性变形杆菌变形酶基因表达的用途

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Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made withgfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5α and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression ofP. mirabilis virulence genes in experimental urinary tract infection.
机译：复杂尿路感染的病因 Mirabilis ，当暴露于尿素时会表达尿素酶。尽管认识到正转录激活因子UreR诱导基因表达，但在实验感染过程中酶的表达水平尚不清楚。为了研究 P的体内表达。编码绿色荧光蛋白（GFP）的基因mirabilis 脲酶被用于构建报告基因融合体。用 gfp （修饰后可表达S65T / V68L / S72A [BP Cormack等人），合成尿素酶辅助基因 ureD 的翻译融合体，该融合体以尿素诱导型启动子为先导。 [Gene 173：33-38，1996]）。通过测序融合接头确认构建体。 UreD-GFP融合蛋白在大肠杆菌DH5α和 P中均被尿素诱导。奇迹 HI4320。通过使用Western blotting和针对GFP产生的抗血清，表明表达水平与尿素浓度相关（测试范围为0至500 mM），在200至500 mM尿素中诱导最高。荧光E.大肠菌和 P。尿素诱导后，用荧光显微镜观察了奇异菌，并用分光光度法测定了细胞裂解液中GFP的荧光强度。 P。将带有UreD-GFP融合质粒的mirabilis HI4320经尿道接种到CBA小鼠的膀胱中。攻击后一周，在膀胱和肾脏样本的薄切片中检测到荧光细菌。膀胱组织中细菌的荧光强度高于肾脏。肾脏主要感染单细胞形式的荧光细菌，而在膀胱中观察到聚集的细菌簇。仅很少观察到细长的群体细胞。这些观察结果表明脲酶在体内表达，并且使用GFP作为报道蛋白是研究em P在体内表达的可行方法。实验性尿路感染中的奇异菌毒力基因。

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