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首页> 外文期刊>Infection and immunity >Expression Analysis of the Yersiniabactin Receptor GenefyuA and the Heme Receptor hemR ofYersinia enterocolitica In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase
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Expression Analysis of the Yersiniabactin Receptor GenefyuA and the Heme Receptor hemR ofYersinia enterocolitica In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase

机译：使用绿色荧光蛋白和荧光素酶报道基因对小肠结肠炎耶尔森氏菌耶尔森菌素受体基因fyuA和血红素受体hemR的表达进行体内和体外表达分析

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The enteropathogenic Yersinia enterocolitica strains have several systems for scavenging iron from their environment. We have studied the expression of the fyuA gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the hemR gene, which encodes the receptor for heme, using the reporter genes gfp (encoding green fluorescent protein) and luc (encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of fyuA andhemR or expression of one gene by agfp-luc tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that fyuA and hemR are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27°C. In the in vivo BALB/C mouse infection model, tissue-specific expression of fyuA andhemR reporter fusions was observed. Surprisingly,fyuA and hemR reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying fyuA orhemR reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of fyuA and hemRunder peritoneal growth conditions. Differential expression of thefyuA and hemR reporter fusions could not be observed, suggesting similar regulation of fyuA andhemR in the mouse infection model.
机译：肠致病性小肠结肠炎耶尔森氏菌菌株具有几种从环境中清除铁的系统。我们研究了 fyuA 基因的表达，该基因编码铁载体耶尔西菌素（Ybt）的外膜受体，而 hemR 基因编码血红素的受体，使用报告基因 gfp （编码绿色荧光蛋白）和 luc （编码萤火虫荧光素酶）。为了研究体内外的基因表达，我们构建了几种翻译报告基因融合体，以同时监控 fyuA 和 hemR 的表达或a < em> gfp - luc 串联报告程序。体外表达分析（液体培养基）的结果表明， fyuA 和 hemR 在铁饥饿条件下被强烈抑制，导致在27°C时强烈的荧光和/或发光。在体内BALB / C小鼠感染模型中，观察到了 fyuA 和 hemR 报告基因融合蛋白的组织特异性表达。令人惊讶的是， fyuA 和 hemR 报告基因构建体在肝和肠腔中的耶尔森菌表达较弱，而在腹膜腔中发现了耶尔森氏菌的强表达，并发现了中等表达在脾脏中。引人注目的是，携带 fyuA 或 hemR 报告基因融合体的耶尔森氏菌在小鼠腹腔中生长时的信号强度要比在体外铁抑制下生长的信号强度高三倍。这种过度表达表明，除了Fur抑制外，在腹膜生长条件下，其他激活剂可能还参与了 fyuA 和 hemR 的增强表达。无法观察到 fyuA 和 hemR 报告基因融合体的差异表达，表明 fyuA 和 hemR 在小鼠中的调控相似。小鼠感染模型。

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