A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues.
A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues.
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DOI：
10.3791/59055
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发表时间：
2019-02
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通讯作者：
R. Behera;K. D. Mlynek;Matthew S Linz;Shaun R. Brinsmade
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文献类型：
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作者：
R. Behera;K. D. Mlynek;Matthew S Linz;Shaun R. Brinsmade
细菌毒力基因通常在转录水平上受到多种因子的调控，这些因子响应不同的环境信号。有些因子直接作用于毒力基因;另一些因子通过调节下游调节因子的表达或影响调节因子活性的信号的积累来控制发病机制。虽然在体外生长过程中已广泛研究了调控，但对感染过程中基因表达如何调节知之甚少。当特定基因产物是治疗干预的候选者时，这样的信息是重要的。定量、实时RT-PCR和RNA-Seq等转录方法是在全球水平上检测基因表达的有力方法，但面临许多技术挑战，包括与宿主RNA相比细菌RNA丰度低，以及RNA酶对样品的降解。使用荧光报告分子评估调控相对容易，并且可以与具有独特光谱特性的荧光蛋白进行复用。该方法允许单细胞，时空分析组织中的基因表达，表现出复杂的三维结构和影响细菌调控网络的生理化学梯度。当数据在总体上平均时，这种信息就丢失了。在此，我们描述了一种用于原位定量细菌病原体中的基因表达的方法。该方法基于简单的组织处理和直接观察报告蛋白的荧光。我们证明了这个系统的效用，通过检查金黄色葡萄球菌热单核酸酶（nuc），其基因产物的表达是所需的免疫逃避和完整的毒力离体和体内。我们发现nuc-gfp在肾肿瘤中强烈表达，并揭示了异质性基因表达，部分原因是在完全参与免疫应答的肿瘤中，nuc启动子活性的明显空间调节。该方法可以应用于任何具有可操纵遗传系统的细菌和任何感染模型，为临床前研究和药物开发提供有价值的信息。
Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.