The Role Of Alternate Sigma Factors In The Transmission Cycle Of B. Burgdorferi

替代西格玛因子在伯氏疏螺旋体传播周期中的作用

• 批准号: 7732571
• 负责人: Frank Gherardini
• 金额: $ 39.04万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732571
• 关键词: ATPase Domain; Bacteria; Bacteria sigma factor KatF protein; Binding; Borrelia burgdorferi; Boxing; C-terminal; Cell Density; Class; DNA; DNA Binding Domain; DNA Sequence; DNA-Directed RNA Polymerase; Elements; Enhancers; Environment; Escherichia coli; Exposure to; Felis catus; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; HK2 gene; Holoenzymes; Human; Human Glandular Kallikrein 2; Integration Host Factors; Ixodes; LacZ Genes; Midgut; Molecular Weight; N-terminal; Nature; Operon; OspA protein; OspC protein; Oxygen; Penetration; Phase; Phosphorylation; Positioning Attribute; Proteins; RNA polymerase sigma 54; Recombinants; Regulation; Relative (related person); Reporter; Reporter Genes; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Salivary Glands; Sigma Factor; Signal Pathway; Signal Transduction; Simulate; Site; System; Temperature; Ticks; Time; Transcription Initiation; Transcriptional Activation; Virulence; Virulence Factors; decorin binding protein C; enhancer binding protein; extracellular; gel mobility shift assay; genetic regulatory protein; inorganic phosphate; mutant; promoter; protein-histidine kinase; research study; response; transmission process

B. burgdorferi is maintained in nature in two very distinct environments, the Ixodes tick and mammalian hosts. Its ability to adapt and survive in these very different environments is attributed to its ability to sense changes in temperature, pH, cell density, oxygen and/or exposure to host factors and alter gene expression accordingly. The capability of bacteria to regulate transcription initiation allows for the coordinated expression of genes at appropriate times. Previous reports have demonstrated that central to the regulation of these responses are SigmaS (encoded by rpoS) and Sigma54 (encoded by rpoN also known as ntrA). In addition, Sigma54-dependent expression of SigmaS is responsible for the expression of key virulence factors e.g., outer surface protein C (OspC), OspA and decorin-binding protein C (DbpC) required for infectivity and transmission during the infective cycle. Promoters recognized by Sigma54-RNA polymerase holoenzyme (Sigma54-holoenzyme) have well conserved GG and GC doublets at Sigma4 and Sigma12 positions, respectively, relative to the transcriptional start site, instead of the typical -35/-10 boxes observed in Sigma70 class promoters. Universally, Sigma54-dependent transcription has been shown to require activator proteins, many of which are response regulators of two-component systems. In most cases, Sigma54-activator proteins bind enhancer-like elements located 100-200 bp upstream of the transcriptional start site of the promoter. Analysis of the genome of B. burgdorferi identified Rrp2 (BBO763) as a predicted Sigma54-dependent activator consisting of an N-terminal receiver domain, a central ATPase domain and a C-terminal DNA-binding domain. The activities of activators of Sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of #m54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. An activator of Sigma54-holoenzyme in B. burgdorferi, Rrp2 (encoded by rrp2), is also a response regulator of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, hk2 (encoded by hk2). The Sigma54-dependent activation of SigmaS requires response regulator, Rrp2 (BB0763). Purified recombinant Rrp2 did not bind to the DNA upstream of B. burgdorferi rpoS in gel-mobility shift assays, nor did Rrp2-delta123, a truncated constitutively active form of Rrp2. Transcriptional activation of a rpoS-lacZ reporter gene in E. coli by Rrp2-delta123 was dependent on sigma54 but did not require DNA sequences upstream of the rpoS promoter. Similarly, quantitative RT-PCR experiments using a cat reporter gene in B. burgdorferi indicated that DNA sequences upstream of the sigma54-dependent rpoS promoter were not needed by Rrp2 to activate transcription of the rpoS P/O-cat reporter construct. Taken together, these findings suggest that unlike expression from most sigma54-dependent promoters, Rrp2 does not utilize an enhancer-like sequence to activate transcription of rpoS. Finally, quantitative RT-PCR showed that rpoN is required for expression of rpoS in B. burgdorferi as cultures enter stationary phase, and that hk2 is needed for optimal expression of rpoS during this time but is not absolutely essential. This suggests that, in addition to Hk2, another histidine kinase or small molecular weight molecule can serve as a phosphate donor to Rrp2, providing multiple signaling pathways for modulating the expression of RpoS and virulence related proteins, such as OspC. The focus of this aspect of our research is to further characterize this regulatory cascade involving SigmaS and Sigma54 in B. burgdorferi and to determine the extracellular signals that promote the survival of the bacterium in the tick midgut and in a human host.

伯氏疏螺旋体在自然界中存在于两种截然不同的环境中：硬蜱和哺乳动物宿主。它在这些截然不同的环境中适应和生存的能力归因于其感知温度、pH、细胞密度、氧气和/或暴露于宿主因素的变化并相应改变基因表达的能力。 细菌调节转录起始的能力允许基因在适当的时间协调表达。之前的报告表明，调节这些反应的核心是SigmaS（由rpoS编码）和Sigma54（由rpoN编码，也称为ntrA）。此外，SigmaS 的 Sigma54 依赖性表达负责关键毒力因子的表达，例如感染周期中感染性和传播所需的外表面蛋白 C (OspC)、OspA 和核心蛋白聚糖结合蛋白 C (DbpC)。 Sigma54-RNA聚合酶全酶（Sigma54-holoenzyme）识别的启动子相对于转录起始位点，分别在Sigma4和Sigma12位置具有非常保守的GG和GC双联体，而不是在Sigma70类启动子中观察到的典型-35/-10框。 一般来说，Sigma54 依赖性转录已被证明需要激活蛋白，其中许多是双组分系统的反应调节因子。在大多数情况下，Sigma54 激活蛋白结合位于启动子转录起始位点上游 100-200 bp 的增强子样元件。对伯氏疏螺旋体基因组的分析发现，Rrp2 (BBO763) 是一种预测的 Sigma54 依赖性激活剂，由 N 端受体结构域、中央 ATP 酶结构域和 C 端 DNA 结合结构域组成。 Sigma54-全酶激活剂的活性根据环境信号进行调节。 #m54-全酶的许多激活剂是双组分调节系统中的反应调节剂，这些蛋白质的磷酸化导致它们的激活。 这些反应调节剂响应环境信号而被其同源蛋白组氨酸激酶磷酸化。 一旦磷酸化，反应调节器就会激活其他基因的转录。 Rrp2（由rrp2编码）是博氏疏螺旋体中Sigma54全酶的激活剂，也是双组分系统的反应调节剂，rrp2位于操纵子中，其基因编码其同源蛋白组氨酸激酶hk2（由hk2编码） ）。 SigmaS 的 Sigma54 依赖性激活需要响应调节因子 Rrp2 (BB0763)。在凝胶迁移率变化分析中，纯化的重组 Rrp2 不与伯氏疏螺旋体 rpoS 上游的 DNA 结合，Rrp2-delta123（Rrp2 的截短组成型活性形式）也不结合。 Rrp2-delta123 对大肠杆菌中 rpoS-lacZ 报告基因的转录激活依赖于 sigma54，但不需要 rpoS 启动子上游的 DNA 序列。类似地，使用伯氏疏螺旋体中的 cat 报告基因进行的定量 RT-PCR 实验表明，Rrp2 不需要 sigma54 依赖性 rpoS 启动子上游的 DNA 序列来激活 rpoS P/O-cat 报告基因构建体的转录。总而言之，这些发现表明，与大多数 sigma54 依赖性启动子的表达不同，Rrp2 不利用增强子样序列来激活 rpoS 的转录。 最后，定量RT-PCR表明，当培养物进入稳定期时，伯氏疏螺旋体中的rpoS表达需要rpoN，并且在此期间rpoS的最佳表达需要hk2，但不是绝对必需的。这表明，除了Hk2之外，另一种组氨酸激酶或小分子量分子可以作为Rrp2的磷酸盐供体，提供多种信号通路来调节RpoS和毒力相关蛋白（例如OspC）的表达。我们这方面研究的重点是进一步表征伯氏疏螺旋体中涉及 SigmaS 和 Sigma54 的调控级联，并确定促进蜱中肠和人类宿主中细菌存活的细胞外信号。

项目成果

Defining plasmids required by Borrelia burgdorferi for colonization of tick vector Ixodes scapularis (Acari: Ixodidae).

定义伯氏疏螺旋体 (Borrelia burgdorferi) 定植蜱载体肩突硬蜱 (蜱螨：硬蜱科) 所需的质粒。

• DOI: 10.1093/jmedent/42.4.676
• 发表时间: 2005
• 期刊: Journal of medical entomology
• 影响因子: 2.1
• 作者: Grimm,Dorothee;Tilly,Kit;Bueschel,DawnM;Fisher,MarkA;Policastro,PaulF;Gherardini,FrankC;Schwan,TomG;Rosa,PatriciaA
• 通讯作者: Rosa,PatriciaA

Insights into the complex regulation of rpoS in Borrelia burgdorferi.

• DOI: 10.1111/j.1365-2958.2007.05813.x
• 发表时间: 2007-07
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Burtnick MN;Downey JS;Brett PJ;Boylan JA;Frye JG;Hoover TR;Gherardini FC
• 通讯作者: Gherardini FC