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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

B. burgdorferi在两个非常不同的环境中保持了自然界，即ixodes tick和哺乳动物宿主。它在这些非常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/或暴露于宿主因子并相应地改变基因表达的能力。细菌调节转录起始的能力允许在适当的时间协调基因的表达。先前的报道表明，这些响应调节的核心是Sigmas（由RPOS编码）和Sigma54（由RPON编码也称为NTRA）。此外，SigMA54依赖性sigmas的表达负责关键毒力因子的表达，例如，外表面蛋白C（OSPC），OSPA和DecorIn结合蛋白C（DBPC）在感染周期中需要进行感染性和传播所需的传播。 SIGMA54-RNA聚合酶全酶（SigMA54-氢酶）认可的启动子在SIGMA4和SIGMA12位置分别具有良好的GG和GC Doublet，相对于转录起始位点，而不是典型的-35/-10盒子在SIGMA70类促销员中。普遍地，已证明SigMA54依赖性转录需要激活蛋白，其中许多是两个组分系统的响应调节剂。在大多数情况下，SIGMA54-激活蛋白结合了启动子转录起始位点上游的100-200 bp的增强子样元素。 B. burgdorferi基因组的分析将RRP2（BBO763）鉴定为由N末端接收器结构域，中央ATPase结构域和C端DNA结合域组成的SIGMA54依赖性激活剂。 Sigma54-氢酶的活化剂的活性受环境信号的响应。＃M54-氢酶的许多活化剂是两组分组中的响应调节剂，这些蛋白质的磷酸化导致它们的激活。这些响应调节剂的同源蛋白组氨酸激酶会响应环境信号而磷酸化。一旦磷酸化，响应调节剂就会激活其他基因的转录。 B. burgdorferi，RRP2（由RRP2编码）中Sigma54-氢酶的激活剂也是两个组件系统的响应调节剂，RRP2的响应调节剂与编码编码其认知蛋白组蛋白激酶的HK2（由HK2编码）的基因中。 Sigma54依赖性辅助激活需要响应调节剂RRP2（BB0763）。纯化的重组RRP2在凝胶型移动转移测定中没有与B. burgdorferi rpos上游的DNA结合，RRP2-DELTA123也没有RRP2的RRP2-DELTA123的RRP2的截短活性形式。 RRP2-DELTA123中RPOS-LACZ报告基因在大肠杆菌中的转录激活取决于Sigma54，但不需要RPOS启动子上游的DNA序列。同样，在B. burgdorferi中使用CAT报告基因的定量RT-PCR实验表明，RRP2不需要SigMA54依赖性RPOS启动子上游的DNA序列激活RPOS P/O-o-cat报告基因构建体的RRP2的转录。综上所述，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，随着培养物进入固定相，RPON在B. burgdorferi中表达RPON需要RPON，并且在此期间需要HK2才能最佳地表达RPO，但并非绝对必要。这表明，除HK2外，另一种组氨酸激酶或小分子量分子可以用作RRP2的磷酸盐供体，为调节RPOS和毒力相关蛋白（例如OSPC）的表达提供了多种信号通路。我们研究的这一方面的重点是进一步表征B. burgdorferi中涉及Sigmas和Sigma54的调节级联，并确定促进细菌在tick中肠和人类宿主中促进细菌存活的细胞外信号。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.