Identifying the Bordetella PlrSR regulon

鉴定博德特氏菌 PlrSR 调节子

• 批准号: 10722876
• 负责人: Robert B. Bourret
• 金额: $ 24.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722876
• 关键词: Acellular Vaccines; Binding Sites; Biochemical; Biochemistry; Bordetella; Bordetella Infections; Bordetella bronchiseptica; Bordetella pertussis; Bypass; Carbon Dioxide; ChIP-seq; Collaborations; Country; Dangerousness; Data; Data Analyses; Disease; Epitopes; Expert Systems; Foundations; Future; Gene Expression; Genes; Goals; Homologous Gene; Human; Immune response; In Vitro; Infant; Infection; Investigation; Knowledge; Laboratories; Link; Lower Respiratory Tract Infection; Lower respiratory tract structure; Mammals; Measures; Methods; Molecular Biology; Mus; Oxidative Stress; Pathogenesis; Pertussis; Pertussis Vaccine; Physiological; Population; Probability; Production; Proteins; Public Health; Quantitative Reverse Transcriptase PCR; Regulation; Regulon; Reporter; Repression; Respiratory Disease; Risk; Role; Sensory; Signal Transduction; Stimulus; Study models; System; Therapeutic; Time; Training; United States; Vaccinated; Vaccinee; Vaccines; Virulence; Virulence Factors; gene product; high reward; improved; in vivo; insight; model organism; novel vaccines; prevent; respiratory; respiratory pathogen; response; therapeutic target; transcriptome sequencing; transmission process; trend; vaccine access; vaccine candidate

PROJECT SUMMARY Pertussis (aka whooping cough) is a serious, re-emerging public health concern despite available vaccines. The acellular vaccine against Bordetella pertussis, used in the U.S. since the 1990s, prevents serious disease, but not colonization or transmission, resulting in a larger reservoir from which infants, who are most vulnerable, can be infected. New vaccines that protect against both colonization and disease are needed. The BvgAS and PlrSR two-component systems control Bordetella virulence. The BvgAS two-component regulatory system (TCS) has long been considered the master regulator of Bordetella virulence. It controls production of all know protein virulence factors, including those in the acellular vaccine. We discovered another TCS, called PlrSR, that is essential for Bordetella viability and for BvgAS activity in the lower respiratory tract (LRT). We found that PlrSR is required for LRT infection even when BvgAS is constitutively active, indicating that PlrSR controls expression of unidentified but critical virulence functions independently of BvgAS. These currently unknown virulence factors could serve as therapeutic targets or new vaccine components, and hence their identification is critical for controlling pertussis in the future. Drs. Cotter and Julio are experts in Bordetella pathogenesis and molecular biology, and Dr. Bourret is an expert in TCS biochemistry. Together, we have characterized PlrS and PlrR proteins biochemically and have discovered a way to bypass the apparent essentiality of plrS in vitro so that we can construct strains to collect gene expression data without knowing the stimuli sensed by PlrS. In Aim 1, we will identify genes regulated by PlrSR using RNA-Seq to reveal positive or negative regulation, and ChIP-Seq to reveal direct or indirect regulation. In Aim 2, we will investigate PlrSR signaling by making reporter fusions to key PlrSR-regulated genes and assessing responses to physiologically relevant stimuli, as well as the consequences of using PlrS lacking PDC or PAS sensory domains. Identification of the PlrSR regulon is low risk/high reward and will lay the foundation for a R01 project. The proposed methods are well-established, suggesting a high probability of achieving our Aims. Identifying the PlrSR regulon will be transformative in understanding Bordetella pathogenesis and enable a future R01 project in which we can determine (i) why PlrR is essential for viability, (ii) the roles of PlrSR regulated gene products in LRT infection by Bordetella, (iii) how PlrSR regulates gene expression, (iv) connections between the PlrSR and BvgAS TCSs, and (v) perhaps gain insight into the stimuli sensed by PlrS.

项目总结 百日咳(又名百日咳)是一个严重的、重新出现的公共卫生问题，尽管 可用的疫苗。百日咳波氏杆菌无细胞疫苗自美国使用以来 20世纪90年代，预防严重疾病，但不能殖民或传播，导致更大的水库 最容易感染的婴儿可能会从这种病毒感染。既能预防这两种疾病的新型疫苗 殖民和疾病是必要的。 BvgAS和PlrSR双组分系统控制波尔德氏菌的毒力。Bvgas 双元调控系统(TCS)长期以来一直被认为是波氏杆菌的主要调控机制 致命性。它控制着所有已知的蛋白质毒力因子的产生，包括那些在非细胞中的那些 疫苗。我们发现了另一种称为PlrSR的TCS，它对波尔德氏菌的生存和 下呼吸道(LRT)BvgAS活性。我们发现PlrSR是LRT感染所必需的 即使当BvgAS具有结构性活性时，也表明PlrSR控制着未鉴定的但 临界毒力独立于BvgAS发挥作用。这些目前未知的毒力因子 可以作为治疗靶点或新的疫苗成分，因此它们的识别 对未来控制百日咳至关重要。 科特博士和胡里奥博士都是波尔德氏菌致病机理和分子生物学方面的专家。 Bourret是TCS生物化学方面的专家。我们共同研究了PLRs和PlrR蛋白 并发现了一种在体外绕过PLRs表面重要性的方法，从而使 我们可以构建菌株来收集基因表达数据，而不需要知道PLR感受到的刺激。 在目标1中，我们将使用rna-seq识别受plrsr调控的基因，以揭示阳性或阴性。 监管，和芯片序列揭示直接或间接的监管。在目标2中，我们将调查PlrSR 通过与PlrSR调节的关键基因进行报告融合并评估对 生理相关刺激，以及使用不含PDC或PAS的PLR的后果 感觉域。 识别PlrSR调节子是低风险/高回报的，并将为 R01项目。建议的方法是成熟的，表明很有可能实现 我们的目标。识别PlrSR调节子将对理解波尔德氏菌产生革命性的影响 发病机制，并支持未来的R01项目，在该项目中，我们可以确定(I)为什么PlrR对 活性，(Ii)PlrSR调控的基因产物在波氏杆菌感染LRT中的作用，(Iii)PlrSR如何 调节基因表达，(Iv)PlrSR和BvgAS TCS之间的连接，以及(V)可能 洞察PLR感知到的刺激。