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.

项目摘要 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。自2000年以来在美国使用的针对百日咳杆菌的无细胞疫苗 20世纪90年代，预防严重疾病，但不能殖民化或传播，导致更大的水库 而婴儿是最容易受到感染的人群。新的疫苗可以预防这两种疾病 殖民化和疾病是必要的。 BvgAS和PlrSR双组分系统控制博德特氏菌的毒力。关于BvgAS 双组分调节系统（TCS）一直被认为是博德特氏菌的主要调节因子 毒性它控制所有已知的蛋白质毒力因子的产生，包括非细胞内的毒力因子。 疫苗我们发现了另一种TCS，称为PlrSR，它对博德特氏菌的生存能力和 下呼吸道（LRT）中的BvgAS活性。我们发现LRT感染需要PlrSR 即使当BvgAS是组成型活性的，表明PlrSR控制未鉴定的但 关键毒力功能独立于BvgAS。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， 是未来控制百日咳的关键。 科特博士和胡里奥博士是博德特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。总之，我们已经表征了PlrS和PlrR蛋白 生物化学，并已发现一种方法，绕过体外plrS的明显必要性， 我们可以构建菌株来收集基因表达数据，而不需要知道PlrS所感知的刺激。 目的1：利用RNA-Seq技术鉴定PlrSR调控的基因， 调控和ChIP-Seq以揭示直接或间接调控。在目标2中，我们将研究PlrSR 通过与关键的PlrSR调节基因进行报告基因融合并评估对 生理相关刺激，以及使用缺乏PDC或PAS的PlrS的后果 感觉域 PlrSR调节子的鉴定是低风险/高回报的，并将为研究PlrSR调节子奠定基础。 R 01项目。所提出的方法是行之有效的，这表明很有可能实现 我们的目标鉴定PlrSR调节子将对理解博德特氏菌具有变革性意义 发病机制，并使未来的R 01项目，我们可以确定（i）为什么PlrR是必不可少的， 存活力，（ii）PlrSR调控的基因产物在博德特氏菌LRT感染中的作用，（iii）PlrSR如何 调节基因表达，（iv）PlrSR和BvgAS TCS之间的联系，以及（v）可能 深入了解PlrS感知的刺激。