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.

项目概要 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。针对百日咳博德特氏菌的无细胞疫苗，自 20世纪90年代，可以预防严重疾病，但不能定植或传播，从而导致更大的储存库 最容易受到感染的婴儿可能会被感染。可预防这两种疾病的新疫苗 需要殖民和疾病。 BvgAS 和 PlrSR 双组分系统控制博德特氏菌毒力。 BvgAS 双组分调控系统（TCS）长期以来被认为是博德特氏菌的主要调控因子 毒力。它控制所有已知蛋白质毒力因子的产生，包括非细胞中的毒力因子 疫苗。我们发现了另一种 TCS，称为 PlrSR，它对于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 处于组成型活性时，表明 PlrSR 控制未识别但 临界毒力功能独立于 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，因此它们的鉴定 对于未来控制百日咳至关重要。 博士。 Cotter 和 Julio 是博德特氏菌发病机制和分子生物学方面的专家。 Bourret 是 TCS 生物化学方面的专家。我们共同鉴定了 PlrS 和 PlrR 蛋白的特征 并发现了一种在体外绕过 plrS 明显必要性的方法，以便 我们可以构建菌株来收集基因表达数据，而无需知道 PlrS 感知的刺激。 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 调节，以及 ChIP-Seq 来揭示直接或间接调节。在目标 2 中，我们将调查 PlrSR 通过与关键 PlrSR 调节基因进行报告基因融合并评估对信号的反应来发出信号 生理相关刺激，以及使用缺乏 PDC 或 PAS 的 PlrS 的后果 感觉域。 PlrSR 调节子的识别是低风险/高回报的，将为后续研究奠定基础。 R01项目。所提出的方法是行之有效的，表明实现的可能性很高 我们的目标。识别 PlrSR 调节子将彻底改变对博德特氏菌的理解 发病机制并启用未来的 R01 项目，在该项目中我们可以确定 (i) 为什么 PlrR 对于 活力，(ii) PlrSR 调节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 之间的连接，以及 (v) 也许 深入了解 PlrS 感受到的刺激。