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 可用的疫苗。针对Bordetella百日咳的细胞疫苗在美国使用 1990年代可以防止严重的疾病，但不能定植或传播，导致较大的储层 最脆弱的婴儿可以从中感染。预防两者的新疫苗 需要定植和疾病。 BVGA和PLRSR两组分系统控制Bordetella毒力。 BVGAS 长期以来，两个组件调节系统（TC）长期以来一直被认为是Bordetella的主要调节器 毒力。它控制着所有已知蛋白毒力因子的生产，包括细胞中的蛋白质毒力因子 疫苗。我们发现了另一个称为PLRSR的TC，这对于Bordetella的生存能力至关重要 下呼吸道（LRT）中的BVGAS活性。我们发现LRT感染需要PLRSR 即使BVGAS具有组成性活动，PLRSR控制着未识别的表达，但 关键毒力独立于BVGA。这些目前未知的毒力因素 可以用作治疗靶标或新的疫苗成分，从而识别 对于未来控制百日咳至关重要。 博士。 Cotter和Julio是Bordetella发病机理和分子生物学方面的专家，博士 Bourret是TCS生物化学专家。我们在一起表征了PLR和PLRR蛋白 从生化上，发现了一种绕过体外PLR的明显本质的方法，以便 我们可以构建菌株以收集基因表达数据，而不必知道PLR所感受到的刺激。 在AIM 1中，我们将使用RNA-Seq鉴定由PLRSR调节的基因，以揭示正面或阴性 调节和chip-seq以揭示直接或间接调节。在AIM 2中，我们将调查PLRSR 通过将记者融合到关键PLRSR调节的基因并评估对 生理上相关的刺激以及缺乏PDC或PAS的PLR的后果 感觉域。 识别PLRSR Regulon的风险低/高奖励，将为A奠定基础 R01项目。所提出的方法是良好的，这表明很有可能实现 我们的目标。识别PLRSR法规将在理解Bordetella方面具有变革性 发病机理和启用未来的R01项目，我们可以确定PLRR为何对 生存力，（ii）PLRSR调节基因产物在Bordetella的LRT感染中的作用，（iii）PLRSR如何 调节PLRSR和BVGAS TCSS之间的基因表达，（iv）连接，也许（V） 深入了解PLR感应的刺激。