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Leptospiral-Phagocyte Dynamics in Leptospirosis

钩端螺旋体病中的钩端螺旋体吞噬细胞动力学

基本信息

批准号：
10643290
负责人：
DAVID A HAAKE
金额：
$ 56.48万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-16 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10643290
关键词：
Accidents Animal Model Antigens Blood Bone Marrow California Candidate Disease Gene Collaborations Data Defect Disease Reservoirs Endothelium Evolution Exposure to Failure Genes Genetic Transcription Genomics Goals Hamsters Health Hemorrhage Human Immune response Impairment In Vitro Individual Infection Inflammasome Interferon Type I Interferon alpha Interferons Interleukin-1 beta Iron Kinetics Leptospira Leptospira interrogans Leptospirosis Libraries Liver Los Angeles Macrophage Membrane Proteins Methods Modeling Mus Myeloid Cells Osmolar Concentration Outcome Pathogenesis Pathogenicity Pathway interactions Peripheral Blood Mononuclear Cell Phagocytes Play Pneumonia Positioning Attribute Production Property Proteins Research Research Personnel Resource-limited setting Role Stimulator of Interferon Genes Stimulus Study models Temperature Testing Time Tissues Universities Virulence Virulence Factors Work acute infection chronic infection fitness human disease improved in vivo in vivo evaluation innate immune pathways interest knock-down mutant neglect pathogen programs response subcutaneous transcriptome sequencing transposon sequencing uptake virulence gene

项目摘要

Abstract: Project 1, University of California, Los Angeles Leptospirosis is a widespread and frequently fatal human health problem that disproportionately impacts individuals living in low resource settings. Research on phagocyte interactions in leptospirosis is significant because little is known about leptospiral virulence factors required for survival within phagocytes or the response of macrophages to leptospires. The proposed studies will involve collaborations between the PI and co- investigator Fayyaz Sutterwala, who is a leader in studies of inflammasome activation. Two related but non- interdependent Aims are proposed. Aim 1 is to examine leptospiral-phagocyte interactions in vitro. We will examine the murine vs human macrophage responses to L. interrogans in terms of type I interferon and inflammasome responses and determine the pathways involved in their activation. We will examine whether leptospiral virulence genes account for lower inflammasome activation in murine relative to human macrophages. We will examine the time course of leptospiral uptake and survival vs killing of leptospiral species and test the hypothesis that macrophage survival is a property of the pathogenic, but not the saprophytic, species. We will identify virulence genes required for survival after exposure to macrophages as a result of phagocytic uptake and examine the leptospiral response to macrophage uptake. Given the finding that LigB plays a role in macrophage infection, we are interested in examining macrophage uptake and killing of a ligAligB knock down mutant that lacks expression of LigA and LigB. Dual RNAseq will be performed with bone marrow-derived macrophages (BMDMs) and L. interrogans including mutants with impaired intracellular survival to identify genes potentially involved in leptospiral-phagocyte interactions. Aim 2 is to examine leptospiral-phagocyte interactions in vivo. We will examine the kinetics of local control vs dissemination after subcutaneous challenge of hamsters vs mice. We will test the hypothesis that L. interrogans survives within phagocytes during infection of the subcutaneous space, blood, and liver. We will identify virulence genes required for intracellular survival within phagocytes after subcutaneous challenge. We are particularly interested in defects in in vivo fitness for local and disseminated infection of our ligAligB knock-down mutant and other virulence gene candidates. We will screen our transposon mutant library for mutants with fitness defects in the subcutaneous space and for dissemination. Using mice with defects in type I interferon response involving cGAS/STING and inflammasome activation involving NLRP3, we will test the hypothesis that these macrophage responses improve local control and reduce dissemination after subcutaneous challenge.

摘要：项目1，加州大学，洛杉矶钩端螺旋体病是一种广泛且经常致命的人类健康问题，生活在低资源环境中的人。钩端螺旋体病吞噬细胞相互作用的研究具有重要意义由于对钩端螺旋体在吞噬细胞内生存所需的毒力因子或应答知之甚少，从巨噬细胞到钩端螺旋体拟议的研究将涉及主要研究者和合作者之间的合作，研究者Fayyaz Sutterwala，他是炎性小体激活研究的领导者。两个相关但不相关的提出了相互依存的目标。目的1：研究钩端螺旋体与吞噬细胞的体外相互作用。我们将检测小鼠与人巨噬细胞对L.就I型干扰素而言，炎症反应，并确定参与其激活的途径。我们将研究是否钩端螺旋体毒力基因解释了相对于人巨噬细胞，小鼠中炎性小体活化较低。我们将检查钩端螺旋体摄取的时间过程和存活与杀灭钩端螺旋体物种的时间过程，并测试假设巨噬细胞存活是致病性物种的特性，而不是嗜血性物种的特性。我们将鉴定由于吞噬摄取而暴露于巨噬细胞后存活所需的毒力基因并检测钩端螺旋体对巨噬细胞摄取的反应。鉴于LigB在其中发挥作用的发现巨噬细胞感染，我们感兴趣的是检查巨噬细胞的摄取和杀死一个ligAligB敲低缺乏LigA和LigB表达的突变体。双重RNAseq将使用骨髓来源的巨噬细胞（BMDM）和L.包括细胞内存活受损的突变体的问号以鉴定基因可能参与钩端螺旋体-吞噬细胞相互作用。目的二是研究钩体与吞噬细胞的相互作用 in vivo.我们将研究仓鼠皮下攻击后局部控制与传播的动力学与小鼠相比。我们将检验L.问号线虫在感染期间在吞噬细胞内存活皮下空间血液和肝脏我们将确定细胞内生存所需的毒力基因，皮下激发后的吞噬细胞。我们特别感兴趣的缺陷，在体内适合当地以及我们的ligAliB敲低突变体和其他毒力基因候选者的播散性感染。我们将筛选我们的转座子突变体库，寻找在皮下空间具有适应性缺陷的突变体，传播。使用具有涉及cGAS/STING和炎性小体的I型干扰素应答缺陷的小鼠激活涉及NLRP 3，我们将测试假设，这些巨噬细胞的反应，提高局部控制并减少皮下攻击后的传播。