Analyzing the expression and activation of Perforin-2 -a bactericidal pore-forming protein- with single domain antibodies

使用单域抗体分析 Perforin-2（一种杀菌性成孔蛋白）的表达和激活

基本信息

批准号：
10320041
负责人：
GEORGE Patrick MUNSON
金额：
$ 19.19万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-17 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10320041
关键词：
Acids Antibodies Antigens Area Bacteria Bacterial Infections Cells Chronic Clinical Complex Data Development Dose EGF gene Flow Cytometry Health Height Host Defense Human Immune Immunologic Techniques Immunologics Immunology procedure In Vitro Individual Infection Invaded Investigation Knockout Mice Knowledge Libraries Membrane Membrane Lipids Modeling Molecular Conformation Mus Mutation Oranges Organ Pathogenesis Peptide Hydrolases Phagocytes Phagocytosis Phagosomes Polymers Process Production Proliferating Proteins Publications Research Research Personnel Resolution Role Rosaniline Dyes Rotation Side Source Structure Tissues VDAC1 gene Validation acid fast bacteria antimicrobial bactericide base chronic infection early screening immune function macrophage microorganism monomer nanobodies pathogen pathogenic bacteria perforin 2 periplasm tool

项目摘要

The phagocytosis and destruction of microorganisms is an essential immunological function of macrophages and other phagocytes that protects us from invading pathogens. However current understanding of this process is incomplete because we have shown that bacteria are able to replicate and survive within macrophages that lack Perforin-2 (PRF2); until recently a largely uncharacterized protein. As expected from cell based studies, when challenged with pathogens PRF2 knockout mice succumb to infectious doses that the majority of their wild-type littermates survive. This is accompanied by replication and dissemination of bacteria to deeper tissues. Further underscoring the importance of PRF2 in host defense is the fact that some pathogens deploy effectors to block the delivery of PRF2 to phagosomes. Recent high resolution structures of PRF2 have shown that it is a pore-forming protein and that the transition from pre-pore-to-pore is driven by low pH; consistent with its role within acidifying phagosomes. This is accompanied by substantial intra- and inter-domain conformational changes. Although the data is not yet extensive, a clinical picture is beginning to emerge that certain mutations within PRF2 correlate with chronic bacterial infections. Further research in this area may well reveal that PRF2 haploinsufficiency is the underlying cause of recurring or persistent infections. These and others studies have established that PRF2 underpins one of the most basic functions of macrophages and other phagocytes. Although considerable advances have been made within this new area of research, progress is hindered by a critical lack of antibodies that are the mainstay of immunological investigations. Thus, the objective of this proposal is to isolate and evaluate heavy chain antibodies (VHHs) against PRF2. We further propose to develop conformation specific VHHs that are able to discriminate between the monomeric and, oligomeric pre-pore and pore conformations. In Aim1 we will capitalize on the recent discovery that pH controls PRF2 conformations to isolate VHHs to both mouse and human pre-pore and pore complexes from a synthetic VHH library. In Aim2 we will produce, rank, and evaluate VHHs in a variety of immunological assays. As an indicator of initial momentum we already have 18 unique clones from a screen against the P2 domain of PRF2; some of which are expected to be specific for PRF2 monomers. We anticipate that anti-PRF2 VHHs will remove current roadblocks that hinder progress to further elucidate the central role of PRF2 in innate immune defense. PRF2 research is highly significant to human health because our preliminary data, recent publications, and characterization of haploinsufficient individuals demonstrate that it underpins an essential function of phagocytes.

对微生物的吞噬和破坏是微生物的重要免疫功能巨噬细胞和其他吞噬细胞保护我们免受病原体入侵。然而目前对这个过程的理解是不完整的，因为我们已经证明细菌能够在缺乏 Perforin-2 (PRF2) 的巨噬细胞内复制并存活；直到最近很大程度上未表征的蛋白质。正如基于细胞的研究所预期的那样，当受到病原体 PRF2 的挑战时基因敲除小鼠死于感染剂量，而其大多数野生型同窝小鼠都存活下来。这伴随着细菌的复制和传播到更深的组织。进一步强调 PRF2 在宿主防御中的重要性在于，一些病原体会部署效应器来阻断 PRF2 将 PRF2 递送至吞噬体。 PRF2 最近的高分辨率结构表明它是成孔蛋白，从前孔到孔的转变是由低 pH 值驱动的；符合它在酸化吞噬体中的作用。这伴随着大量的域内和域间构象变化。尽管数据还不广泛，但临床情况已开始显现 PRF2 内的某些突变与慢性细菌感染相关。更远该领域的研究很可能揭示 PRF2 单倍体不足是导致反复或持续感染。这些和其他研究已经证实 PRF2 是巨噬细胞和其他吞噬细胞最基本的功能之一。尽管这一新的研究领域已经取得了相当大的进展，但进展还很缓慢。由于严重缺乏抗体而受到阻碍，而抗体是免疫学研究的支柱。因此，该提案的目的是分离和评估针对 PRF2 的重链抗体 (VHH)。我们进一步建议开发特定构象的 VHH，能够区分单体和寡聚预孔和孔构象。在 Aim1 中，我们将利用最近的发现 pH 控制 PRF2 构象以将 VHH 分离到小鼠和人类前孔中和来自合成 VHH 文库的孔复合物。在 Aim2 中，我们将生成、排名和评估 VHH 在各种免疫学测定中。作为初始势头的指标，我们已经有 18 个独特的指标来自针对 PRF2 的 P2 结构域的筛选的克隆；其中一些预计将专门针对 PRF2 单体。我们预计抗 PRF2 VHH 将消除目前阻碍进一步阐明 PRF2 在先天免疫防御中的核心作用的进展。 PRF2研究是对人类健康非常重要，因为我们的初步数据、最近的出版物和单倍体不足个体的表征表明它支持了一个基本功能吞噬细胞。