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.

微生物的吞噬作用和破坏是巨噬细胞和其他保护我们免受入侵病原体的吞噬细胞。但是目前对这个过程的理解是不完整的，因为我们已经表明细菌能够在缺乏穿孔蛋白-2的巨噬细胞中复制和生存（PRF2）；直到最近未表征的蛋白质。正如基于细胞的研究所预期的那样，当对病原体PRF2挑战时淘汰小鼠屈服于传染剂量，大多数野生型窝窝夫人都能生存。这伴随着将细菌复制和传播到更深的组织中。进一步强调 PRF2在宿主防御中的重要性是，某些病原体部署了阻止将PRF2传递到吞噬体。 PRF2最近的高分辨率结构表明它是一个孔形成的蛋白质和从孔前孔的过渡是由低pH驱动的；与它在酸化的吞噬体中的作用。这伴随着大量的内域和域间构象变化。尽管数据尚未广泛，但临床情况开始出现PRF2中的某些突变与慢性细菌感染相关。更远在该领域的研究很可能表明PRF2单倍性是导致的根本原因反复感染或持续感染。这些和其他研究已经确定PRF2的基础巨噬细胞和其他吞噬细胞的最基本功能之一。尽管在这一新的研究领域中已经取得了很大进步，但进展是受到免疫研究的支柱的严重缺乏抗体的阻碍。因此，该提案的目的是隔离和评估针对PRF2的重链抗体（VHHS）。我们进一步建议开发特定于构象的VHHS 单体和寡聚的前孔和孔构象。在AIM1中，我们将利用最近的发现pH控制PRF2构象以将VHH分离为小鼠和人类预孔和合成VHH库中的孔复合物。在AIM2中，我们将生产，排名和评估VHHS 在各种免疫学测定中。作为初始动量的指标，我们已经有18个独特的屏幕上的克隆针对PRF2的P2域；预计其中一些是特定的 PRF2单体。我们预计抗PRF2 VHHS将消除阻碍的当前障碍进步以进一步阐明PRF2在先天免疫防御中的核心作用。 PRF2研究是对于人类健康非常重要，因为我们的初步数据，最新出版物和单倍宽松的个体的表征表明，它是基础的基础吞噬细胞。