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

微生物的吞噬和破坏是免疫系统的基本免疫功能。 巨噬细胞和其他吞噬细胞保护我们免受入侵的病原体。然而目前 对这一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（PRF 2）的巨噬细胞内复制和存活;直到最近， 未知蛋白质正如基于细胞的研究所预期的，当用病原体PRF 2挑战时 基因敲除小鼠死于其大多数野生型同窝仔存活的感染剂量。这 伴随着细菌的复制和传播到更深的组织。进一步强调 PRF 2在宿主防御中的重要性在于，一些病原体部署效应器来阻断PRF 2， 将PRF 2递送至吞噬体。最近的高分辨率结构的PRF 2已经表明，它是一个 孔形成蛋白，并且从预孔到孔的转变是由低pH驱动的;与 其在酸化吞噬体内的作用。这是伴随着大量的内部和内部域 构象变化虽然数据还不广泛，但临床图片已开始 PRF 2中的某些突变与慢性细菌感染相关。进一步 这一领域的研究很可能揭示，PRF 2单倍不足是 复发或持续感染。这些和其他研究已经确定，PRF 2支持 巨噬细胞和其他吞噬细胞的最基本功能之一。 虽然在这一新的研究领域取得了相当大的进展，但进展仍然很缓慢。 由于严重缺乏作为免疫学研究支柱的抗体而受阻。因此，在本发明中， 本发明的目的是分离和评价抗PRF 2的重链抗体（VHH）。 我们进一步建议开发构象特异性VHH，其能够区分 单体和低聚物前孔和孔构象。在目标1中，我们将利用最近的 发现pH控制PRF 2构象以将VHH分离至小鼠和人前孔 和来自合成VHH文库的孔复合物。在目标2中，我们将产生、排名和评估VHH 在各种免疫学测定中。作为初始动力的指标，我们已经有18个独特的 来自针对PRF 2的P2结构域的筛选的克隆;其中一些预期对PRF 2具有特异性。 PRF 2单体。我们预计，抗PRF 2 VHH将消除目前阻碍 进一步阐明PRF 2在先天免疫防御中的核心作用。PRF 2研究 对人类健康非常重要，因为我们的初步数据，最近的出版物， 单倍不足个体的特征表明，它支持了一个重要的功能， 吞噬细胞

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.