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

吞噬和破坏微生物是一种基本的免疫功能。 巨噬细胞和其他吞噬细胞，保护我们免受入侵的病原体。无论如何，当前 对这一过程的理解是不完整的，因为我们已经证明细菌能够 在缺乏穿孔素-2(PRF2)的巨噬细胞内复制并存活；直到最近， 未鉴定的蛋白质。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 基因敲除的小鼠会屈服于大多数野生型后代存活的感染性剂量。这 伴随着细菌的复制和传播到更深的组织。进一步强调 PrF2在宿主防御中的重要性在于，一些病原体部署效应器来阻止 将PrF2递送到吞噬小体。最近PRF2的高分辨结构表明，它是一种 成孔蛋白，从前孔道到孔道的转变是由低pH值驱动的；与 它在酸化噬菌体中的作用。这伴随着大量的域内和域间 构象变化。尽管数据还不是很广泛，但临床图景已经开始 发现PrF2中的某些突变与慢性细菌感染相关。进一步 这方面的研究可能会很好地揭示PrF2单倍体不足是导致 反复或持续的感染。这些和其他研究已经确定了PRF2是 巨噬细胞和其他吞噬细胞最基本的功能之一。 尽管在这一新的研究领域已经取得了相当大的进展，但进展是 由于严重缺乏抗体而受到阻碍，而抗体是免疫学研究的主要内容。因此， 本提案的目的是分离和评估抗PRF2的重链抗体(VHH)。 我们进一步建议开发构象特定的VHH，能够区分 单体和寡聚体的预孔和孔道构象。在Aim1中，我们将利用最近的 PH控制PrF2构象以分离小鼠和人类前孔的VHH的发现 以及来自合成VHH库的孔道复合体。在AIM2中，我们将对VHH进行生产、排名和评估 在各种免疫学检测中。作为初始动量的指标，我们已经有18个独特的 来自屏幕的针对PRF2的P2结构域的克隆；其中一些预计是针对 PRF2单体。我们预计，反对PRF2的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 对人类健康具有非常重要的意义，因为我们的初步数据、最近的出版物和 单倍体缺陷个体的特征表明，它支持了单倍体缺陷的一个基本功能 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.