Molecular mechanisms of C.albicans - leukocyte integrin interactions

描述（由申请人提供）：CDC估计自1980年以来真菌感染的发生率增加了500倍。白色念珠菌是一种常见的机会性真菌病原体，是免疫功能低下个体侵袭性真菌疾病的主要原因；是院内感染的主要原因；占所有感染性心内膜炎的20%，其死亡率高达70%。诱导细胞介导的对白色念珠菌的免疫在宿主防御和先天免疫系统细胞的主要任务中至关重要。先前，已经证明整合素1M22 （CD11b/CD18）是参与白色念珠菌识别的主要中性粒细胞（PMN）受体。申请人已证明从白色念珠菌中释放出抑制PMN粘附和向真菌迁移的活性。利用两种独立的亲和方法，通过质谱和氨基酸序列纯化了负责这些活性的同一单一蛋白，并明确确定为ph调节抗原1 (Pra1p)，也称为纤维蛋白原结合蛋白1 （Fbp1）。纯化的Pra1p阻断了PMN对真菌的粘附，并阻止了PMN介导的表达Pra1p的杀死，但对Pra1p缺乏的白色念珠菌没有作用。我们假设1M22-Pra1p与纤维蛋白原的相互作用在白色念珠菌的发病机制中起关键作用：真菌结合的Pra1p是PMN对白色念珠菌的主要靶点；可溶形式的Pra1p作为诱饵，帮助真菌逃脱宿主的监视；纤维蛋白原不仅与Pra1p相互作用，还与1M22相互作用，调节相互作用。为了验证这一假设，这些相互作用的生物学意义将在PMN杀伤试验和使用缺乏Pra1p的白色念珠菌菌株和缺乏1M22和密切相关的整合素1X22的转基因小鼠体内小鼠模型中进行评估（目的1）。可溶性Pra1p对这些反应的影响将在体外和体内进行测定（目的2）。纤维蛋白原作为调节剂的作用将在生物化学上进行研究，并使用纤维蛋白原结合位点1M22被破坏的小鼠（目的3）。这些相互作用的机制将通过鉴定1M22和白色念珠菌中介导它们相互作用的位点来确定（目的4）。这些研究将为白色念珠菌与宿主相互作用的基本机制提供见解，并可能阐明控制真菌感染的新策略。公共卫生相关性：多形核白细胞已被证明是宿主对白色念珠菌感染的先天免疫防御的主要成分，它们在微生物识别中利用的最重要的受体是整合素1M22。申请人已经确定Pra1p是真菌蛋白中1M22的主要配体。本研究将探讨1M22 - Pra1p在转基因小鼠体内和离体模型中相互作用的分子机制。这项研究的数据有望突出真菌与宿主相互作用的机制，并有助于设计新的抗真菌肽。

DESCRIPTION (provided by applicant): The CDC has estimated that the incidence of fungal infections has increased by 500-fold since 1980. Candida albicans, a common opportunistic fungal pathogen, is the leading cause of invasive fungal disease in immunocompromised individuals; is the major cause of in-hospital infections; and accounts for 20% of all infective endocarditis, which has a mortality rate as high as 70%. The induction of cell-mediated immunity to C. albicans is critical in host defense and the prime task of cells of the innate immune system. Previously, it has been demonstrated that the integrin 1M22 (CD11b/CD18) is the major neutrophil (PMN) receptor involved in C. albicans recognition. The applicant has shown that an activity is released from C. albicans that inhibits PMN adhesion and migration to the fungus. Utilizing two independent affinity approaches, the same single protein responsible for these activities was purified and identified unequivocally as pH-regulated Antigen 1 (Pra1p), also known as Fibrinogen Binding Protein 1, Fbp1, by mass spectrometry and amino acid sequence. Purified Pra1p blocked PMN adhesion to the fungus and prevented PMN-mediated killing of Pra1p-expressing, but not of Pra1p-deficient strains of C. albicans. We hypothesize that the 1M22-Pra1p- fibrinogen interactions are pivotal in C. albicans pathogenesis: fungal-bound Pra1p is a main target of PMN on C. albicans; the soluble form of Pra1p serves as decoy to assist the fungus in escaping host surveillance; and fibrinogen, which interacts not only with Pra1p but also with 1M22, modulates the interactions. To test this hypothesis, the biological significance of these interactions will be assessed in PMN killing assays and in vivo murine models of systemic candidiasis utilizing C. albicans strains, which lack Pra1p and transgenic mice which lack 1M22, and a closely related integrin 1X22 (Aim 1). The effects of soluble Pra1p on these responses will be assays in vitro and in vivo (Aim 2). The role of fibrinogen as a modulator will be investigated biochemically and using mice in which the fibrinogen binding site for 1M22 has been disrupted (Aim 3). The mechanism underlying these interactions will be defined by identifying sites in 1M22 and in C. albicans which mediate their engagement (Aim 4). These studies will provide insights into the basic mechanism underlying C. albicans interactions with the host and may elucidate new strategies to control fungal infections. PUBLIC HEALTH RELEVANCE: Polymorphonuclear leukocytes have been shown to be the primary components of the host's innate immune defenses against C. albicans infections, and the most prominent receptor that they utilize in microbial recognition is integrin 1M22. The applicant has identified Pra1p as the major ligand of 1M22 among fungal proteins. The proposed study will investigate the molecular mechanism of 1M22 - Pra1p interactions in models ex vivo and in vivo in transgenic mice. Data from this study are expected to highlight mechanisms which are involved in fungal-host interactions and to help design new antifungal peptides.