Biofilm Formation by Pneumocystis

描述(由申请人提供)：肺孢子虫属真菌可在免疫系统衰弱的宿主中引起致命性肺炎(PCP)，例如艾滋病毒感染者和艾滋病患者；接受免疫抑制治疗的患者；以及最近接受靶向免疫治疗的患者。感染的传播方式；哺乳动物肺内的生命周期；以及在这种不适宜居住的环境中生存所使用的策略在很大程度上是未知的，这在很大程度上是因为缺乏持续的培养方法。我们的长期目标是了解肺孢子虫的感染过程，以此作为一种手段来确定其生存策略，然后利用这些策略来阻断感染。生产生物膜是许多微生物用来保护免受环境攻击、成员之间的交流和区别以及作为传播中心的一种战略。我们推测肺孢子虫在肺泡内的附着和生长类似于生物膜的形成。一个支持卡氏肺孢子虫(来自大鼠)和P.Murina(来自小鼠)明显生物膜形成的体外系统被鉴定出来。生物膜表现出与其他真菌生物膜形成相似的生长动力学、共聚焦属性和形态变化。在目前的提案中，这一系统将被优化，以提供一种新的工具来理解肺孢子虫的生存策略，然后将被用来解决关于这一过程的生物学问题。提出了以下具体目标：(1)确定和表征形成肺孢子虫生物膜的最佳条件。将使用一种系统的方法来评估基质、条件和添加剂，从而形成强大的生物膜。将使用显微和定量方法来绘制生物膜形成的进程。(2)确定与生物膜形成相关的生物过程。肺孢子虫在形成生物膜时所经历的形态变化是戏剧性的。利用分子和生物化学方法鉴定和验证生物膜形成过程中涉及的基因和基因产物，将提供有关生物膜产生生物学的基本信息，并导致对这一过程的新的认识和理解。(3)评价体外生物膜在体内的致病性。我们将用显微和定量的方法比较生物膜衍生生物与标准模型之间的感染进展。体内的形态和基因表达将与体外生物膜进行比较，以验证体外系统的有效性。生物膜工艺的优化和评价将从根本上推进肺孢子虫的研究。公共卫生相关性：微生物病原体利用生物膜逃避受感染宿主的免疫防御和抗菌治疗。我们为真菌病原体肺孢子虫确定了一种生物膜系统，这将被用来了解它在肺中生存并导致肺炎的策略。

DESCRIPTION (provided by applicant): Members of the fungal genus Pneumocystis can cause a lethal pneumonia (PCP) in hosts with debilitated immune systems, such as HIV-infected persons with AIDS; patients undergoing immunosuppressive therapy; and more recently in those patients received targeted immunotherapy. The manner in which the infection is disseminated; the life cycle within the mammalian lung; and the strategies used for survival within this inhospitable environment are largely unknown due in large part to the lack of a continuous cultivation method. It is our long term goal to understand the infection process of Pneumocystis as a means to identify its survival strategies which could then be exploited for interdiction of infection. Production of biofilms is a strategy used by many microbes for protection against environmental assaults; for communication and differentiation among members; and as foci for dissemination. We posit that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system was identified that supports apparent biofilm formation by P. carinii (from rat) and P. murina (from mouse). The biofilms showed similar growth kinetics, confocal attributes, and morphological changes compatible with biofilm formation by other fungi. In the present proposal, this system will be optimized to provide a novel tool for understanding the survival strategies of Pneumocystis and then will be used to address questions about the biology of the process. The following specific aims are proposed: (1) Define and characterize the optimal conditions for formation of Pneumocystis biofilms. A systematic approach will be used to evaluate matrices, conditions, and additives leading to robust biofilm formation. Microscopic and quantitative methods will be used to chart the progression of biofilm formation. (2) Identify the biological process associated with biofilm formation. The morphologic changes Pneumocystis undergoes as it forms a biofilm are dramatic. Identification and verification of the genes and gene products involved in the progression using molecular and biochemical methods will provide basic information on the biology of biofilm production and lead to a new appreciation and understanding of this process. (3) Assess the pathogenicity of in vitro biofilms in vivo. The progression of infection initiated by biofilm-derived organisms vs. the standard model will be compared by microscopic and quantitative methods. The in vivo morphology and gene expression will compared to in vitro biofilms for validation of the in vitro system. Optimization and evaluation of the biofilm process will fundamentally advance the study of Pneumocystis. PUBLIC HEALTH RELEVANCE: Microbial pathogens use biofilms to escape the infected host's immune defenses and antimicrobial therapy. We identified a biofilm system for the fungal pathogen, Pneumocystis, that will be used to understand the strategies it employs to survive in the lung and cause pneumonia.