Biofilm Formation by Pneumocystis

描述（由申请人提供）：肺囊虫真菌属的成员可以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；最近，这些患者接受了靶向免疫治疗。感染传播的方式；哺乳动物肺内的生命周期；在这种恶劣的环境中生存的策略在很大程度上是未知的，这在很大程度上是由于缺乏持续的培养方法。我们的长期目标是了解肺囊虫的感染过程，作为确定其生存策略的一种手段，从而可以用于阻断感染。生产生物膜是许多微生物用来抵御环境攻击的一种策略；促进成员之间的沟通和区分；作为传播的焦点。我们认为肺囊虫在肺泡内的附着和生长类似于生物膜的形成。鉴定了一种体外系统，支持卡氏假单胞菌（大鼠）和鼠假单胞菌（小鼠）形成明显的生物膜。生物膜的生长动力学、共聚焦特性和形态变化与其他真菌形成的生物膜相似。在目前的建议中，该系统将被优化，为了解肺囊虫的生存策略提供一种新的工具，然后将用于解决有关该过程的生物学问题。提出以下具体目标：(1)定义和表征肺囊虫生物膜形成的最佳条件。系统的方法将用于评估基质，条件和添加剂导致稳健的生物膜的形成。显微镜和定量方法将用于绘制生物膜形成过程的图表。(2)识别与生物膜形成相关的生物过程。肺囊虫在形成生物膜时所经历的形态变化是显著的。利用分子和生物化学方法鉴定和验证参与这一过程的基因和基因产物将为生物膜产生的生物学提供基本信息，并导致对这一过程的新的认识和理解。(3)评估体外生物膜在体内的致病性。将通过显微镜和定量方法比较由生物膜衍生生物与标准模型引起的感染进展。体内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。生物膜工艺的优化与评价将从根本上推进肺囊虫病的研究。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。我们确定了一个真菌病原体的生物膜系统，肺囊虫，这将被用来了解它在肺中生存和引起肺炎的策略。

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.