Surface sensing, memory, and motility control in biofilm formation

项目摘要 生物膜是表面附着的微生物群落，造成了重大的临床问题，部分原因是 生物膜细胞具有高度的抗生素耐受性。与器械相关的生物膜感染导致的成本超过10亿美元 每年。此外，致死性铜绿假单胞菌生物膜感染在囊性纤维化（CF）和纤维化中是常见的。 其他呼吸道疾病。更好地了解微生物群落是如何形成的， 逆转生物膜形成。我们报道的研究表明，铜绿假单胞菌可以检测表面接触，通过 途径需要IV型皮利（TFP）和膜结合的信号复合物，产生第二个 信使cAMP。我们最近的研究结果使用单细胞分辨率的整个社区的细胞跟踪， 结合cAMP报告基因，导致我们的中心假设：表面传感是基于cAMP-TFP的。 基于记忆，并且不会通过逐渐增加附着细胞及其 胞内cAMP。相反，表面诱导细胞内cAMP水平的相移时间波， TFP活动构成了表面的“记忆”。这段记忆，是多代人的， 鲁棒，允许表面暴露细胞的后代适应表面并增加表面 细胞数量的数量级比它们的祖先更快。为了理解这个关键的 我们将使用群体水平和单细胞分析，结合分子遗传学方法 在严格的生物物理学理论框架内，探索这些最早的机械基础， 生物膜形成中的事件。我们将（1）检验表面接触引起相移波的假设， 编码表面记忆的cAMP水平和TFP活性，允许表面适应， 极大地改变了细胞在表面上的行为，（2）测试表面感知通过 整合TFP的机械收缩，PilA的内膜动力学，以及内 膜PilJ-PilA复合物是cAMP信号传导所需的。待建议的研究完成后，我们会 已经建立了铜绿假单胞菌感知并不可逆地附着于表面的机制。给定 不可逆的附着是生物膜形成的第一步，我们的工作揭示了一个关键方面， 细菌生物学

PROJECT SUMMARY Biofilms are surface-attached microbial communities that pose a significant clinical problem, in part because biofilm cells are highly antibiotic tolerant. Device-related biofilm infections incur costs of >1 billion dollars annually. Moreover, lethal Pseudomonas aeruginosa biofilm infections are common in cystic fibrosis (CF) and other respiratory diseases. A better understanding of how microbial communities form is required to prevent or reverse biofilm formation. Our reported studies show that P. aeruginosa can detect surface contact via a pathway requiring Type IV pili (TFP) and a membrane-bound signaling complex that generates the second messenger cAMP. Our recent findings using cell tracking of entire communities at single-cell resolution and combined with a cAMP reporter lead to our Central Hypothesis: Surface sensing is predicated on cAMP-TFP- based memory, and does not occur by gradually increasing surface residence times of attached cells and their intracellular cAMP. Rather, the surface induces phase-shifted temporal waves of intracellular cAMP levels and TFP activity that constitute a `memory' of the surface. This memory, which is multigenerational and surprisingly robust, allows planktonic descendants of surface-exposed cells to adapt to the surface and increase surface cell populations orders of magnitude faster than their ancestors upon attachment. To understand this pivotal event, we will use population-level and single-cell analyses, combined with molecular genetic approaches within a rigorous biophysical theoretical framework, to explore the mechanistic underpinnings of these earliest events in biofilm formation. We will (1) test the hypothesis that surface contact induces phase-shifted waves of cAMP levels and TFP activity that encode a memory of the surface, allowing for surface adaptation that drastically modifies behavior of cells on surfaces, (2) test the hypothesis that surface sensing is transmitted via integrating TFP mechanical retraction, inner membrane dynamics of PilA, and the formation of an inner membrane PilJ-PilA complex required for cAMP signaling. Upon completion of the proposed studies, we will have established the mechanism by which P. aeruginosa senses and irreversibly attaches to a surface. Given that irreversible attachment is the first committed step in biofilm formation, our work uncovers a key aspect of bacterial biology.