Surface sensing, memory, and motility control in biofilm formation

项目总结 生物膜是附着在表面的微生物群落，它们构成了重大的临床问题，部分原因是 生物膜细胞对抗生素有高度的耐受性。设备相关生物膜感染造成10亿美元的损失 每年一次。此外，致死性铜绿假单胞菌生物被膜感染在囊性纤维化(CF)和 其他呼吸道疾病。需要更好地了解微生物群落是如何形成的，以防止或 反生物膜形成。我们报告的研究表明，铜绿假单胞菌可以通过一种 需要IV型菌毛的途径(TFP)和产生第二种蛋白的膜结合信号复合体 信使营地。我们最近的发现是在单细胞分辨率下对整个社区进行细胞跟踪，并 结合一位坎普记者，得出了我们的中心假设：表面感知是基于cAMP-TFP- 基于记忆，而不是通过逐渐增加附着细胞及其表面停留时间而发生的 胞内cAMP。相反，表面诱导细胞内cAMP水平的相移时间波和 TFP活动构成了对表面的“记忆”。这种记忆是多代人的，令人惊讶的是 坚固耐用，使暴露在表面的浮游细胞后代适应表面并增加表面 细胞数量在附着时比它们的祖先快几个数量级。要理解这一关键 我们将使用种群水平和单细胞分析，结合分子遗传学方法 在严格的生物物理理论框架内，探索这些最早的 生物膜形成中的事件。我们将(1)检验表面接触产生相移波的假设。 CAMP水平和TFP活性编码表面的记忆，允许表面适应 极大地改变了细胞在表面上的行为，(2)检验了表面感觉通过 结合TFP的机械回缩、PilA的内膜动力学和内膜形成 膜PilJ-PilA是cAMP信号转导所必需的复合体。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转地附着在表面的机制。vt.给出 这种不可逆转的附着是生物膜形成的第一步，我们的工作揭示了 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.