How do bacteria sense and navigate chemical gradients within biofilms?

大多数细菌附着在表面上，形成密集的群落，称为生物膜。虽然其中一些组合在我们的生活中发挥着有益的作用，但人体内的感染通常很难治疗，因为生物膜保护细胞免受抗生素和免疫系统的侵害。了解促进生物膜形成的基本过程对于开发破坏或操纵这些重要细菌群落的新方法至关重要。铜绿假单胞菌会在烧伤患者和囊性纤维化患者中引起危险的感染，它使用一种叫做菌毛的微小的抓钩状附属物在生物膜中移动。我们的团队最近证明了单个铜绿假单胞菌细胞可以利用基于毛的运动性在发育中的生物膜中导航到更有利的营养环境。我们发现这个过程被称为趋化性，因为当细胞检测到自己正在远离营养源时，它们会将自己拉向相反的方向。这种非凡的能力可能使趋化细胞在生物膜中具有优势，并为控制生物膜的形成开辟了新的途径。虽然其他形式的微生物趋化性已被广泛研究，但对基于毛的趋化性知之甚少。这项研究将解决我们知识中的两个主要空白：首先，我们不知道细菌实际上是如何感知它们是朝向还是远离化学引诱剂的来源的。一般来说，有两种不同的可能性：细胞可以从一个位置移动到另一个位置并测量浓度随时间的变化（时间传感），或者它们可以直接感知浓度随身体长度的变化（空间传感）。这项工作将结合新型微流体实验、基于计算机的细胞跟踪和细菌遗传学来直接测试这两种不同的可能性。我们的初步实验表明，随着时间的推移，当细胞经历化学引诱剂浓度的降低时，细胞逆转的可能性不会增加，这表明表面附着的细胞不使用时间感知来指导趋化。未来的实验将使用新的细菌菌株，当荧光标记的蛋白质反向时，它们将重新定位到它们的另一端，这将使我们能够测试细胞是否能够感知它们长度上浓度的变化。其次，我们的实验表明，细胞倾向于直接沿着化学梯度移动，但这种观察结果不能用已知的细菌运动形式来解释。表面附着的细菌被认为只有一种行为可以促进趋化性：反向。然而，单靠倒转只允许细胞探索一维线，所以如果一个细胞碰巧落在垂直于梯度的表面上，它将无法转向营养来源。在我们的初步工作中，我们发现了一种表面附着细胞重新定向运动的新方法。我们基于计算机的图像分析软件显示，细胞经常进行类似翻筋斗的动作，我们称之为“旋转”。当细胞在几分钟到几小时的时间内顺时针或逆时针方向移动时，就会发生这些重新定向。这项研究将使用新的细菌菌株和尖端的超分辨率显微镜来了解细胞是如何产生旋转的。此外，我们将结合从跟踪数以万计的细胞运动中获得的数据与数学模型，以量化逆转和旋转如何共同产生趋化性。综上所述，这项研究将从根本上为细胞如何调节其在生物膜内的运动提供新的理解，可能为我们提供新的工具来抑制生物膜的发育或控制工业应用中的细胞运动。

Most bacteria live attached to surfaces where they form dense communities called biofilms. While some of these assemblages play beneficial roles in our lives, infections within the human body are often very difficult to treat because biofilms protect cells from both antibiotics and the immune system. Understanding the fundamental processes that contribute to biofilm formation is essential to developing new ways to disrupt or manipulate these important bacterial communities.The bacterial species Pseudomonas aeruginosa, which causes dangerous infections in burn victims and cystic fibrosis patients, use tiny grappling hook-like appendages called pili move within biofilms. Our group recently demonstrated that single P. aeruginosa cells can use pili-based motility to navigate to more favourable nutrient environments within a developing biofilm. We found that this process, called chemotaxis, arises because cells pull themselves in the opposite direction when they detect they are moving away from a nutrient source. This remarkable ability likely gives chemotactic cells an advantage in biofilms and opens a new way to control biofilm formation. While other forms of microbial chemotaxis have been extensively studied, relatively little is known about pili-based chemotaxis. This study will address two major gaps in our knowledge:First, we do not know how bacteria actually sense whether they are going towards or away from the source of chemoattractant. In general, there are two different possibilities: cells could either move from one location to another and measure the change in concentration over time (temporal sensing) or they could directly sense changes in concentration over the length of their bodies (spatial sensing). The proposed work will use a combination of novel microfluidic experiments, computer based cell tracking, and bacterial genetics to directly test these two different possibilities. Our preliminary experiments indicate that cells do not increase their probability of reversing when they experience a decrease in the concentration of chemoattractants over time, suggesting that surface attached cells do not use temporal sensing to guide chemotaxis. Future experiments will use novel bacterial strains that relocalise fluorescently labelled proteins to their opposite pole when they reverse direction, which will allow us to test if cells can sense changes in concentration over their length. Second, our experiments indicate that cells tend to travel directly up chemical gradients, yet this observation cannot be explained by known forms of bacterial motility. Surface attached bacteria are thought to have only one behaviour in their repertoire to facilitate chemotaxis: reverse direction. However, reversals alone only allow cells to explore a one-dimensional line, so if a cell happened to land on a surface perpendicular to the gradient, it would be incapable of steering towards the nutrient source. In our preliminary work, we have discovered a new way in which surface attached cells can reorient their motility. Our computer-based image analysis software reveals that cells frequently perform somersault-like manoeuvres we call "twiddles". These reorientations occur when cells steer in either a clockwise or counter-clockwise direction over a period of minutes to hours. This study will use novel bacterial strains and cutting edge super-resolution microscopy to understand how cells generate twiddles. In addition, we will combine data obtained from tracking the movement of tens of thousands of cells with mathematical models to quantify how reversals and twiddles work together to generate chemotaxis.Taken together, this study will provide fundamentally new understanding of how cells regulate their movement within biofilms, potentially giving us new tools to inhibit biofilm development or control the motility of cells in industrial applications.