粘细菌生物膜中基于核酸-多糖相互作用的复合型胞外基质的生物学特性和构建机理

Bacteria in natural surroundings are able to grow as biofilms, which comprises highly structured matrix-enclosed communities and differs profoundly from their planktonic counterparts at least in terms of physiological responses and developmental dynamics. This multicellular and polymer-encased mode of growth is now accepted as a preferred lifestyle option for prokaryotes. The extracellular matrix (ECM) is considered as the physical base of biofilm integration and structure-forming, which is defined as a mixture of extracellular polysaccharides (EPS), nucleic acids, proteins and other cell-released components. It has been noticed that some bacteria can produce substantial quantities of DNA to the ECM, which might be involved in biofilm formation. While little is known about present state of extracellular DNA (eDNA) in the biofilms, especially about its interactions with other macromolecules in the matrix and participation in the establishment of biofilm architecture. According to our preliminary studies, for the first time, it was recognized that all EPS-based biofilms formed by Myxococcus xanthus contained extensive amounts of eDNA molecules as part of the ECM. Most intriguingly, unlike the eDNA reported to be present in other bacteria that release free DNA into their environment, the eDNA in M. xanthus is intimately interwoven with the fibrillar EPS network and follows the same structural pattern. Based on further data presented in this application, it is indicated that DNA excretion in M. xanthus might be an active and coordinated event, which strengthens the EPS mechanical properties. Therefore, we developed the following working hypothesis: "In addition to be released by cell lysis associated with program cell death, M. xanthus cells may actively secrete eDNA in an organized fashion to bind EPS to build a stronger extracellular matrix network for improved biological activities". The study starts form elucidating the molecular and biochemical basis of interaction between eDNA and EPS, and their roles in establishment of a 'super' complex ECM. Subsequently, the biological features associated with the integrated DNA-EPS ECM will be analyzed. Moreover, the molecular components involved in actively producing eDNA and building the integrated ECM will be identified and characterized. The aims proposed in this application are designed to test our hypothesis which has the potential to provide insightful information on the role of ECM in biofilm formation as well as assign novel and important biological function to DNA beyond its role as genetic material. Furthermore, since eDNA and EPS do exist in various other pathogens, the finding derived from this study may help to further elucidate key virulence factors during biofilm formation, tissue adherence and invasion for these bacteria.

在自然条件下，细菌能够形成高度结构化和群落化的生物膜结构，胞外DNA（eDNA）在这一过程中起到了重要作用。但是，仍然还有许多关键科学问题没有解决。我们首次证明了粘细菌的代表类群黄色粘球菌（Myxococcus xanthus）形成的生物膜胞外基质中存在大量高度组织化的eDNA，并提出eDNA与胞外多糖（EPS）发生了复杂的大分子相互作用。这一相互关系导致了eDNA以一种化学/物理有序的方式与EPS紧密交织，遵循类似的构建和分布模式，形成复合型胞外基质的网络结构。本项目将综合利用微生物学、生物化学、生物物理学和分子生物学等多种方法，从不同角度、多层面地研究M. xanthus胞外基质中eDNA与EPS相互作用的物化机制及其所赋予生物膜的重要生理功能，试图探求复合型胞外基质的构建原理。这将扩展对于eDNA和EPS在生物膜中履行功能的认知，有助于了解生物膜形成过程中所涉及的特殊分子机制。

在自然条件下，细菌附着在物体表面形成高度结构化和群落化的生物膜并成为一种更为优势化的生活方式。通过我们的前期研究，首次证实了黄色粘球菌（Myxococcus xanthus）生物膜中还存在高度组织化的胞外DNA（eDNA），并且与胞外多糖（EPS）分子呈现了明显的共定位分布，形成了复合型的胞外基质（ECM）。本项目依据从简单到复杂，从体外到体内，从化学分子水平到细胞系统水平的原则开展了相关研究，以M. xanthus形成的发育性和非发育性生物膜为基本模式和研究对象，阐明了ECM中DNA-EPS相互作用的化学实质及其赋予生物膜的生理功能，并探索了DNA-EPS复合型ECM构建的分子机制。首先，获取的直接实验证据表明DNA与EPS之间存在着复杂的大分子相互作用，从化学分子水平上阐明了静电相互作用在这一过程中的主导地位，并且进一步证明了这一作用介导了eDNA与EPS在生物膜ECM中的分布与定位。其次，我们表征了eDNA-EPS的相互作用所赋予复合型ECM的相关生物学功能和特性。分析缺失了eDNA或者EPS的M. xanthus生物膜在各种生物特性和功能方面发生的改变，证明了复合型ECM在机械强度、粘附力和对于细胞的保护作用等方面具备明显的优势；确认了在复合型ECM中，EPS对DNA具有保护作用能够抵抗各种核酸水解酶的攻击，从而保证eDNA在基质中持续稳定的存在；实现了自动追踪并分析大量细胞的运动行为，表征了M. xanthus形成生物膜时，eDNA-EPS复合型ECM对于细胞的S运动起到了粘附调节和具体行为选择的作用。进一步探讨了DNA-EPS复合型ECM对于M. xanthus的生物膜各种生物过程的影响，如捕食，子实体发育，粘孢子形成等，从而通过系统的实验证明了eDNA与EPS的协调产生并发生相互作用对于构建DNA-EPS复合型ECM及其功能的履行是十分重要的。最后，我们通过遗传学和生物化学手段鉴定和表征了在M. xanthus生物膜形成过程中参与eDNA产生和基质形成的部分调控元件。本项目取得的结果不但可以帮助我们更加深入的了解eDNA和EPS在细菌生物膜中的功能以及复杂细菌生物膜的形成机制，还可为研究细菌生物膜抗性和感染以及寻找治疗相关疾病的药物提供借鉴。