黄色粘球菌四型菌毛与胞外多糖特异性相互作用的分子机制与生物学意义

Myxococcus xanthus (Mx) is a Gram-negative soil bacterium capable of sophisticated social behaviors, e.g. fruiting body formation and specific predatory behavior. A crucial feature of these behaviors is the ability to move on solid surfaces by a flagella-independent gliding motility, which is regulated by the adventurous and social (S) motility systems. The S motility, that is mechanistically equivalent to the twitching motility in P. aeruginosa and N. gonorrhoeae, is typically employed for coordinated group movement of cells and powered by cycles of type IV pili (TFP) extension and retraction triggered by interaction of TFP with exopolysaccharide (EPS). As the molecular engines, TFP are composed of thousands of subunits called type IV pilin (PilA). As the binding target, EPS also forms a complex network within Mx biofilms to direct motility and morphogenesis. Although specific interactions between TFP and EPS have been proposed, the key moieties, both in TFP and EPS, involved in the interaction remain elusive...In this study, we propose to explore the specific interaction between TFP and EPS in Mx with combined approaches. Several biochemical/biophysical techniques will be employed to investigate the detailed characterizations of interaction between the PilA and saccharides, which may reveal the potential moieties in EPS responsible for the interaction. By screening a large number of mutant PilA proteins for their recognition of specific EPS moiety, the residues in PilA crucial for the interaction may be determined. Then we will try to further understand molecular mechanisms underlying some multi-cellular behaviors: how the TFP-EPS interaction dominates S motility; how EPS provides the specific guiding information to establish the complicated architectures of biofilms and fruiting bodies; if there are any small molecules can specifically inhibit the TFP-EPS recognition. Furthermore, it has been discovered that a variety of adjacent albeit distinct fruiting bodies of different Myxococcus appear on the same piece of soil, implying that these strains discriminate between different individuals in a mixed community. Based on our preliminary results, we seek to validate the hypothesis that TFP may act as feller for Myxococcus self-recognition through strains/species specific interactions with their corresponding EPS, which will greatly expand our capacity for the biological significance of TFP-EPS specific interactions on the ecological and evolutionary level...Mx is an excellent model system for this study because the involvement of TFP and EPS on motility was first discovered in this bacterium. The possible findings are likely to add significantly to the biological knowledge about regulatory events during social behaviors. TFP/EPS and their associated interactions are also key virulence factors for various pathogens, and this study will potentially lead to an increased understanding of new therapeutic approaches that target TFP-EPS.

黄色粘球菌（M. xanthus）细胞表面的四型菌毛（TFP）和胞外多糖（EPS）之间的特异性识别及相互作用在多个细胞群体行为过程中发挥了十分关键的作用。本项目将综合运用微生物学、分子生物学、生物化学和生物物理学等手段和方法，从不同角度、多层面地分析TFP-EPS相互作用的物理/化学实质，揭示它们参与特异性结合的关键位点，并进一步探究其在介导和调控群体性细胞行为过程中发挥的核心作用，例如：主导细胞群体运动的分子机制；为细胞提供导向性信息继而指导生物膜/子实体的空间定位与结构构建的机理；检验粘球菌依靠特异性的TFP-EPS相互识别在混合群体中实现种群分割并形成单一细胞来源子实体的可能性。这对于深入理解细菌复杂的细胞群体行为以及微观生态过程都有着重要的科学价值。同时，由于TFP和EPS在众多病原微生物的感染过程中不可或缺，本研究的结果也可以为深刻理解细菌的致病机制提供帮助。

黄色粘球菌（M. xanthus）细胞表面的四型菌毛（TFP）和胞外多糖（EPS）之间的特异性识别及相互作用在群体运动、捕食、自我/异体细胞识别、生物被膜形成以及多细胞分化发育等多个细胞群体行为过程中发挥了十分关键的作用，但是TFP和EPS中参与特异性识别和结合的关键位点仍然未知，极大地阻碍了深刻的理解这一相互作用的生化本质和生理功能。通过本项目的实施，我们在建立M. xanthus细胞运动行为的自动化追踪与分析、TFP的单体PilA蛋白与EPS相互作用的定性及定量分析、捕食以及分化发育等复杂细胞行为过程中的细胞荧光定位分析等技术方法的基础之上，综合利用微生物学、分子生物学、生物化学和生物物理学等多种手段，从不同角度、多层面地揭示了TFP-EPS相互作用的物理/化学特征。寻找和鉴定了PilA与EPS中参与特异性识别和结合的相关位点，证明了M. xanthus EPS中与PilA相互作用的关键残基是以氨基葡萄糖为代表的氨基己糖，筛选得到了Tyr69和Trp146两个氨基酸残基是PilA中与EPS发生相互作用的可能关键位点。进一步，通过系统分析基于TFP-EPS 相互作用的S-运动在M. xanthus捕食过程中的整合细胞行为功能，揭示了这一相互作用在介导捕食者-猎物这一种间相互作用过程中的可能作用。同时，发现相对近缘的不同粘球菌菌株能够在混和培养中实现种群分割并形成独立的子实体，并且其PilA蛋白只与各自的EPS发生特异性结合，进而提示了不同菌株依靠TFP-EPS的特异性相互识别区分自我/非我细胞，进而实现种群分割和“纯洁遗传”的可能性。本项目所获得的实验结果，有助于深入理解M. xanthus中TFP-EPS生物大分子特异性相互作用的分子机制，并且进一步拓展了这一相互作用的生物学意义，揭示其在介导粘细菌复杂细胞行为过程和微生物种间相互作用等方面的可能功能。