The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm.
The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm.
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DOI：
10.1371/journal.ppat.1002623
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Koo H
中科院分区：
文献类型：
--
作者：
Xiao J;Klein MI;Falsetta ML;Lu B;Delahunty CM;Yates JR 3rd;Heydorn A;Koo H
毒性生物膜可导致一系列感染，包括口腔疾病。所有生物膜都含有微生物衍生的细胞外基质。牙膜和细菌表面形成的外多糖（EPS）为微生物提供了结合位点；最终，积累的EPS与微生物细胞结合在一起。细菌在基质中的代谢活动导致环境酸化。我们利用混合细菌物种系统，探索了变形链球菌产生的eps -基质在唾液涂层-磷灰石表面生物膜形态发生过程中调节三维结构和种群变化的机制。同时，我们使用一种新的3D原位ph定位技术检测了基质是否会影响完整生物膜内ph微环境的发展。数据显示，eps基质的产生有助于通过局部细胞-基质相互作用形成胞外多糖缠结的细菌-胰岛（微菌落）的复杂网络，从而产生空间异质性。这种复杂的3D结构在整个生物膜中创造了分隔的酸性和富含eps的微环境，从而引发了致病变形链球菌在混合物种系统中的优势地位。3d基质和eps包埋微菌落的建立主要由变形链球菌gtfB/gtfC基因介导，该基因在naeslundii放线菌和口腔链球菌存在下表达增强。酸性口袋仅在受EPS保护的细菌胰岛内部被发现，这阻碍了缓冲液（pH 7.0）的快速中和。结果，在附着表面的特定位置检测到低pH（<5.5）区域。与生物膜内这种结构外的细胞相比，eps微集落复合物内的细胞对氯己定的抗性增强。我们的研究结果说明了三维环境中基质结构和pH异质性之间的关键相互作用。在靠近磷灰石表面的结构酸性微环境的形成是与龋生生物膜毒力相关的重要因素。这些观察结果可能具有口腔以外的相关性，因为基质是所有生物膜所固有的。表面形成的毒性生物膜与许多人类感染有关。龋齿，即蛀牙，是细菌与牙齿表面的糖相互作用的一个典型例子。当变形链球菌代谢糖时，它们会产生一种叫做葡聚糖的胶状聚合物，帮助它们牢固地附着在牙齿上。葡聚糖也会在口腔内的细菌表面形成，并会聚集和缠绕额外的微生物，形成被称为牙菌斑生物膜的凝胶状结构。我们在这些生物膜中发现了独特的细菌岛，特别是靠近牙齿表面的地方，为细菌的繁殖提供了安全的避风港，并产生了腐蚀牙齿的酸。一个有趣的谜团是，当牙齿周围有大量中性ph值的唾液时，为什么酸会积聚在牙齿表面。我们发现细菌胰岛特别受到葡聚糖的保护，它延缓了中和。我们注意到，在生物膜内，这些胰岛的内部是酸性的，只有耐酸细菌才能在那里繁殖，确保持续的局部酸生产。我们的研究表明，由葡聚糖介导的生物膜的构建形成了复杂的3D结构，创造了多种对毒力表达至关重要的酸性微环境。这些结果可能有助于开发调节生物膜形成的增强方法。
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms. Virulent biofilms formed on surfaces are associated with many human infections. The disease dental caries, expressed as cavities, is a prime example of the consequences arising from interactions between bacteria and sugars on tooth-surfaces. When Streptococcus mutans metabolize sugars, they produce a glue-like polymer termed glucan, helping them to adhere firmly to teeth. Glucan is also formed on bacterial surfaces in the mouth, and will accumulate and enmesh additional microorganisms creating the gelatinous formation known as dental plaque-biofilm. We found unique islets of bacteria within these biofilms, particularly close to the tooth-surface, providing safe havens in which bacteria thrive and produce acids that erode teeth. One intriguing mystery is why acids accumulate on the tooth-surface when there is an abundance of neutral-pH saliva surrounding the teeth. We found that bacterial-islets are particularly protected by glucan, which retards neutralization. We noticed that, within biofilms, the interiors of these islets are acidic, where only acid-tolerant bacteria can prosper, ensuring continued localized acid production. Our study demonstrates that construction of biofilms mediated by glucans forms complex 3D architectures, creating a variety of acidic-microenvironments that are essential for virulence expression. These results may aid in the development of enhanced methods to modulate biofilm formation.
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