S. mutans-C. albicans interactions synergize the virulence of cariogenic biofilms

 描述（由申请人提供）：幼儿牙菌斑生物膜的微生物学研究揭示了幼儿龋齿（ECC）与C.白色念珠菌，沿着S.变异人这种关联如何与疾病的发病机制有关仍然是一个谜。我们的初步数据提供了惊人的证据，S。mutans和C.白色念珠菌发展出一种共生关系，在蔗糖存在下协同牙菌斑-生物膜的毒力，放大了体内龋损的严重性。使用我们的ECC啮齿动物模型，我们观察到感染水平随着S。mutans和C.与单独感染任一种的那些相比，来自共感染动物的噬菌斑生物膜内的白色念珠菌。重要的是，在共感染动物中菌斑-生物膜的毒力协同增强，导致牙齿光滑表面上猖獗的龋损的发展（类似于在ECC中发现的那些）。观察到的协同作用是迷人的，因为C。白色念珠菌通常与S.变异人然而，进一步的体外研究表明，S。突变体衍生的胞外酶称为GtfB，与蔗糖作用，在S. mutans和C.白色念珠菌相互结合并形成同种生物膜。随着同种生物膜的形成，C.白色念珠菌通过增强高度不溶性和扩散限制性EPS基质的组装和S.变形菌小菌落因此，C.白色念珠菌衍生的因子有助于同种生物膜的形成。 我们假设S.通过GtfB和C.白色念珠菌因子调节牙齿上高度致龋生物膜的发展。可以想象的是，含有密集的产酸微生物群体的细胞外基质的改变增强了局部的酸积累，这对于龋齿病变的发展是至关重要的。为了验证我们的假设，我们将关注三个目标：在目标1中，我们将确定C。白念珠菌突变体与S. 利用无偏遗传学方法（通过筛选转录因子缺失突变体的可用文库）结合生物化学和共聚焦显微镜方法在体外对突变体进行了研究。梭白色念珠菌和gtfB缺陷型S.然后将检查变异株（及其亲本菌株）对体外生物膜pH微环境（Aim 2）和体内生物膜毒力表达（Aim 3）的影响。在目标2中，我们将研究细菌-真菌相互作用如何调节完整生物膜结构内酸性小生境的时空发展，使用我们的时间推移pH映射。最后，在目标3中，我们将研究GtfB和C的作用。白念珠菌在龋病发病机制中的作用。白色念珠菌和gtfB缺陷型S.变异株（以其亲本菌株作为对照）与我们的啮齿动物模型。从实验室研究到体内研究的全面计划提供了对这种高度依赖性的分子机制的重要见解。 毒性跨王国相互作用及其在ECC中的意义。

 DESCRIPTION (provided by applicant): Microbiological studies of plaque-biofilms from toddlers reveal an association between early-childhood caries (ECC) and the presence of C. albicans, along with elevated populations of S. mutans. How this association is implicated in the pathogenesis of the disease remains an enigma. Our preliminary data provide striking evidence that S. mutans and C. albicans develop a symbiotic relationship that synergizes virulence of plaque- biofilms in the presence of sucrose, amplifying the severity of carious lesions in vivo. Using our rodent model of ECC, we observed enhanced levels of infection with elevated carriage of both S. mutans and C. albicans within plaque-biofilms from co-infected animals compared to those infected with either species alone. Importantly, the virulence of plaque-biofilm in co-infected animal was synergistically enhanced, leading to the development of rampant carious lesions on smooth-surface of teeth (similar to those found in ECC). The observed synergism is fascinating because C. albicans usually does not associate well with S. mutans. However, further in vitro studies revealed that S. mutans-derived exoenzyme termed GtfB, acting with sucrose, plays a central role in the ability of S. mutans and C. albicans to associate with each other and form co-species biofilms. As co- species biofilm is initiated, the presence of C. albicans dramatically modifies the physical environment of the biofilm by enhancing the assembly of highly insoluble and diffusion-limiting EPS matrix and the growth of S. mutans microcolonies. Thus, C. albicans-derived factors contribute with co-species biofilm development. We hypothesize that S. mutans-C.albicans association mediated via GtfB and C. albicans factors modulate the development of hypercariogenic biofilms on teeth. It is conceivable that the alterations in the extracellular matrix containing a dense population of acidogenic microorganisms enhance acid accumulation locally, which is critical for the development of carious lesions. To test our hypothesis, we will focus on three aims: In Aim 1, we will identify C. albicans mutants defective in the ability to develop co-species biofilm with S. mutans using unbiased genetic approach (by screening an available library of transcription factor deletion mutants) combined with biochemical and confocal microscopy methods in vitro. The C. albicans defective in co-species biofilm and gtfB-defective S. mutans (and their parental strains) will be then examined for their influence on biofilm pH microenvironment in vitro (Aim 2) and expression of biofilm virulence in vivo (Aim 3). In Aim 2, we will investigate how bacterial-fungal interactions modulate the spatio-temporal development of acidic niches within intact biofilm architecture using our time-lapsed pH mapping. Lastly, in Aim 3, we will examine the role of GtfB and C. albicans factors in the pathogenesis of dental caries in vivo using C. albicans defective in co-species biofilm and gtfB-defective S. mutans (with their parental strains as controls) with our rodent model. A comprehensive program from laboratory studies to in vivo investigations is offered to provide critical insights into the molecular mechanisms of this highly virulent cross-kingdom interaction and their implications in ECC.