Amylase Binding Streptococci Dental Plaque and Caries

描述：牙菌斑是导致龋齿和牙周病的口腔生物膜。许多唾液蛋白已被证明与菌斑中的细菌相互作用，并且这种相互作用可能在菌斑形成中起关键作用。其中一种相互作用是淀粉酶（唾液中最丰富的酶）与淀粉酶结合链球菌（ABS）之间的相互作用，ABS在牙菌斑中数量众多。基于我们在上一个资助期内产生的最新发现，这个曾经修改过的竞争性续期申请旨在继续我们对淀粉酶与ABS结合的生物化学，生理学，龋齿学和生态学后果的研究。我们发现，虽然淀粉酶结合缺陷的突变体坚持不太好的淀粉酶包被的表面，并在体外表现出有缺陷的生物膜形成，他们殖民的大鼠牙齿比野生型菌株，竞争超过他们的父母在大鼠喂蔗糖/淀粉饮食的菌株。这些令人惊讶的发现使人们认识到AbpA抑制蔗糖依赖性定殖决定子，例如但可能不限于S. gordonii。因此，我们下一个资助期的目标是：1）研究参与淀粉酶结合的基因和蛋白质（abpA和abpB）以及参与葡聚糖合成的基因（rgg和gtfG）之间的潜在相互作用。我们将评估突变株中abpA、abpB和gtfG的转录，以确定Abp是否在转录水平调节gtfG表达（反之亦然）。2)在标准体外粘附和生物膜模型中比较野生型和突变株。3)使用蛋白质组学方法评估Gtf和淀粉酶结合蛋白之间的物理相互作用，例如蛋白质印迹-配体结合测定、Maldi-Tof（基质辅助、激光解吸-电离/飞行时间）质谱法或噬菌体展示。4)确定Gtf缺陷或Gtf熟练的S. gordonii调节S.戈登氏菌在大鼠口腔定植和致龋性。5)确定S. gordonii-淀粉酶相互作用调节S.变形链球菌定殖竞争和致龋性;表达AbpA的变异株显示出改变的定殖和/或致龋能力。整合体外与体内研究对于了解牙菌斑形成的机制至关重要。

DESCRIPTION: Dental plaque is the oral biofilm responsible for the etiology of Dental caries and periodontal disease. A number of salivary proteins have been shown to interact with bacteria in plaque, and such interactions likely play critical roles in plaque formation. One such interaction is that between amylase, the most abundant enzyme in saliva, and the amylase-binding streptococci (ABS), which are numerous in plaque. Based on our recent findings generated during the previous funding period, this once-amended competitive renewal application seeks to continue our studies of the biochemical, physiological, cariological and ecological consequences of amylase-binding to ABS. We found that while amylase binding-deficient mutants adhere less well to amylase-coated surfaces and demonstrate defective biofilm formation in vitro, they colonize rat teeth better than wild type strains, out-compete their parental strains in rats fed sucrose/starch diet. These surprising findings led to the realization that AbpA inhibits sucrose-dependent colonization determinants such as, but perhaps not limited to, GtfG of S. gordonii. Thus, our Aims for the next funding period are to: 1) investigate potential interactions of genes and proteins involved in amylase binding (abpA and abpB) and genes involved in glucan synthesis (rgg and gtfG). We will evaluate the transcription of abpA, abpB and gtfG in mutant strains to determine if Abp modulates gtfG expression (or vice versa) at the transcriptional level. 2) compare wildtype and mutant strains in standard in vitro adhesion and biofilm models. 3) assess the physical interaction between Gtf and amylase-binding proteins using proteomic approaches such as Western blot-ligand binding assays, Maldi-Tof (Matrix-assisted, Laser-Desorption-Ionization/Time of Flight) mass spectrometry, or phage display. 4) determine if amylase-binding mutations in Gtf-deficient or -proficient S. gordonii modulate S. gordonii oral colonization and cariogenicity in rats. 5) determine if S. gordonii-amylase interactions modulate S. mutans colonization competition and cariogenicity and if strains of S. mutans made to express AbpA show altered colonization and/or cariogenic abilities. Integration of in vitro with in vivo studies is crucial for mechanistic understanding of Dental plaque formation.