Internalization of S. mutans in vascular endothelial cells

描述(申请人提供)：变形链球菌(Sm)长期以来一直被认为是龋病的主要病原体，龋病是人类最常见的传染病。但人们对它在口腔外感染中的作用却知之甚少。SM是一种机会致病菌，常见于人类细菌血症中，是感染性心内膜炎(IE)的病原体。IE是一种严重的心血管疾病，通常与牙科手术直接相关，是一种瓣膜内皮细胞肥大。现在有强有力的证据表明，Sm与出血性中风有关，出血性中风发生在大脑中的血管破裂时。此外，流行病学和DNA测序证据支持Sm在动脉粥样硬化中的作用。动脉粥样硬化是一种复杂的炎症性疾病，困扰着大中型动脉，在美国是主要的死亡原因。这些疾病的共同之处在于它们都是心血管系统的疾病。病原体附着和侵袭内皮组织的能力是IE致病所必需的，侵袭内皮细胞的能力已被证明与心内膜炎的严重程度相关。我们之前已经报道过特定的Sm菌株可以侵袭人的心血管主动脉内皮细胞(HCAEC)。新的数据表明，HCAEC的侵袭表型与编码胶原结合蛋白CNM的基因的存在有关，CNM的缺失使菌株无法侵袭。SM OMZ175是一种表达CNM的菌株，与对照组相比，在加速的动脉粥样硬化小鼠模型中，SM OMZ175产生了显著的病理变化。细胞内病原体已经进化出多种途径和机制进入、运输和存活于真核细胞内。因此，进入的方式在决定细菌命运的过程中起着重要作用。目前还没有关于Sm进入细胞的细胞事件的报道。我们的总体假设是，表达CNM的Sm菌株，如我们的模式生物OMZ175，能够在HCAEC中黏附、进入并持续存在，进入机制对Sm在细胞内的运输和生存至关重要。我们的工作假设将在这项拟议的项目中得到验证，即侵袭性Sm通过特定的机制进入内皮细胞，而CNM在这一进入中发挥核心作用。了解一种普遍存在的口腔病原体的特定菌株获得的将其毒力扩展到口腔以外部位的机制，将有助于进一步深入了解粘附素介导的链球菌与其宿主相互作用的分子细节。这项申请的具体目的是：1)确定Sm加入HCAEC的机制；2)确定CNM在加入过程中的贡献。我们自己和其他人的数据表明，Sm的侵袭性菌株也会入侵口腔细胞，并可以在有牙齿的儿童中检测到。此外，现已在血链球菌、猪链球菌和鼠链球菌中发现了CNM。因此，这些研究将具有超越CVD病理学的意义。此外，该项目还将有助于将CNM作为超强毒力Sm菌株的生物标志物的应用。

DESCRIPTION (provided by applicant): Streptococcus mutans (Sm) has long been recognized as a major pathogen of dental caries, the most common infectious disease in humans. What is much less appreciated is its role in extra-oral infections. Sm is an opportunistic pathogen frequently identified in human bacteremias and is an etiologic agent of infectious endocarditis (IE). IE, a valvular vegetation of the endothelial layer, is a serious cardiovascular disease with often fatal outcomes directly related to dental procedures. There is now strong evidence that Sm is involved in hemorrhagic stroke, which occurs when a blood vessel ruptures in the brain. Additionally, epidemiological and DNA sequencing evidence supports a contributing role of Sm to atherosclerosis. Atherosclerosis is a complex inflammatory disease afflicting medium and large sized arteries and is the leading cause of death in the USA. The commonality of these diseases is that they are diseases of the cardiovascular system. The ability of a pathogen to attach to and invade endothelial tissues has previously been shown to be required for virulence in IE and the ability to invade endothelial cells has been shown to correlate with endocardititis severity. We have previously reported that specific strains of Sm can invade human cardiovascular aortic endothelial cells (HCAEC). New data demonstrate that the HCAEC invasion phenotype correlates with the presence of a gene encoding a collagen binding protein, Cnm, and that deletion of cnm renders the strains unable to invade. Sm OMZ175, a strain that expresses Cnm, produces significant pathology compared to control mice in a mouse model of accelerated atherosclerosis. Intracellular pathogens have evolved multiple pathways and mechanisms to enter, traffic and survive within eukaryotic cells. Thus, the mode of entry plays a major role in determining bacterial fate. There are no reports about the cellular events that govern Sm entry into cells. Our overall hypothesis is that strains of Sm expressing Cnm, such as our model organism OMZ175, are able to adhere to, enter, and persist in HCAECs and that the mechanism of entry is critical to the trafficking and survival of Sm intracellularly. Our workig hypothesis to be tested in this proposed project is that invasive Sm enters endothelial cells using specific mechanisms and that Cnm plays a central role in this entry. Understanding the mechanisms that specific strains of a ubiquitous oral pathogen have acquired that expand its virulence to sites outside of the oral cavity would provide further insight into the molecular details of adhesin mediated streptococci interactions with its host. The specific aims of this application are 1) Determine the mechanism of entry of Sm into HCAEC and 2) Determine the contribution of Cnm in the entry process. Our own data and that of others indicate that invasive strains of Sm also invade oral cells and can be detected in predentate children. In addition, cnm has now been found in strains of S. sanguinis, S. suis and S. rattus. Therefore these studies will have significance beyond CVD pathology. In addition, this project would contribute information concerning the application of cnm as a biomarker for hypervirulent strains of Sm.