Intracellular Invasion by Streptococcus mutans: Significance in Disease

变形链球菌的细胞内侵袭：在疾病中的意义

• 批准号: 8436799
• 负责人: Jacqueline Abranches
• 金额: $ 35.59万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-17 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436799
• 关键词: Active Immunization; Adhesions; Antibodies; Arterial Fatty Streak; Atherosclerosis; Attenuated; Bacteria; Binding; Binding Proteins; Biological Markers; Blood Circulation; Blood Platelets; Brain hemorrhage; Cardiac; Cerebrum; Clinical; Collagen; Communicable Diseases; Coronary artery; Cytoplasm; DNA; Data; Defect; Dental; Dental caries; Detection; Development; Disease; Endocarditis; Endothelial Cells; Endothelium; Environmental Risk Factor; Event; Extracellular Matrix; Genes; Genomics; Glycoproteins; Goals; Heart; Heart Diseases; Heart Valves; Human; Immunoblot Analysis; In Vitro; Infection; Infective endocarditis; Invaded; Laminin; Larva; Lesion; Link; Mediating; Microbial Biofilms; Modeling; Molecular; Mus; Mutagenesis; Names; Operon; Oral cavity; Organism; Oryctolagus cuniculus; Pathogenesis; Patients; Phenotype; Platelet Activation; Prevention strategy; Preventive; Property; Prophylactic treatment; Proteins; Publications; Publishing; Recombinants; Research; Risk; Role; Site; Streptococcus mutans; Surface; System; Systemic infection; Threonine; Tissues; Translating; Variant; Virulence; Virulence Factors; Work; antimicrobial; design; efficacy testing; experience; glycosylation; glycosyltransferase; human tissue; in vitro Model; in vivo; mutant; novel; oral bacteria; oral infection; pathogen; prevent; public health relevance; research study; trait

DESCRIPTION (provided by applicant): There is growing evidence that certain facets of heart disease may be influenced by infections arising from the oral cavity. Streptococcus mutans, a major etiologic agent of dental caries, is also a leading causative agent of infective endocarditis (IE) and has been implicated in the development and progression of atherosclerosis. We have recently demonstrated that certain S. mutans strains invade and persist in the cytoplasm of Human Coronary Artery Endothelial Cells (HCAEC). Invasion of HCAEC was associated with an ability to avidly bind to collagen and laminin, and invasive strains were significantly more virulet than non-invasive strains in the Galleria mellonella model for systemic infection. A collagen-binding protein (Cnm) was found only in invasive strains, and inactivation of the cnm gene significantly impaired collagen and laminin binding capacity, abolished HCAEC invasion, and attenuated virulence in G. mellonella. Characterization of a gene encoding a putative glycosyltransferase (CsbB) co-transcribed with cnm suggested that CsbB is involved in Cnm glycosylation. Preliminary data using a rabbit endocarditis model suggested that expression of Cnm enables infection of the underlying endothelium by S. mutans whereas a cnm mutant was trapped in the heart valve vegetation. Collectively, our data suggest that the ability to invade an persist in the host cytoplasm is an important, previously unrecognized, virulence trait of S. mutans. Our working hypothesis is that S. mutans strains expressing Cnm have an enhanced capacity to colonize human tissues, and that this ability is a major virulence trait in systemic infections. The major goals of this application are to conduct the functional analysis of Cnm and to characterize the role of Cnm in host-bacteria interactions. To achieve these goals, we propose three well-integrated Specific Aims. In Aim 1 (Molecular characterization of the cnm locus), we will (i) conduct the transcriptional characterization of the cnm operon, (ii) employ mutagenesis approaches to continue the characterization of the Cnm and CsbB, and (iii) identify the essential machinery for intracellular invasion using an heterologous host system. In Aim 2 (Functional analysis of Cnm), we will (i) use recombinant variant forms of Cnm to identify the functional domains of Cnm, and (ii) determine whether Cnm is glycosylated and, if so, how Cnm is modified. In Aim 3 (Streptococcus mutans-host interactions), we will (i) use in vitro models to assess Cnm-platelet interactions, (ii) use an ex vivo adhesion model to examine the ability of invasive and non-invasive strains to colonize heart tissues, and (iii) use a rabbit endocarditis model to establish the contribution of Cnm to the pathogenesis of IE and to test the efficacy of active immunization with Cnm to prevent S. mutans infections. This study has strong potential to be readily translated into clinical settings as Cnm could serve as a biomarker for detection of hypervirulent strains in patients in need to receive prophylactic treatment, or as a potential target for the development of effective strategies for prevention and treatment of S. mutans extra-oral infections.

描述（由申请人提供）：越来越多的证据表明，心脏病的某些方面可能会受到口腔感染的影响。变形链球菌是龋齿的主要病原体，也是感染性心内膜炎的主要病原体 （IE）并与动脉粥样硬化的发生和进展有关。我们最近证明某些变形链球菌菌株侵入并持续存在于人冠状动脉内皮细胞（HCAEC）的细胞质中。 HCAEC 的侵袭与强烈结合胶原蛋白和层粘连蛋白的能力相关，并且在大蜡螟全身感染模型中，侵袭性菌株的毒性明显高于非侵袭性菌株。仅在侵入菌株中发现了胶原结合蛋白（Cnm），cnm 基因失活显着损害了胶原和层粘连蛋白的结合能力，消除了 HCAEC 侵袭，并减弱了大蜡螟的毒力。与 cnm 共转录的编码假定糖基转移酶 (CsbB) 的基因的表征表明，CsbB 参与了 Cnm 糖基化。使用兔心内膜炎模型的初步数据表明，Cnm 的表达使得变形链球菌能够感染底层内皮，而 cnm 突变体则被困在心脏瓣膜赘生物中。总的来说，我们的数据表明，侵入并持续存在于宿主细胞质中的能力是变形链球菌的一个重要的、以前未被识别的毒力特征。我们的工作假设是，表达 Cnm 的变形链球菌菌株具有增强的定植人体组织的能力，并且这种能力是全身感染的主要毒力特征。该应用的主要目标是进行 Cnm 的功能分析并表征 Cnm 在宿主-细菌相互作用中的作用。为了实现这些目标，我们提出了三个整合良好的具体目标。在目标 1（cnm 位点的分子表征）中，我们将 (i) 进行 cnm 操纵子的转录表征，(ii) 采用诱变方法继续表征 Cnm 和 CsbB，以及 (iii) 使用异源宿主系统识别细胞内入侵的基本机制。在目标 2（Cnm 的功能分析）中，我们将 (i) 使用 Cnm 的重组变体形式来鉴定 Cnm 的功能域，以及 (ii) 确定 Cnm 是否糖基化，如果是，则确定 Cnm 是如何修饰的。在目标 3（变形链球菌与宿主相互作用）中，我们将（i）使用体外模型评估 Cnm-血小板相互作用，（ii）使用离体粘附模型来检查侵入性和非侵入性菌株定植心脏组织的能力，以及（iii）使用兔心内膜炎模型来确定 Cnm 对 IE 发病机制的贡献并测试活性菌株的功效 使用 Cnm 进行免疫接种以预防变形链球菌感染。这项研究具有很容易转化为临床环境的巨大潜力，因为 Cnm 可以作为检测需要接受预防性治疗的患者的高毒力菌株的生物标志物，或者作为制定预防和治疗变形链球菌口腔外感染的有效策略的潜在目标。