Mechanisms of Pneumococcal Adherence

描述（由申请人提供）：S。肺炎链球菌（S. p.）是一种重要的人类病原体，每年在全球造成超过100万人死亡。此外，仅在美国，S.p每年就造成约700万例中耳炎。目前可用的结合疫苗包括90种已知胶囊类型中的7种。虽然该疫苗显著减少了由疫苗血清型引起的侵袭性疾病和定殖，但由非疫苗血清型引起的侵袭性疾病和定殖却意外增加。此外，疫苗不能有效预防中耳炎。呼吸道的定植是疾病的重要先兆。尽管粘附定植的重要性，因此，发病机制，S.P粘附气道上皮细胞的机制仍然不清楚。我们已经确定了一种新的外切糖苷酶依赖性粘附机制，需要SP神经氨酸酶，NanA，和2-半乳糖苷酶，BgaA。该机制与最近临床分离株的粘附和S. p.对人原代上皮细胞的粘附相关，表明其将在体内相关。我们的初步数据表明，虽然NanA暴露的受体粘附，BgaA作为粘附素。为了进一步定义这种粘附机制，我们有2个具体目的：1）阐明BgaA结合位点。我们将定义BgaA区域，介导粘附人类上皮细胞。BgaA是由S. p.表达的第二大蛋白，并且具有未知功能的C-末端区域。我们将进行分子和结构分析，以阐明如何BgaA作为一个粘附素。蛋白质的不同区域将被表达并用于结合和抑制研究，以鉴定介导粘附的区域。随后通过诱变扫描鉴定有助于结合的残基。BgaA的结构将被用作鉴定结合位点和鉴定蛋白质C-末端区域功能的替代方法。2)鉴定上皮细胞表面的BgaA受体。已提出S. p.的初始附着通过神经氨酸酶暴露的聚糖受体发生;然而，粘附素是未知的，并且存在不同受体的相互矛盾的报告。我们将使用全面的技术来确定BgaA是否与聚糖结合，以及这是否是蛋白质或脂质的结构。在识别受体后，我们将进一步表征其与BgaA的相互作用。这些具体目标的成功完成将提供对S. p.粘附机制的更多理解。这些知识可能会大大有助于开发更有效的疫苗和/或治疗方法，以减少S. p.疾病的负担的长期目标。公共卫生相关性：人类气道的定植是肺炎球菌疾病的重要前兆;然而，对细菌最初附着于气道的机制知之甚少。本研究将描述一种新的糖苷酶依赖性粘附机制。我们将鉴定细菌粘附素和上皮表面的受体。我们对肺炎球菌粘附性的进一步了解将有助于我们实现开发更有效的疫苗或治疗以减轻肺炎球菌疾病负担的长期目标。

DESCRIPTION (provided by applicant): S. pneumoniae (S.p.) is an important human pathogen causing more than 1 million deaths per year world-wide. In addition, S.p is responsible for an estimated 7 million cases of otitis media each year in the US alone. The currently available conjugate vaccine includes 7 of the 90 known capsule types. While this vaccine has significantly reduced invasive disease and colonization caused by vaccine serotypes, there has been an unexpected increase in both invasive disease and colonization caused by non-vaccine serotypes. In addition, the vaccine does not effectively protect against otitis media. Colonization of the respiratory tract is an essential precursor to disease. Despite the importance of adherence to colonization and, therefore, pathogenesis, the mechanisms by which S.p adheres to airway epithelia remain unclear. We have identified a novel exoglycosidase-dependent adherence mechanism requiring the S.p. neuraminidase, NanA, and 2- galactosidase, BgaA. This mechanism is relevant to adherence of recent clinical isolates and S.p. adherence to human primary epithelial cells, suggesting that it will be relevant in vivo. Our preliminary data demonstrate that while NanA exposes a receptor for adherence, BgaA acts as an adhesin. To further define this mechanism of adherence we have 2 Specific Aims: 1) Elucidate the BgaA binding site. We will define the BgaA region that mediates adherence to human epithelial cells. BgaA is the 2nd largest protein expressed by S.p. and possesses a C-terminal region of unknown function. We will perform molecular and structural analyses to elucidate how BgaA acts as an adhesin. Different regions of the protein will be expressed and used in binding and inhibition studies to identify the region(s) that mediate(s) adherence. The residues that contribute to binding will subsequently be identified by mutagenesis scanning. Structuring of BgaA will be utilized as an alternative approach to identify the binding site and identify the function of the C-terminal region of the protein. 2) Identify the BgaA receptor on the epithelial cell surface. The initial attachment of S.p. has been proposed to occur through a glycan receptor exposed by neuraminidase; however, the adhesin is unknown and there have been conflicting reports of different receptors. We will use a comprehensive range of techniques to determine if BgaA binds to a glycan and if this is structure in the context of a protein or lipid. Following identification of the receptor we will further characterize its interaction with BgaA. The successful completion of these Specific Aims will provide an increased understanding of this mechanism of S.p. adherence. This knowledge is likely to contribute significantly to the long-term goal of developing more effective vaccines and/or treatments to reduce the burden of S.p. disease. PUBLIC HEALTH RELEVANCE: Colonization of the human airway is an essential precursor to pneumococcal disease; however the mechanisms by which the bacteria initially attach to the airway are poorly understood. This study will characterize a novel glycosidase-dependent mechanism of adherence. We will identify the bacterial adhesin and the receptor on the epithelial surface. Our increased understanding of pneumococcal adherence will help us achieve the longer term goal of developing a more effective vaccine or treatment to reduce the burden of pneumococcal disease.