Mechanisms of Pneumococcal Adherence

描述（由申请人提供）：肺炎链球菌（s.p.）是一种重要的人类病原体，每年在世界范围内造成100多万人死亡。此外，仅在美国，标准普尔每年就造成了大约700万例中耳炎病例。目前可用的结合疫苗包括90种已知胶囊类型中的7种。虽然这种疫苗显著减少了由疫苗血清型引起的侵袭性疾病和定植，但由非疫苗血清型引起的侵袭性疾病和定植却意外增加。此外，该疫苗不能有效预防中耳炎。呼吸道的定植是疾病的重要前兆。尽管粘附定殖的重要性，因此，发病机制，S.p粘附气道上皮的机制尚不清楚。我们已经确定了一种新的依赖外糖苷酶的粘附机制，需要S.p.神经氨酸酶，NanA和2-半乳糖苷酶，BgaA。这一机制与近期临床分离株的粘附性和S.p.对人原代上皮细胞的粘附性有关，表明它在体内也是相关的。我们的初步数据表明，虽然NanA暴露了粘附受体，但BgaA充当粘附素。为了进一步明确这种粘附机制，我们有两个具体目标：1)阐明BgaA的结合位点。我们将定义介导人上皮细胞粘附的BgaA区域。BgaA是S.p.表达的第二大蛋白，具有功能未知的c端区域。我们将通过分子和结构分析来阐明BgaA作为粘附素的作用机制。蛋白质的不同区域将被表达并用于结合和抑制研究，以确定介导粘附的区域。有助于结合的残基随后将通过诱变扫描确定。BgaA的结构将被用作鉴定结合位点和鉴定蛋白质c端区域功能的替代方法。2)鉴定上皮细胞表面的BgaA受体。S.p.的初始附着被认为是通过神经氨酸酶暴露的聚糖受体发生的；然而，粘附素是未知的，并且有不同受体的相互矛盾的报道。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背景下的结构。在确定受体后，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增加对S.p.依从性机制的理解。这一知识可能对开发更有效的疫苗和/或治疗方法以减轻S.p.疾病负担的长期目标作出重大贡献。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，细菌最初附着在气道上的机制尚不清楚。本研究将描述一种新的依赖糖苷酶的粘附机制。我们将鉴定细菌粘附素和上皮表面的受体。我们对肺炎球菌依从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方法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.