Mechanisms of Pneumococcal Adherence

描述(申请人提供)：肺炎链球菌(S.P.)是一种重要的人类病原体，每年在全世界造成100多万人死亡。此外，仅在美国，S.P.每年估计就有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，疫苗并不能有效预防中耳炎。呼吸道定植是疾病的重要前兆。尽管坚持定植和致病机制很重要，但S.P附着于呼吸道上皮细胞的机制仍不清楚。我们已经确定了一种新的依赖外糖苷酶的黏附机制，需要S.P.神经氨酸酶NAA和2-半乳糖苷酶BGaA。这种机制与新近临床分离株和S.P.的粘附性有关。与人类原代上皮细胞的黏附，表明它在体内将是相关的。我们的初步数据表明，当NaNA暴露出黏附的受体时，BgaA则扮演粘附素的角色。为了进一步确定这种黏附机制，我们有两个具体的目标：1)阐明BGaA的结合部位。我们将定义介导与人上皮细胞黏附的BgaA区域。BGaA是S.P.表达的第二大蛋白。并具有未知功能的C-端区。我们将进行分子和结构分析，以阐明BGaA作为粘附素的作用。该蛋白的不同区域将被表达并用于结合和抑制研究，以确定介导(S)黏附的区域(S)。随后将通过突变扫描来鉴定有助于结合的残基。BGaA的结构将被用作确定结合位点和确定蛋白质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。S.P.的初始附着。已被认为是通过神经氨酸酶暴露的葡聚糖受体发生的；然而，粘附素尚不清楚，关于不同受体的报道也相互矛盾。我们将使用一系列广泛的技术来确定BgaA是否与糖链结合，以及这是否是蛋白质或脂类背景下的结构。在鉴定受体之后，我们将进一步鉴定它与BgaA的相互作用。这些具体目标的成功完成将有助于加深对S.P.这一机制的理解。坚持不懈。这些知识很可能对开发更有效的疫苗和/或治疗以减轻S.P.负担的长期目标做出重大贡献。疾病。与公共卫生相关：人类呼吸道的定植是肺炎球菌疾病的重要先兆；然而，细菌最初附着在呼吸道的机制却知之甚少。这项研究将描述一种新的糖苷酶依赖的黏附机制。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更多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.