Mechanism of PsrP mediated adhesion

描述(申请人提供)：PSRP-secY2A2是一个37kb的致病岛，其存在与肺炎链球菌导致人类疾病的能力呈正相关。PsrP-secY2A2编码PsrP，是一种520 kDa的细胞壁蛋白，我们已经确定它是一种粘附素，是下呼吸道疾病发展所必需的，影响生物膜的产生，并受温度和氧气的调节。重要的是，抗PsrP的抗体在体外抑制细菌黏附，保护小鼠免受肺炎球菌的攻击。我们的长期目标是确定和描述导致侵袭性肺炎球菌疾病发展的宿主：：病原体的相互作用。这项建议的目的是表征PsrP介导的黏附，并确定PsrP的BR结构域是否是保护性抗原。我们将：目标1.确定psrP-secY2A2的操纵子结构和转录调控。PSRP-secY2A2编码18个基因，分为6个可能的操纵子。实验表明，PsrP的产生对氧气和温度的变化做出了响应。我们将确定psrP-secY2A2的操纵子结构。我们将确定温度和氧气是否调节psrP-secY2A2的转录。我们将确定删除ciaRH(一个对氧反应的双组分系统)、hrcA&ctsrA(热激反应调节因子)和stkP(全局应激调节因子)对psrP-secY2A2表达的影响。目的2.确定SRR1和SRR2在PsrP功能中的作用。成熟的PsrP由一个富含丝氨酸的区域(SRR1)、一个介导黏附的碱性区域(BR)、第二个极长的富含丝氨酸的区域(SRR2)和一个细胞壁锚定区组成。我们的实验模型预测，SRR2结构域的功能是将BR结构域向外延伸到囊外，以调节黏附。此外，BR与其他细菌上的PsrP的SRR1结构域结合。我们将确定BR相对于胶囊的位置。我们将确定减少SRR2重复数(即减少PsrP长度)对粘附性和毒力的影响。我们将确定缺失SRR1和BR对PsrP黏附、毒力和生物被膜产生的影响。目的3.确定重组PsrP基区疫苗是否能抵抗攻击。针对BR的抗体在体外抑制细菌黏附，保护小鼠免受被动免疫后的攻击。我们将确定负责PsrP介导结合的BR片段。我们将开发抗BR的单抗，并确定它们是否抑制细菌黏附，影响生物被膜的形成，并防止肺炎球菌的攻击。我们将确定mAbs被动免疫和重组BR构建的主动免疫是否能保护小鼠免受肺炎球菌攻击。公共卫生相关性：这项提案将确定Psrp促进肺炎链球菌引起下呼吸道疾病的机制(S)。拟议实验的完成将有助于更好地了解肺炎链球菌的毒力；此外，还将确定PsrP是否为包括在多组分肺炎球菌疫苗中的可行候选者。

DESCRIPTION (provided by applicant): psrP-secY2A2 is a 37-kb pathogenicity island whose presence has been positively correlated with the ability of Streptococcus pneumoniae to cause human disease. psrP-secY2A2 encodes PsrP, a 520-kDa cell wall protein, which we have determined is an adhesin, is required for the development of lower respiratory tract disease, affects biofilm production, and is temperature and oxygen regulated. Importantly, antibodies against PsrP inhibit bacteria adhesion in vitro and protect mice against pneumococcal challenge. Our long-term goal is to identify and characterize the host::pathogen interactions that are responsible for the development of invasive pneumococcal disease. The goal of this proposal is to characterize PsrP-mediated adhesion and to determine if the BR domain of PsrP is a protective antigen. We will: Aim 1. Determine the operon structure and transcriptional regulation of psrP-secY2A2. psrP-secY2A2 encodes 18 genes divided into 6 putative operons. Experiments indicate that PsrP production is responsive to changes in oxygen and temperature. We will determine the operon structure of psrP-secY2A2. We will determine whether temperature and oxygen regulate psrP-secY2A2 transcription. We will determine the effects of deleting ciaRH (a two-component system responsive to oxygen), hrcA & ctsrA (heat-shock response regulators), and stkP (global stress regulator) on psrP-secY2A2 expression. Aim 2. Determine the role of SRR1 and SRR2 on PsrP function. Mature PsrP is composed of a serine-rich region (SRR1), a basic region (BR) which mediates adhesion, a second extremely long serine-rich region (SRR2), and a cell wall anchor domain. Our experimental model predicts that the SRR2 domain functions to extend the BR domain outward beyond the capsule to mediate adhesion. Also that BR binds to the SRR1 domain of PsrP on other bacteria. We will determine the location of BR relative to capsule. We will determine the effects of reducing the number of SRR2 repeats (i.e. reducing PsrP length) on adhesion and virulence. We will determine the effect of deleting SRR1 and BR on PsrP adhesion, virulence, and biofilm production. Aim 3. Determine if vaccination with recombinant PsrP Basic Region protects against challenge. Antibodies against the BR inhibit bacteria adhesion in vitro and protect mice from challenge following passive immunization. We will determine the segment of BR that is responsible for PsrP-mediated binding. We will develop monoclonal antibodies (mAbs) against BR and determine if they inhibit bacterial adhesion, affect biofilm formation, and protect against pneumococcal challenge. We will determine if passive vaccination with mAbs and active vaccination with recombinant BR construct protects mice against pneumococcal challenge. PUBLIC HEALTH RELEVANCE: This proposal will determine the mechanism(s) by which PsrP contributes towards the ability of Streptococcus pneumoniae to cause lower respiratory tract disease. Completion of the proposed experiments will lead to a better understanding of S. pneumoniae virulence; moreover, will determine if PsrP is a viable candidate for inclusion in a multi-component pneumococcal vaccine.