Mechanism of PsrP mediated adhesion

描述（由申请人提供）：psrP-secY 2A 2是一个37-kb的致病岛，其存在与肺炎链球菌引起人类疾病的能力正相关。psrP-secY 2A 2编码PsrP，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需的，影响生物膜的产生，并且是温度和氧气调节的。重要的是，针对PsrP的抗体在体外抑制细菌粘附并保护小鼠免受肺炎球菌攻击。我们的长期目标是确定和表征宿主：：病原体相互作用，负责发展的侵袭性肺炎球菌疾病。本提案的目的是表征PsrP介导的粘附，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-secY 2A 2的操纵子结构和转录调控。psrP-secY 2A 2编码18个基因，分为6个操纵子。实验表明，PsrP的生产是响应于氧气和温度的变化。我们将确定psrP-secY 2A 2的操纵子结构。我们将确定是否温度和氧气调节psrP-secY 2A 2转录。我们将确定删除ciaRH（一种对氧有反应的双组分系统）、hrcA & ctsrA（热休克反应调节因子）和stkP（全局应激调节因子）对psrP-secY 2A 2表达的影响。目标2.确定SRR 1和SRR 2对PsrP功能的作用。成熟PsrP由富含丝氨酸的区域（SRR 1）、介导粘附的碱性区域（BR）、第二个极长的富含丝氨酸的区域（SRR 2）和细胞壁锚结构域组成。我们的实验模型预测SRR 2结构域的功能是将BR结构域向外延伸到包膜之外以介导粘附。BR还与其他细菌上PsrP的SRR 1结构域结合。我们将确定BR相对于太空舱的位置。我们将确定减少SRR 2重复序列的数量（即减少PsrP长度）对粘附和毒力的影响。我们将确定删除SRR 1和BR对PsrP粘附、毒力和生物膜产生的影响。目标3.确定重组PsrP碱性区疫苗接种是否可保护抵抗攻毒。针对BR的抗体在体外抑制细菌粘附并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。我们将开发针对BR的单克隆抗体（mAb），并确定它们是否抑制细菌粘附，影响生物膜形成，并保护免受肺炎球菌的攻击。我们将确定被动接种单克隆抗体和主动接种重组BR构建体是否保护小鼠免受肺炎球菌攻击。公共卫生相关性：本提案将确定PsrP促进肺炎链球菌引起下呼吸道疾病的能力的机制。这些实验的完成将有助于更好地理解S。此外，将确定PsrP是否是用于包含在多组分肺炎球菌疫苗中的可行候选物。

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.