Mechanism of PsrP mediated adhesion

项目总结/摘要 psrP-secY2A2是一个37-kb的致病岛，其存在与细菌的致病性正相关。 肺炎链球菌引起人类疾病的能力。psrP-secY2A2编码PsrP，一个520-kDa的细胞 我们已经确定壁蛋白是一种粘附素，是下呼吸道发育所必需的 疾病，影响生物膜的产生，并受到温度和氧气的调节。重要的是， PsrP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，这是负责发展侵入性 肺炎球菌病该提案的目标是表征PsrP介导的粘附， 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个假定的操纵子。实验表明，PsrP的产生是响应于 氧气和温度的变化。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转录。我们将确定 删除ciaRH（对氧反应的双组分系统）、hrcA和ctsrA（热休克反应 调节子）和stkP（全局应激调节子）对psrP-secY2A2表达的影响。 目标2.确定SRR 1和SRR 2对PsrP功能的作用。成熟的PsrP由富含丝氨酸的 区域（SRR 1）、介导粘附的碱性区域（BR）、第二极长的富含丝氨酸的区域（SRR 2）、第二极长的富含丝氨酸的区域（SRR 3）、第三极长的富含丝氨酸的区域（SRR 4）、第四极长的富含丝氨酸的区域（SRR 5）、第四极长的富含丝氨酸的区域（SRR 6）、第四极长的富含丝氨酸的区域（SRR 7）、第五极长的富含丝氨酸的区域（SRR 7）、第五极长的富含丝氨酸的区域（SRR 8）、第五极长的富含丝氨酸的区域（SRR 8）、第六极长的富含丝氨酸的区域（SRR 8）、第五极长的富含丝氨酸的区域（SRR 9）、第六极长的富含丝氨酸的区域（SRR 8）、第五极长的富含丝氨酸的区域（SRR 9）、第六极长的富含丝氨酸的区域（SRR 9）、第六极长的富含丝氨酸的区域（S10）、第六极长的富含丝氨酸的 （SRR 2）和细胞壁锚结构域。我们的实验模型预测，SRR 2结构域的功能， 使BR结构域向外延伸超过包膜以介导粘附。BR还与SRR1结合， PsrP对其他细菌的作用。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。 抗BR抗体在体外抑制细菌粘附并保护小鼠免受被动感染后的攻击。 次免疫我们将确定负责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攻击。我们将确定是否被动接种 mAb和用重组BR构建体主动接种保护小鼠免受肺炎球菌攻击。

PROJECT SUMMARY/ABSTRACT 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 constructs protects mice against pneumococcal challenge.