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.
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.
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