Yaffa Mizrachi Nebenzahl 1,2

1. Deciphering the Function of Selected Novel Streptococcus pneumoniae Proteins in Pathogenesis and Development of Target Derived Antagonists Yaffa Mizrachi Nebenzahl1,2 Understanding the sequential molecular interactions of S. pneumoniae with its human host may lead to the identification of molecules crucial for disease development. We have previously identified proteins in the cell-wall with known enzymatic activities in the cytoplasm Fructose bisphosphatealdolase (FBA), Phosphoenolpyruvate protein phosphotransferase (PtsA), GlutamyltRNAsynthetase (GtS), NADH oxidase (NOX), Aspartate Carbamoyltransferase catalytic subunit (ATCase), PTS system, mannose-specific IIAB components (PTSMAN) and ABC transporter substrate-binding protein (ABC) Bioinformatic analysis demonstrated that these proteins are conserved among the sequenced strains of S. pneumoniae We hypothesized that some of these proteins in the cell-wall may function as adhesins

2. Identification of Putative receptors to bacterial adhesins Inhibition of adhesion to A549 Inserted peptides were sequenced and aligned against the human genome A549 A549 A549 A549 rPtsA rPtsA Peptide synthesis from the human homology region Inhibition of adhesion In vitro Inhibition of colonization In vivo

3. Target molecules in the Host A549 cells

4. Target molecules in the Host U251 cells

5. Summary • A group of S. pneumoniae cell-wall proteins were identified and found to function as putative adhesins • Null mutant bacteria lacking those proteins were found to have reduced adhesion in vitro and reduced virulence in vivo in the mouse models • Screening a combinatorial peptide library expressed in filamentous phages identified protein binding peptides • Sequences homologous to these peptides were identified in cell membrane and ECM proteins • Peptides derived from the human membrane and extracellular matrix proteins, homologous to the insert peptides in the inhibitory phages, were found to • Inhibit bacterial adhesion to the lung derived A549 cells • Reduce bacterial load in the nasopharynx and the lung in intranasal inoculation model and mortality in IN and the intraperitoneal inoculation mouse model • Five out of the six proteins suspected to be the adhesin’s putative targets molecules in the host were found to be expressed in the A549 cells and 4 were found in U251 cells • These peptides may be considered for future treatment of S. pneumoniae clinical infections