Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.

SummaryFor most bacteria, adherence to human cells is achieved by bacterial lectins binding to mammalian surface glyconjugates. 6-Phosphogluconate dehydrogenase (6PGD) was identified by us as one of Streptococcus pneumoniae cell wall lectin proteins, which elicits an age-dependent immune response in humans. This study assesses the role of 6PGD in S. pneumoniae pathogenesis as an adhesin and its ability to elicit a protective immune response in mice. Recombinant 6PGD (r6PGD) was cloned from S. pneumoniae serotype 3 (strain WU2). r6PGD interference in adhesion of three genetically unrelated unencapsulated pneumococcal strains (3·8, 14·8 and R6) and two genetically unrelated encapsulated pneumococcal strains (WU2 and D39) to A549 type II lung carcinoma cell was tested. BALB/c mice were immunized with r6PGD and boosted after 3 weeks. Immunized mice were challenged intranasally with a lethal dose of S. pneumoniae . r6PGD inhibited 90% and 80% of pneumococcal adhesion to the A549 cells of three unencapsulated S. pneumoniae strains and two encapsulated S. pneumoniae strains, respectively, in a concentration-dependent manner ( P < < < < 0·05). Antibodies to r6PGD produced in mice significantly inhibited bacterial adhesion to A549 cell ( P < < < < 0·05). Immunization of mice with r6PGD protected 60% ( P < < < < 0·001) of mice for 5 days and 40% ( P < < < < 0·05) of the mice for 21 days following intranasal lethal challenge. We have identified 6PGD as a surface-located immunogenic lectin protein capable of acting as an adhesin. 6PGD importance to bacterial pathogenesis was demonstrated by the ability of r6PGD to elicit a protective immune response in mice.