Streptococcus pneumoniae Cell Wall-Localized Trigger Factor Elicits a Protective Immune Response and Contributes to Bacterial Adhesion to the Host.

Trigger factor (TF) has a known cytoplasmic function as a chaperone. In a previous study we showed that pneumococcal TF is also cell-wall localized and this finding combined with the immunogenic characteristic of TF, has led us to determine the vaccine potential of TF and decipher its involvement in pneumococcal pathogenesis. Bioinformatic analysis revealed that TF is conserved among pneumococci and has no human homologue. Immunization of mice with recombinant (r)TF elicited a protective immune response against a pneumococcal challenge, suggesting that TF contributes to pneumococcal pathogenesis. Indeed, rTF and an anti-rTF antiserum inhibited bacterial adhesion to human lung derived epithelial cells, indicating that TF contributes to the bacterial adhesion to the host. Moreover, bacteria lacking TF demonstrated reduced adhesion, in vitro, to lung-derived epithelial cells, neural cells and glial cells. The reduced adhesion could be restored by chromosomal complementation. Furthermore, bacteria lacking TF demonstrated significantly reduced virulence in a mouse model. Taken together, the ability of rTF to elicit a protective immune response, involvement of TF in bacterial adhesion, conservation of the protein among pneumococcal strains and the lack of human homologue, all suggest that rTF can be considered as a future candidate vaccine with a much broader coverage as compared to the currently available pneumococcal vaccines.

Flavin Reductase Contributes to Pneumococcal Virulence by Protecting from Oxidative Stress and Mediating Adhesion and Elicits Protection Against Pneumococcal Challenge.

Pneumococcal flavin reductase (FlaR) is known to be cell-wall associated and possess age dependent antigenicity in children. This study aimed at characterizing FlaR and elucidating its involvement in pneumococcal physiology and virulence. Bioinformatic analysis of FlaR sequence identified three-conserved cysteine residues, suggesting a transition metal-binding capacity. Recombinant FlaR (rFlaR) bound Fe2+ and exhibited FAD-dependent NADP-reductase activity, which increased in the presence of cysteine or excess Fe2+ and inhibited by divalent-chelating agents. flaR mutant was highly susceptible to H2O2 compared to its wild type (WT) and complemented strains, suggesting a role for FlaR in pneumococcal oxidative stress resistance. Additionally, flaR mutant demonstrated significantly decreased mice mortality following intraperitoneal infection. Interestingly, lack of FlaR did not affect the extent of phagocytosis by primary mouse peritoneal macrophages but reduced adhesion to A549 cells compared to the WT and complemented strains. Noteworthy are the findings that immunization with rFlaR elicited protection in mice against intraperitoneal lethal challenge and anti-FlaR antisera neutralized bacterial virulence. Taken together, FlaR's roles in pneumococcal physiology and virulence, combined with its lack of significant homology to human proteins, point towards rFlaR as a vaccine candidate.

Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice.

Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freund's adjuvant (CFA) in the initial immunization and with incomplete Freund's adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA­pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA­immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1 involvement. In addition, rabbit and mouse anti-rFBA antisera significantly protected the mice against a lethal S. pneumoniae challenge in comparison with preimmune sera. Our results emphasize the mixed involvement of the Th1, Th2 and Th17 arms of the immune system in response to immunization with pneumococcal rFBA, a potential vaccine candidate.

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinß4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice.

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.

Streptococcus pneumoniae surface-exposed glutamyl tRNA synthetase, a putative adhesin, is able to induce a partially protective immune response in mice.

Glutamyl tRNA synthetase (GtS) has been found to be among the Streptococcus pneumoniae cell wall-derived proteins that have age-dependent immunogenicity in children. Here, GtS was cloned, expressed, and purified and then was used to immunize 7-week-old BALB/c OlaHsd mice. Serum obtained from mice immunized with recombinant (r) GtS cross-reacted with a 55.9-kDa protein, identified as GtS, in the cell wall fraction derived from genetically and capsularly unrelated strains of S. pneumoniae. Surface localization of GtS was further confirmed using flow cytometry analysis. The rGtS and anti-rGtS antiserum significantly inhibited the adhesion of 3 pairs of encapsulated and unencapsulated strains of S. pneumoniae to A549 cells. Thirty-nine percent of rGtS-immunized mice survived a lethal bacterial challenge, whereas no control mice survived. These results suggest that GtS, an age-dependent S. pneumoniae antigen, is a surface-located adhesin that is capable of inducing a partially protective immune response against S. pneumoniae in mice.

Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae.

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 vaccine potential of Streptococcus pneumoniae surface lectin- and non-lectin proteins.

We have fractionated S. pneumoniae surface proteins into lectin and non-lectin fractions and tested their ability to elicit protective immune responses in the mouse model system. The total cell wall protein fraction (CW-T) was separated into lectin (CW-L), and non-lectin (CW-NL) fractions and used for immunization of mice. Immunized mice were challenged intranasally or intraperitoneally with S. pneumoniae strain WU2 (serotype 3). CW-T, CW-NL and CW-L and adjuvant only vaccination protected 55%, 43%, 44% and 0% of the intranasally challenged mice, respectively and 67%, 86%, 11% and 0% of mice challenged intraperitoneally, respectively. Immunogenic proteins in each fraction were sequenced and identified using MALDI-TOF. CW-L proteins provided a significantly better protection against intranasal inoculation and CW-NL proteins provided a significantly better protection from intraperitoneal inoculation. Proteins identified by sera from mice immunized with the cell-wall derived fractions may constitute candidates for future development of anti S. pneumoniae vaccines.