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1.
Sci Rep ; 8(1): 314, 2018 01 10.
Article in English | MEDLINE | ID: mdl-29321514

ABSTRACT

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.


Subject(s)
Bacterial Adhesion , Bacterial Proteins/genetics , FMN Reductase/genetics , Oxidative Stress , Streptococcus pneumoniae/pathogenicity , Animals , Bacterial Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , FMN Reductase/metabolism , Female , Humans , Macrophages, Peritoneal/microbiology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mutation , Phagocytosis , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/genetics , Virulence/genetics
2.
PLoS One ; 11(3): e0150320, 2016.
Article in English | MEDLINE | ID: mdl-26990554

ABSTRACT

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.


Subject(s)
Cell Wall/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphotransferases (Nitrogenous Group Acceptor)/metabolism , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/enzymology , Adhesins, Bacterial/physiology , Cell Line, Tumor , Child, Preschool , Flow Cytometry , Humans , Streptococcus pneumoniae/immunology
3.
Int J Mol Med ; 37(4): 1127-38, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26935978

ABSTRACT

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.


Subject(s)
Cytokines/immunology , Fructose-Bisphosphate Aldolase/therapeutic use , Pneumococcal Infections/prevention & control , Streptococcal Vaccines/therapeutic use , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/therapeutic use , Animals , Female , Freund's Adjuvant/immunology , Freund's Adjuvant/therapeutic use , Fructose-Bisphosphate Aldolase/immunology , Immunization , Lipids/immunology , Lipids/therapeutic use , Mice , Mice, Inbred BALB C , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Rabbits , Streptococcal Vaccines/immunology , T-Lymphocytes, Helper-Inducer/microbiology , Th1 Cells/immunology , Th17 Cells/immunology , Th2 Cells/immunology
4.
PLoS One ; 8(4): e61128, 2013.
Article in English | MEDLINE | ID: mdl-23577197

ABSTRACT

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.


Subject(s)
Adhesins, Bacterial/metabolism , Immunity , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/metabolism , Streptococcus pneumoniae/enzymology , Adhesins, Bacterial/genetics , Adhesins, Bacterial/immunology , Aerobiosis , Aging/immunology , Anaerobiosis , Animals , Bacterial Adhesion/drug effects , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Bacterial Vaccines/metabolism , Cell Line , Child, Preschool , Female , Gene Deletion , Gene Expression Regulation, Bacterial/drug effects , Humans , Infant , Mice , Multienzyme Complexes/deficiency , Multienzyme Complexes/genetics , Multienzyme Complexes/immunology , NADH, NADPH Oxidoreductases/deficiency , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/immunology , Peptides/pharmacology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/immunology
5.
PLoS One ; 6(8): e23472, 2011.
Article in English | MEDLINE | ID: mdl-21887255

ABSTRACT

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligand(high) lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-ligand(dull) macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.


Subject(s)
Antigens, Ly/metabolism , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 1/metabolism , Streptococcus pneumoniae/immunology , Administration, Intranasal , Animals , Bacterial Load/immunology , Bone Marrow Cells/cytology , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/microbiology , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Ligands , Lung/immunology , Lung/microbiology , Lung/pathology , Lymphocyte Activation/immunology , Macrophage Activation/immunology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis
6.
Int Immunol ; 20(9): 1139-46, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18596024

ABSTRACT

The role of IL-1 in susceptibility to Streptococcus pneumoniae infection was studied in mice deficient in genes of the IL-1 family [i.e. IL-1alpha-/-, IL-1beta-/-, IL-1alpha/beta-/- and IL-1R antagonist (IL-1Ra)-/- mice] following intra-nasal inoculation. Intra-nasal inoculation of S. pneumoniae of IL-1beta-/- and IL-1alpha/beta-/- mice displayed significantly lower survival rates and higher nasopharyngeal and lung bacterial load as compared with control, IL-1alpha-/- and IL-1Ra-/- mice. Treatment of IL-1beta-/- mice with rIL-1beta significantly improved their survival. A significant increase in blood neutrophils was found in control, IL-1alpha-/- and IL-1Ra-/- but not in IL-1beta-/- and IL-1alpha/beta-/- mice. Local infiltrates of neutrophils and relatively preserved organ architecture were observed in the lungs of IL-1alpha-/- and control mice. However, S. pneumoniae-infected IL-1beta-/-, IL-1alpha/beta-/- and IL-1Ra-/- mice demonstrated diffuse pneumonia and tissue damage. Altogether, all three isoforms contribute to protection against S. pneumoniae; our results point to differential role of IL-1alpha and IL-1beta in the pathogenesis and control of S. pneumoniae infection and suggest that IL-1beta has a major role in resistance to primary pneumococcal infection while the role of IL-1alpha is less important.


Subject(s)
Interleukin-1alpha/metabolism , Interleukin-1beta/metabolism , Pneumonia, Pneumococcal/immunology , Streptococcus pneumoniae/pathogenicity , Animals , Female , Humans , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1alpha/administration & dosage , Interleukin-1alpha/genetics , Interleukin-1alpha/immunology , Interleukin-1beta/administration & dosage , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Nasopharynx/microbiology , Pneumonia, Pneumococcal/drug therapy , Pneumonia, Pneumococcal/microbiology , Pneumonia, Pneumococcal/mortality , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Streptococcus pneumoniae/isolation & purification
7.
J Infect Dis ; 195(12): 1828-37, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17492599

ABSTRACT

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.


Subject(s)
Adhesins, Bacterial/isolation & purification , Bacterial Adhesion/physiology , Cadherins/metabolism , Fructose-Bisphosphate Aldolase/immunology , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/metabolism , Adhesins, Bacterial/metabolism , Animals , Antibodies, Bacterial/metabolism , Cadherins/isolation & purification , Cell Line, Tumor , Female , Fructose-Bisphosphate Aldolase/genetics , Fructose-Bisphosphate Aldolase/metabolism , Humans , Mice , Mice, Inbred BALB C , Nasopharynx/microbiology , Pneumococcal Infections/prevention & control , Rabbits , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Streptococcus pneumoniae/genetics , Time Factors , Virus Attachment
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