scholarly journals Characterization of the Streptococcus pneumoniae BgaC Protein as a Novel Surface β-Galactosidase with Specific Hydrolysis Activity for the Galβ1-3GlcNAc Moiety of Oligosaccharides

2009 ◽  
Vol 191 (9) ◽  
pp. 3011-3023 ◽  
Author(s):  
Jae Kap Jeong ◽  
Ohsuk Kwon ◽  
Yun Mi Lee ◽  
Doo-Byoung Oh ◽  
Jung Mi Lee ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Signal Sequence ◽  
Surface Protein ◽  
Fluorescein Isothiocyanate ◽  
Initial Contact ◽  
High Morbidity ◽  
Surface Localization ◽  
Microscopy Analysis ◽  
Hydrolysis Activity

ABSTRACT Streptococcus pneumoniae is a causative agent of high morbidity and mortality. Although sugar moieties have been recognized as ligands for initial contact with the host, only a few exoglycosidases have been reported to occur in S. pneumoniae. In this study, a putative β-galactosidase, encoded by the bgaC gene of S. pneumoniae, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from Escherichia coli, was found to have a highly regiospecific and sugar-specific hydrolysis activity for the Galβ1-3-GlcNAc moiety of oligosaccharides. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of S. pneumoniae, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequence or membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC and by a reassociation assay with fluorescein isothiocyanate-labeled BgaC. Although the bgaC deletion mutation did not significantly attenuate the virulence of S. pneumoniae in vivo, the bgaC mutant strain showed relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo, whereas the mutant showed higher colonization levels at 6 and 24 h postinfection in vivo. Our data strongly indicate for the first time that S. pneumoniae bgaC encodes a surface β-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.

ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2818-2828 ◽  
Author(s):  
Maria Laura Ferrando ◽  
Susana Fuentes ◽  
Astrid de Greeff ◽  
Hilde Smith ◽  
Jerry M. Wells
Keyword(s):  
Signal Sequence ◽  
Surface Protein ◽  
Normal Growth ◽  
Streptococcus Suis ◽  
Carbohydrate Utilization ◽  
Binding Domains ◽  
A Cell ◽  
Anchoring Motif

We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved α-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal α-amylase domain of ApuA was shown to have α-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase α-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.

scholarly journals Role of Pneumococcal Surface Protein C in Nasopharyngeal Carriage and Pneumonia and Its Ability To Elicit Protection against Carriage of Streptococcus pneumoniae

2002 ◽  
Vol 70 (5) ◽  
pp. 2526-2534 ◽  
Author(s):  
Priya Balachandran ◽  
Alexis Brooks-Walter ◽  
Anni Virolainen-Julkunen ◽  
Susan K. Hollingshead ◽  
David E. Briles
Keyword(s):  
Streptococcus Pneumoniae ◽  
Protein C ◽  
Surface Protein ◽  
Wild Type ◽  
Nasal Tissue ◽  
Cholera Toxin Subunit B ◽  
Colonization Model ◽  
Subunit B

ABSTRACT Previous studies suggested that PspC is important in adherence and colonization within the nasopharynx. In this study, we conducted mutational studies to further identify the role PspC plays in the pathogenesis of pneumococci. pspC and/or pspA was insertionally inactivated in a serotype 2 Streptococcus pneumoniae strain and in a serotype 19 S. pneumoniae strain. In the mouse colonization model, pneumococcal strains with mutations in pspC were significantly attenuated in their abilities to colonize. In a mouse pneumonia model, strains with mutations in pspC were unable to infect or multiply within the lung. Using reverse transcriptase PCR we were able to demonstrate that pspC is actively transcribed in vivo, when the bacteria are growing in the nasal cavity and in the lungs. In the bacteremia model, a strain mutated for pspC alone behaved like the wild type, but the absence of both pspC and pspA caused accelerated clearance of the bacteria. Intranasal immunization with PspC with cholera toxin subunit B as an adjuvant protected against intranasal challenge. Evidence was also obtained that revertants that spontaneously acquired PspC expression could multiply and colonize the nasal tissue. This latter finding strongly indicates that pneumococci are actively metabolizing and growing while in the nasopharynx.

scholarly journals Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization

2015 ◽  
Vol 22 (11) ◽  
pp. 1146-1153 ◽  
Author(s):  
Lance E. Keller ◽  
Xiao Luo ◽  
Justin A. Thornton ◽  
Keun-Seok Seo ◽  
Bo Youn Moon ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mouse Model ◽  
Pneumococcal Disease ◽  
Surface Protein ◽  
Antibody Isotype ◽  
Content Type ◽  
Mouse Immunization ◽  
Escape Mutants

ABSTRACTCurrent vaccinations are effective against encapsulated strains ofStreptococcus pneumoniae, but they do not protect against nonencapsulatedStreptococcus pneumoniae(NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in anin vivomouse model and in anin vitrocell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK+) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease.

scholarly journals Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2

2005 ◽  
Vol 73 (1) ◽  
pp. 298-307 ◽  
Author(s):  
Abdul Q. Khan ◽  
Quanyi Chen ◽  
Zheng-Qi Wu ◽  
James C. Paton ◽  
Clifford M. Snapper
Keyword(s):  
Streptococcus Pneumoniae ◽  
Humoral Immunity ◽  
Protein A ◽  
Surface Protein ◽  
Lethal Challenge ◽  
Humoral Immune ◽  
Extracellular Bacterium

ABSTRACT Little is known regarding the role of Toll-like receptors (TLRs) in regulating protein- and polysaccharide-specific immunoglobulin (Ig) isotype production in response to an in vivo challenge with an extracellular bacterium. In this report we demonstrate that MyD88−/−, but not TLR2−/−, mice are markedly defective in their induction of multiple splenic proinflammatory cytokine- and chemokine-specific mRNAs after intraperitoneal (i.p.) challenge with heat-killed Streptococcus pneumoniae capsular type 14 (S. pneumoniae type 14). This is correlated with analogous responses in splenic cytokine protein release in vitro following addition of S. pneumoniae type 14. Consistent with these data, naïve MyD88−/−, but not TLR2−/−, mice are more sensitive to killing following i.p. challenge with live S. pneumoniae type 14, relative to responses in wild-type mice. However, prior immunization of MyD88−/− mice with heat-killed S. pneumoniae type 14 protects against an otherwise-lethal challenge with live S. pneumoniae type 14. Surprisingly, both MyD88−/− and TLR2−/− mice exhibit striking and equivalent defects in elicitation of type 1 IgG isotypes (IgG3, IgG2b, and IgG2a), but not the type 2 IgG isotype, IgG1, specific for several protein and polysaccharide antigens, in response to i.p. challenge with heat-killed S. pneumoniae type 14. Of note, the type 1 IgG isotype titers specific for pneumococcal surface protein A are reduced in MyD88−/− mice but not TLR2−/− mice. These data suggest that distinct TLRs may differentially regulate innate versus adaptive humoral immunity to intact S. pneumoniae and are the first to implicate a role for TLR2 in shaping an in vivo type 1 IgG humoral immune response to a gram-positive extracellular bacterium.

scholarly journals Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export

2006 ◽  
Vol 188 (19) ◽  
pp. 6943-6952 ◽  
Author(s):  
Małgorzata Marczak ◽  
Andrzej Mazur ◽  
Jarosław E. Król ◽  
Wiesław I. Gruszecki ◽  
Anna Skorupska
Keyword(s):  
Outer Membrane ◽  
Rhizobium Leguminosarum ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Fluorescein Isothiocyanate ◽  
Surface Expression ◽  
Periplasmic Space ◽  
Intact Cells ◽  
Protease Digestion

ABSTRACT Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of β-structure; however, α-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.

scholarly journals Mucosal Infections and Invasive Potential of Nonencapsulated Streptococcus pneumoniae Are Enhanced by Oligopeptide Binding Proteins AliC and AliD

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jessica L. Bradshaw ◽  
Haley R. Pipkins ◽  
Lance E. Keller ◽  
James K. Pendarvis ◽  
Larry S. McDaniel
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Treatment Strategies ◽  
Surface Protein ◽  
Proteome Analysis ◽  
High Morbidity ◽  
Pneumococcal Vaccines ◽  
Virulence Determinants ◽  
Pneumococcal Infections ◽  
Pathogenic Potential

ABSTRACT Nonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen that colonizes the nasopharynx and is associated with noninvasive diseases such as otitis media (OM), conjunctivitis, and nonbacteremic pneumonia. Since capsule expression was previously thought to be necessary for establishment of invasive pneumococcal disease (IPD), serotype-specific polysaccharide capsules are targeted by currently licensed pneumococcal vaccines. Yet, NESp expressing oligopeptide binding proteins AliC and AliD have been isolated during IPD. Thus, we hypothesize AliC and AliD are major NESp virulence determinants that facilitate persistence and development of IPD. Our study reveals that NESp expressing AliC and AliD have intensified virulence compared to isogenic mutants. Specifically, we demonstrate AliC and AliD enhance murine nasopharyngeal colonization and pulmonary infection and are required for OM in a chinchilla model. Furthermore, AliC and AliD increase pneumococcal survival in chinchilla whole blood and aid in resistance to killing by human leukocytes. Comparative proteome analysis revealed significant alterations in protein levels when AliC and AliD were absent. Virulence-associated proteins, including a pneumococcal surface protein C variant (CbpAC), were significantly downregulated, while starvation response indicators were upregulated in the double mutant relative to wild-type levels. We also reveal that differentially expressed CbpAC was essential for NESp adherence to epithelial cells, virulence during OM, reduction of C3b deposition on the NESp surface, and binding to nonspecific IgA. Altogether, the rise in NESp prevalence urges the need to understand how NESp establishes disease and persists in a host. This study highlights the roles of AliC, AliD, and CbpAC in the pathogenesis of NESp. IMPORTANCE Despite the effective, widespread use of licensed pneumococcal vaccines over many decades, pneumococcal infections remain a worldwide burden resulting in high morbidity and mortality. NESp subpopulations are rapidly rising in the wake of capsule-targeted vaccine strategies, yet there is very little knowledge on NESp pathogenic potential and virulence mechanisms. Although NESp lacks a protective capsule, NESp lineages expressing AliC and AliD have been associated with systemic infections. Furthermore, higher antibiotic resistance rates and transformation efficiencies associated with emerging NESp threaten treatment strategies needed to control pneumococcal infections and transmission. Elucidating how NESp survives within a host and establishes disease is necessary for development of broadened pneumococcal prevention methods. Our study identifies virulence determinants and host survival mechanisms employed by NESp with a high pathogenic potential. Moreover, our study also identifies virulence determinants shared by NESp and encapsulated strains that may serve as broad prevention and therapeutic targets.

scholarly journals Pneumococcal Surface Protein A Inhibits Complement Activation by Streptococcus pneumoniae

1999 ◽  
Vol 67 (9) ◽  
pp. 4720-4724 ◽  
Author(s):  
Anh-Hue T. Tu ◽  
Robert L. Fulgham ◽  
Mark A. McCrory ◽  
David E. Briles ◽  
Alexander J. Szalai
Keyword(s):  
Streptococcus Pneumoniae ◽  
Defense Mechanisms ◽  
Alternative Pathway ◽  
Protein A ◽  
Surface Protein ◽  
Serum Complement ◽  
Factor B ◽  
Pneumococcal Surface Protein A

ABSTRACT Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the α chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

scholarly journals 4-1BB (CD137) Differentially Regulates Murine In Vivo Protein- and Polysaccharide-Specific Immunoglobulin Isotype Responses to Streptococcus pneumoniae

2003 ◽  
Vol 71 (1) ◽  
pp. 196-204 ◽  
Author(s):  
Zheng-Qi Wu ◽  
Abdul Q. Khan ◽  
Yi Shen ◽  
Karen M. Wolcott ◽  
Wojciech Dawicki ◽  
...  
Keyword(s):  
T Cells ◽  
Streptococcus Pneumoniae ◽  
Protein A ◽  
Teichoic Acid ◽  
Surface Protein ◽  
Cell Receptor ◽  
Primary Response ◽  
Humoral Immune ◽  
Specific Immunoglobulin

ABSTRACT 4-1BB (CD137) is induced on activated CD4+ and CD8+ T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4+ TCRαβ+ T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8+ T cells and is associated with the expansion of CD4+ T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.

scholarly journals Animal Models of Streptococcus pneumoniae Disease

2008 ◽  
Vol 21 (4) ◽  
pp. 666-685 ◽  
Author(s):  
Damiana Chiavolini ◽  
Gianni Pozzi ◽  
Susanna Ricci
Keyword(s):  
Streptococcus Pneumoniae ◽  
Animal Models ◽  
Otitis Media ◽  
High Mortality ◽  
Research Effort ◽  
Risk Groups ◽  
In Vivo Studies ◽  
High Morbidity ◽  
Pneumococcal Infections

SUMMARY Streptococcus pneumoniae is a colonizer of human nasopharynx, but it is also an important pathogen responsible for high morbidity, high mortality, numerous disabilities, and high health costs throughout the world. Major diseases caused by S. pneumoniae are otitis media, pneumonia, sepsis, and meningitis. Despite the availability of antibiotics and vaccines, pneumococcal infections still have high mortality rates, especially in risk groups. For this reason, there is an exceptionally extensive research effort worldwide to better understand the diseases caused by the pneumococcus, with the aim of developing improved therapeutics and vaccines. Animal experimentation is an essential tool to study the pathogenesis of infectious diseases and test novel drugs and vaccines. This article reviews both historical and innovative laboratory pneumococcal animal models that have vastly added to knowledge of (i) mechanisms of infection, pathogenesis, and immunity; (ii) efficacies of antimicrobials; and (iii) screening of vaccine candidates. A comprehensive description of the techniques applied to induce disease is provided, the advantages and limitations of mouse, rat, and rabbit models used to mimic pneumonia, sepsis, and meningitis are discussed, and a section on otitis media models is also included. The choice of appropriate animal models for in vivo studies is a key element for improved understanding of pneumococcal disease.

scholarly journals Enterococcal Leucine-Rich Repeat-Containing Protein Involved in Virulence and Host Inflammatory Response

2007 ◽  
Vol 75 (9) ◽  
pp. 4463-4471 ◽  
Author(s):  
Sophie Brinster ◽  
Brunella Posteraro ◽  
Hélène Bierne ◽  
Adriana Alberti ◽  
Samira Makhzami ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Surface Protein ◽  
Surface Proteins ◽  
Host Cells ◽  
High Morbidity ◽  
Leucine Rich Repeat ◽  
Repeat Domain ◽  
Underlying Diseases ◽  
Host Inflammatory Response

ABSTRACTEnterococcus faecalisis an important nosocomial pathogen associated with high morbidity and mortality for patients who are immunocompromised or who have severe underlying diseases. TheE. faecalisgenome encodes numerous surface-exposed proteins that may be involved in virulence. This work describes the characterization of the first internalin-like protein inE. faecalis, ElrA, belonging to the recently identified WxL family of surface proteins. ElrA contains an N-terminal signal peptide for export, a leucine-rich repeat domain that may interact with host cells, and a C-terminal WxL domain that interacts with the peptidoglycan. Disruption of theelrAgene significantly attenuates bacterial virulence in a mouse peritonitis model. TheelrAdeletion mutant also displays a defect in infection of host macrophages and a decreased interleukin-6 response in vivo. Finally,elrAexpression is induced in vivo. Altogether, these results demonstrate a role for ElrA in theE. faecalisinfectious process in vivo and suggest that this surface protein may contribute toE. faecalisvirulence by stimulating the host inflammatory response.

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