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L Jeannine Brady

Publications and source records attributed to L Jeannine Brady.

13 recordsLinked to original sources
PubMed2006

Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants.

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Deltaffh, DeltascRNA, and DeltaftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the beta subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Deltaffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

Adaptation, Physiological↗
PubMed2006

A unique serine-rich repeat protein (Srr-2) and novel surface antigen (epsilon) associated with a virulent lineage of serotype III Streptococcus agalactiae.

Group B streptococci (GBS) are pathogens of both neonates and adults, with serotype III strains in particular being associated with invasive disease and meningitis. In this study, a novel GBS surface antigen, epsilon, was found to be co-expressed with the previously reported delta antigen on an identical subset of serotype III GBS. Expression of delta/epsilon on the surface of serotype III GBS was shown to distinguish the restriction digest pattern (RDP) III-3 and multilocus sequence typing (ST)-17 lineage. epsilon-Specific antibodies were reactive with a unique, high-molecular-mass, serine-rich repeat protein (Srr-2) found exclusively in RDP III-3 strains. The gene encoding Srr-2 was located within a putative accessory secretory locus that included secY2 and secA2 homologues and had a genetic organization similar to that of the secY2/A2 locus of staphylococci. In contrast, serotype III delta/epsilon-negative strains and strains representative of serotypes Ia, Ib, Ic and II shared a common Srr-encoding gene, srr-1, and an organization of the secY2/A2 locus similar to that of previously reported serotype Ic, delta/epsilon-negative serotype III and serotype V GBS strains. Representative serotype III delta/epsilon-positive strains had LD(90) values 3-4 logs less than those of serotype III delta/epsilon-negative strains in a neonatal mouse model of infection. These results indicate that the RDP III-3/ST-17 lineage expresses Srr-2 and is highly virulent in an in vivo model of neonatal sepsis.

Adenosine Triphosphatases↗
PubMed2005

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2.

The signal recognition particle (SRP)-translocation pathway is conserved in all three domains of life and delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum. We determined the requirement in the cariogenic oral pathogen Streptocococcus mutans of the three universally conserved elements of the SRP pathway: Ffh/SRP54, scRNA, and FtsY/SRalpha. Previously, we reported that insertional interruption of S. mutans ffh was not lethal, but resulted in acid sensitivity. To test whether S. mutans could survive extensive disruption of the SRP pathway, single and double deletions of genes encoding Ffh, scRNA, and FtsY were generated. Without environmental stressors, all mutant strains were viable, but unlike the wild-type, none could initiate growth at pH 5.0 or in 3.5% NaCl. Survival of challenge with 0.3 mM H(2)O(2) was also diminished without ffh. Members of the YidC/Oxa1/Alb3 family are also ubiquitous, involved in the translocation and assembly of membrane proteins, and have been identified in prokaryotes/mitochondria/chloroplasts. Two genes encoding YidC homologs, YidC1 and YidC2, are present in streptococcal genomes with both expressed in S. mutans. Deletion of YidC1 demonstrated no obvious phenotype. Elimination of YidC2 resulted in a stress-sensitive phenotype similar to SRP pathway mutants. Mutants lacking both YidC2 and SRP components were severely impaired and barely able to grow, even in the absence of environmental stress. Here, we report the dispensability of the cotranslational SRP protein translocation system in a bacterium. In S. mutans, this pathway contributes to protection against rapid environmental challenge and may overlap functionally with YidC2.

Bacterial Proteins↗
PubMed2005

A whole cell BIAcore assay to evaluate P1-mediated adherence of Streptococcus mutans to human salivary agglutinin and inhibition by specific antibodies.

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.

Agglutinins↗
PubMed2005

Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

Chloroform↗
PubMed2004

An ffh mutant of Streptococcus mutans is viable and able to physiologically adapt to low pH in continuous culture.

Previously, we described in Streptococcus mutans strain NG8 a 5-gene operon (sat) that includes ffh, the bacterial homologue of the eukaryotic signal recognition particle (SRP) protein, SR54. A mutation in ffh resulted in acid sensitivity but not loss of viability. In the present study, chemostat-grown cells of the ffh mutant were shown to possess only 26% and 39% of the parental membrane F-ATPase activity and 55% and 75% of parental glucose-phosphotransferase (PTS) activity when pH-7 and pH-5-grown cells, respectively, were assayed. Two-dimensional-gel electrophoretic analyses revealed significant differences in protein profiles between parent and ffh-mutant strains at both pH 5 and pH 7. It appears that the loss of active SRP (Ffh) function, while not lethal, results in substantial alterations in cellular physiology that includes acid tolerance.

Acclimatization↗
PubMed2004

Further characterization of immunomodulation by a monoclonal antibody against Streptococcus mutans antigen P1.

We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion with N-chlorosuccinimide but not in isotype distribution or overall reactivity with S. mutans or intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with either S. mutans alone or S. mutans coated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlapping spaP subclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface of S. mutans alters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.

Agglutinins↗
PubMed2004

Redirecting the humoral immune response against Streptococcus mutans antigen P1 with monoclonal antibodies.

The adhesin P1 of Streptococcus mutans has been studied as an anticaries vaccine antigen. An anti-P1 monoclonal antibody (MAb) bound to S. mutans prior to mucosal immunization of mice was shown previously to alter the amount, specificity, isotype, and biological activity of anti-P1 antibodies. The present study was undertaken to screen this and four additional anti-P1 MAbs for immunomodulatory activity when complexed with S. mutans and administered by a systemic route and to evaluate sera from immunized mice for the ability to inhibit adherence of S. mutans to immobilized human salivary agglutinin. All five MAbs tested influenced murine anti-P1 serum antibody responses in terms of subclass distribution and/or specificity. The effects varied depending on which MAb was used and its coating concentration. Two MAbs promoted a more effective, and two others a less effective, adherence inhibition response. An inverse relationship was observed between the ability of the MAbs themselves to inhibit adherence and the ability of antibodies elicited following immunization with immune complexes to inhibit adherence. Statistically significant correlations were demonstrated between the levels of anti-P1 serum immunoglobulin G2a (IgG2a) and IgG2b, but not of IgG1 or IgG3, and the ability of sera from immunized animals to inhibit bacterial adherence. These results indicate that multiple anti-P1 MAbs can mediate changes in the immune response and that certain alterations are potentially more biologically relevant than others. Immunomodulation by anti-P1 MAbs represents a useful strategy to improve the beneficial immune response against S. mutans.

Adhesins, Bacterial↗
PubMed2004

Characterization of the Streptococcus mutans P1 epitope recognized by immunomodulatory monoclonal antibody 6-11A.

Monoclonal antibody (MAb) 6-11A directed against Streptococcus mutans surface adhesin P1 was shown previously to influence the mucosal immunogenicity of this organism in BALB/c mice. The specificity of anti-P1 serum immunoglobulin G (IgG) and secretory IgA antibodies and the subclass distribution of anti-P1 serum IgG antibodies were altered, and the ability of elicited serum antibodies to inhibit S. mutans adherence in vitro was in certain cases increased. MAb 6-11A is known to recognize an epitope dependent on the presence of the proline-rich region of the protein, although it does not bind directly to the isolated P-region domain. In this report, we show that MAb 6-11A recognizes a complex discontinuous epitope that requires the simultaneous presence of the alanine-rich repeat domain (A-region) and the P-region. Formation of the core epitope requires the interaction of these segments of P1. Residues amino terminal to the A-region also contributed to recognition by MAb 6-11A but were not essential for binding. Characterization of the MAb 6-11A epitope will enable insight into potential mechanisms of immunomodulation and broaden our understanding of the tertiary structure of P1.

Animals↗
PubMed2004

Contribution of the alanine-rich region of Streptococcus mutans P1 to antigenicity, surface expression, and interaction with the proline-rich repeat domain.

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.

Alanine↗
PubMed2003

Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization, enzymatic activity, and protein-protein interactions.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.

Actins↗
PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗