A conserved C-terminal 13-amino-acid motif of Gap1 is required for Gap1 function and necessary for the biogenesis of a serine-rich glycoprotein of Streptococcus parasanguinis.

Adhesion of Streptococcus parasanguinis to saliva-coated hydroxyapatite (SHA), an in vitro tooth model, is mediated by long peritrichous fimbriae. Fap1, a fimbria-associated serine-rich glycoprotein, is required for fimbrial assembly. Biogenesis of Fap1 is controlled by an 11-gene cluster that contains gly, nss, galT1 and -2, secY2, gap1 to -3, secA2, and gtf1 and -2. We had previously isolated a collection of nine nonadherent mutants using random chemical mutagenesis approaches. These mutants fail to adhere to the in vitro tooth model and to form fimbriae. In this report, we further characterized these randomly selected nonadherent mutants and classified them into three distinct groups. Two groups of genes were previously implicated in Fap1 biogenesis. One group has a mutation in a glycosyltransferase gene, gtf1, that is essential for the first step of Fap1 glycosylation, whereas the other group has defects in the fap1 structural gene. The third group mutant produces an incompletely glycosylated Fap1 and exhibits a mutant phenotype similar to that of a glycosylation-associated protein 1 (Gap1) mutant. Analysis of this new mutant revealed that a conserved C-terminal 13-amino-acid motif was missing in Gap1. Site-directed mutagenesis of a highly conserved amino acid tryptophan within this motif recapitulated the deletion phenotype, demonstrating the importance of the Gap1 C-terminal motif for Fap1 biogenesis. Furthermore, the C-terminal mutation does not affect Gap1-Gap3 protein-protein interaction, which has been shown to mediate Fap1 glycosylation, suggesting the C-terminal motif has a distinct function related to Fap1 biogenesis.

A conserved domain of previously unknown function in Gap1 mediates protein-protein interaction and is required for biogenesis of a serine-rich streptococcal adhesin.

Fap1-like serine-rich proteins are a new family of bacterial adhesins found in a variety of streptococci and staphylococci that have been implicated in bacterial pathogenesis. A gene cluster encoding glycosyltransferases and accessory Sec components is required for Fap1 glycosylation and biogenesis in Streptococcus parasanguinis. Here we report that the glycosylation-associated protein, Gap1, contributes to glycosylation and biogenesis of Fap1 by interacting with another glycosylation-associated protein, Gap3. Gap1 shares structural homology with glycosyltransferases. The gap1 mutant, like the gap3 mutant, produced an aberrantly glycosylated Fap1 precursor and failed to produce mature Fap1, suggesting that Gap1 and Gap3 might function in concert in the Fap1 glycosylation and biogenesis. Indeed, Gap1 interacted with Gap3 in vitro and in vivo. A Gap1 N-terminal motif, within a highly conserved domain of unknown function (DUF1975) identified in many bacterial glycosyltransferases, was required for the Gap1-Gap3 interaction. Deletion of one, four and nine amino acids within the conserved motif gradually inhibited the Gap1-Gap3 interaction and diminished production of mature Fap1 and concurrently increased production of the Fap1 precursor. Consequently, bacterial adhesion to an in vitro tooth model was also reduced. These data demonstrate that the Gap1-Gap3 interaction is required for Fap1 biogenesis and Fap1-dependent bacterial adhesion.

Identification of critical residues in Gap3 of Streptococcus parasanguinis involved in Fap1 glycosylation, fimbrial formation and in vitro adhesion.

BACKGROUND: Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation. RESULTS: Deletion and site-directed mutagenesis were employed to dissect the regions within Gap3 that were important for its function in Fap1 glycosylation and biogenesis. A deletion of 6 consecutive amino acids, PDLPIL, eliminated the production of the mature 200 kDa Fap1 protein and gave rise instead to a 470 kDa Fap1 intermediate that was only partially glycosylated. Site-directed mutagenesis of the 6 amino acids revealed that only three of these amino acids were required. Mutants in these amino acids (L64R, P65R and L67T) produced the premature 470 kDa Fap1 intermediate. Mutants in the remaining amino acids produced the mature form of Fap1. Cell surface expression of the Fap1 precursor among L64R, P65R and L67T mutants was reduced to levels consistent with that of a gap3 insertional mutant. Electron micrographs showed that these 3 mutants lost their long peritrichous fimbriae. Furthermore, their in vitro adhesion ability to saliva-coated hydroxylapatite (SHA) was inhibited. CONCLUSION: Our data suggest that 3 highly conserved, hydrophobic residues L64, P65 and L67 in Gap3 are essential for Gap3 function and are important for complete glycosylation of Fap1, fimbrial formation and bacterial adhesion.

The utility of affinity-tags for detection of a streptococcal protein from a variety of streptococcal species.

There is no systematic examination of affinity tag utility in Gram-positive bacteria, which limits the investigation of protein function in this important group of bacteria as specific antibodies for many of native proteins are generally not available. In this study, we utilized an E. coli-streptococcal shuttle vector pVT1666 and constructed two sets of expression plasmids pVPT-CTag and pVPT-NTag, with each set containing five affinity tags (GST, GFP, HSV, T7 and Nano) that can be fused to either the C- or N-terminus of a target protein. A putative glycosyltransferase (Gtf2) essential for Fap1 glycosylation was used to demonstrate the utility of the cassettes in detection of Gtf2 fusion proteins, and the biological relevance of the proteins in our working strain Streptococcus parasanguinis. GFP and T7 tags were readily expressed in S. parasanguinis as either an N- or C-terminal fusion to Gtf2. Only the C- terminal fusion of GST and HSV were able to be identified in S. parasanguinis. The Nano tag was not detected in either E. coli or S. parasanguinis. Genetic complementation experiments indicated that all the tagged Gtf2 fusion proteins could restore the Gtf2 function in the null mutant except for the Nano-tagged Gtf2 at its N-terminal fusion. Using a T7-tagged Gtf2 fusion construct, we demonstrated that the fusion cassette is also useful in detection of the fusion tag expression in other streptococci including S. mutans, S. pneumoniae and S. sanguinis. Therefore, the expression cassettes we constructed will be a useful tool not only to investigate protein-protein interactions in Fap1 biogenesis in S. parasanguinis, but also to study protein functions in other gram-positive bacteria in which pVT1666 replicates.

Interaction between two putative glycosyltransferases is required for glycosylation of a serine-rich streptococcal adhesin.

Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of Streptococcus parasanguinis (formerly S. parasanguis). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that N-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both S. parasanguinis as well as in the Fap1 glycosylation system in Escherichia coli. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione S-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.

Differential roles of individual domains in selection of secretion route of a Streptococcus parasanguinis serine-rich adhesin, Fap1.

Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.

The glycan moieties and the N-terminal polypeptide backbone of a fimbria-associated adhesin, Fap1, play distinct roles in the biofilm development of Streptococcus parasanguinis.

Fap1, a fimbria-associated glycoprotein, is essential for biofilm formation of Streptococcus parasanguinis and mediates bacterial attachment to saliva-coated hydroxylapatite, an in vitro tooth model (E. H. Froeliger and P. M. Fives-Taylor, Infect. Immun. 69:2512-2519, 2001; H. Wu and P. M. Fives-Taylor, Mol. Microbiol. 34:1070-1081, 1999; H. Wu et al., Mol. Microbiol. 28:487-500, 1998). Fap1 belongs to a growing family of high-molecular-weight serine-rich proteins found in streptococcal and staphylococcal species and possesses two serine-rich repeat regions. The glycan moiety of Fap1 appears to be O linked within the repeat regions (A. E. Stephenson et al., Mol. Microbiol. 43:147-157, 2002). In the present study, we identified a gene cluster immediately upstream of fap1 that encodes three putative glycosyltransferases and one nucleotide-sugar synthetase-like protein. Inactivation of one glycosyltransferase gene galT2 abolished the expression of two glycan epitopes; however, it did not alter bacterial ability to adhere to both SHA and saliva-conditioned biofilm surfaces. In contrast, the biofilms formed by the galT2 mutant were shallow and had a 70% decrease in biomass accumulation, suggesting that these glycan moieties mediated by GalT2 are not required for the initial adhesion but are important for biofilm formation. A recombinant N-terminal Fap1 polypeptide was shown to interact with a 53-kDa salivary protein and block and displace bacterial attachment, further demonstrating the role of the Fap1 polypeptide in bacterial adhesion. Taken together, these results suggest that Fap1 glycosylation plays an important role in bacterial biofilm formation, whereas the nonglycosylated Fap1 peptide mediates bacterial initial attachment during the process of biofilm formation.

Two gene determinants are differentially involved in the biogenesis of Fap1 precursors in Streptococcus parasanguis.

Mature Fap1, a 200-kDa fimbria-associated adhesin, is required for fimbrial biogenesis and biofilm formation in Streptococcus parasanguis. Fap1-like proteins are found in the genomes of many streptococcal and staphylococcal species. Fap1 is a serine-rich glycoprotein modified by O-linked glycan moieties. In this study, we identified a seven-gene cluster including secY2, orf1, orf2, orf3, secA2, gtf1, and gtf2 that is localized immediately downstream of fap1. The lower G+C contents and the presence of a putative transposase element suggest that this gene cluster was horizontally transferred from other bacteria and represents a genomic island. At least two genes in this island mediated Fap1 biogenesis. Mutation of a glucosyltransferase (Gtf1) gene led to accumulation of a Fap1 precursor, which had no detectable glycan moieties. Inactivation of a gene coding for an accessory Sec protein (SecY2) resulted in expression of a distinct Fap1 precursor, which reacted with one glycan-specific Fap1 antibody but not with another glycan-specific antibody. Furthermore, partially glycosylated Fap1 was detected on the cell surface and in the culture supernatant. These data suggest that SecY2 has a role in complete glycosylation of Fap1 and imply that SecY2 is not the only translocation channel for the Fap1 precursor and that alternative secretion machinery exists. Together, Gtf1 and SecY2 are involved in biogenesis of two distinct Fap1 precursors in S. parasanguis. Discovery of the effect of an accessory Sec protein on Fap1 glycosylation suggests that Fap1 secretion and glycosylation are coupled during Fap1 biogenesis.

SecA2 is distinct from SecA in immunogenic specificity, subcellular distribution and requirement for membrane anchoring in Streptococcus parasanguis.

A secA2 gene is present in the genomes of a wide variety of Gram-positive bacteria. In Streptococcus parasanguis, a primary colonizer of the tooth surface, secA2 is involved in the secretion of a small group of proteins including the fimbrial adhesin, Fap1. Although the substrate specificity is different, SecA2 is predicted to be similar to SecA in structure and function based on the homology between these two proteins. In this study, polyclonal antibodies against SecA2 and SecA did not cross-react with each other, indicating that these two proteins possessed distinct immunogenic epitopes. Fractionation analysis demonstrated that SecA2 was not evenly distributed between the cytoplasmic membrane and the cytoplasm as was noted for SecA. SecA2 was associated with the membrane in the wild type and in secA2 mutants with different regions deleted. The subcellular distribution of SecA2 was not dependent on secY2, suggesting that the membrane association is not through SecY2. These data suggested that SecA2 is distinct from SecA in many respects such as substrate specificity, immunogenic specificity, subcellular distribution and requirement for membrane anchoring.

Influence of nicotine and cotinine on epithelial colonization by periodontopathogens.

BACKGROUND: Since smoking is an established risk factor for the development of periodontitis, the present study investigated whether nicotine and cotinine can make epithelial cells more prone to colonization by periodontopathogens. METHODS: Primary epithelial cell mono-layers were inoculated with nicotine and cotinine prior to adhesion experiments with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The number of bacteria associated with cells inoculated or not with nicotine or cotinine were assessed by an indirect culture viability assay. The same experimental set-up was used for assessing HeLa cells exposed to cigarette smoke extract (CSE). RESULTS: Primary epithelial cells inoculated with concentrations of nicotine and cotinine, found in smokers and non smokers, did not show significant differences (P>0.05) in colonization susceptibility to A. actinomycetemcomitans. When these concentrations were increased to 1 mg/ml, a significant (P<0.05) and species-specific effect of the colonization susceptibility of epithelial cells was observed: It increased for A. actinomycetemcomitans, while it decreased for P. gingivalis. For both species the effects were more pronounced for nicotine, although this was not statistically significant. The change in colonization susceptibility did not result from alterations of the bacterial viability due to nicotine or cotinine. Treatment of HeLa cells with CSE also led to a species-specific variation in colonization tendency; i.e., increased for A. actinomycetemcomitans (P<0.05), but not for P. gingivalis. CONCLUSIONS: The susceptibility of epithelial cells to become colonized by either A. actinomycetemcomitans or P. gingivalis could be altered by nicotine, cotinine, or CSE in a time-dependent, species-specific manner. Whether these findings that support the hypothesis of an increased patient susceptibility for bacterial adhesion to epithelial cells in smokers are clinically relevant remains to be proven.

Investigating the role of secA2 in secretion and glycosylation of a fimbrial adhesin in Streptococcus parasanguis FW213.

Adhesion of Streptococcus parasanguis FW213, a primary colonizer, to the tooth surface is mediated mainly by peritrichous long fimbriae. The fimbrial structural unit, Fap1, is indispensable for fimbriae biogenesis, adhesion to an in vitro tooth model and biofilm formation. Mature Fap1 is a glycoprotein with an apparent molecular mass of 200 kDa. Glycosylated Fap1 is not present in some mutants screened from a transposon mutant library. Localization of the transposition sites revealed a gene determined to be secA2, which is distinct from the canonical secA gene. In FW213, glycosylated Fap1 was present in all the subcellular fractions including the cytoplasm. In VT1574, a non-polar mutant of secA2 generated by in frame deletion, Fap1 was not secreted. Glycosylated Fap1 was present in the membrane and cytoplasm of the mutant, although in greatly reduced amounts. Fap1 secretion and abundance were restored when VT1574 was complemented by a plasmid-borne secA2. The secretion defect of the secA2 mutation appears to be limited to a small group of proteins such as Fap1 and FimA. These data suggested that Fap1 secretion rather than glycosylation was the major effect of the deletion of secA2; however, this deletion also had an impact on Fap1 abundance. Two more secA2 mutants with different regions deleted were tested for their ability to secrete Fap1. One mutant was completely unable to secrete Fap1 while the other was able to secrete, but in a decreased amount. These data suggest that the region deleted in the latter mutant (nucleotides 2032-2337) is dispensable for Fap1 secretion.

Aae, an autotransporter involved in adhesion of Actinobacillus actinomycetemcomitans to epithelial cells.

The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.

Interaction of human salivary mucin MG2, its recombinant N-terminal region and a synthetic peptide with Actinobacillus actinomycetemcomitans.

The antimicrobial properties of human salivary mucin MG2 against the periodontal pathogen, Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), were investigated using purified MG2, rNMUC7 (a recombinant polypeptide containing residue 1-144 of MG2) and synthetic peptides PEP1 (residue 1-17) and PEP2 (residue 47-63). MG2 and rNMUC7 bound to A. actinomycetemcomitans strains SUNY75, SUNY465, SUNY523, 652 and JP2 in a liquid phase binding assay. The bactericidal activities of rNMUC7, PEP1 and PEP2 against A. actinomycetemcomitans SUNY523 were examined in a colony forming unit killing assay. The LD50 for rNMUC7 was 9 microM, for PEP2 was 20 microM and PEP1 did not exhibit bactericidal activity. The primary structure of these polypeptides was analyzed and a direct relationship between net positive charge and bactericidal activity was found. Screening of saliva samples from 60 individuals on Western blots probed with an anti-MG2 antibody against PEP2 revealed that a 20 kDa MG2 fragment was present in 66% of subjects and that this fragment was not present in glandular secretions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides derived from the 20 kDa fragment confirmed that this fragment contained a portion of the amino terminal region of MG2. The present study showed that the N-terminal region of MG2 and a subdomain within this region are microbicidal against A. actinomycetemcomitans and that a 20 kDa fragment of MG2 occurs in whole saliva. This suggests that cleavage of MG2 in vivo may produce fragments with microbicidal properties and that this may represent a novel mechanism of host defense.

Peptide methionine sulfoxide reductase (MsrA) is not a major virulence determinant for the oral pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is an oral pathogen that is a causative agent for periodontal disease as well as other non-oral infections. The chronic inflammation associated with periodontal diseases suggests that the bacterium must be able to neutralize oxygen intermediates to survive in the host tissues. Methionine sulfoxide reductase (MsrA) is an enzyme that has been demonstrated to have a role in protection against oxidative damage and has also been identified to be required for the proper expression or maintenance of functional adhesins on the surface of several pathogenic bacteria. The A. actinomycetemcomitans homologue of msrA has been isolated and a chromosomal insertion mutant constructed by allele replacement mutagenesis. Inactivation of the gene led to a complete loss of enzymic activity toward a synthetic substrate. However, the isogenic mutant was not more sensitive to oxidative stress or less adherent to epithelial cells as compared with the parent strain. These data suggest that this strain of A. actinomycetemcomitans has redundant systems that compensate for the MsrA activities ascribed for other organisms.

Construction of a novel transposon mutagenesis system useful in the isolation of Streptococcus parasanguis mutants defective in Fap1 glycosylation.

Streptococcus parasanguis, a primary colonizer of the tooth surface, has long, peritrichous fimbriae. A fimbria-associated protein, Fap1, is identified as an adhesin of S. parasanguis FW213. The mature Fap1 protein is glycosylated, and the glycosylation is required for fimbria biogenesis and bacterial adhesion. Little is known about the mechanism of Fap1 glycosylation due to the lack of identifiable mutants. A novel transposon mutagenesis system was established and used to generate a mutant library. Screening of the library with a monoclonal antibody specific for a glycan epitope of Fap1 yielded six mutants with decreased expression levels of surface-associated glycosylated Fap1 protein. Southern blot analyses revealed that three of the mutants had the transposon inserted in the fap1 locus, whereas the other three mutants had insertions in other genes. Among the latter three mutants, two expressed Fap1 polypeptides on which no glycosylation was detected by glycan-specific antibodies; the other mutant expressed a partially glycosylated Fap1 polypeptide. These data suggest that three mutants were isolated with defects in genes implicated in Fap1 glycosylation.

The divergently transcribed Streptococcus parasanguis virulence-associated fimA operon encoding an Mn(2+)-responsive metal transporter and pepO encoding a zinc metallopeptidase are not coordinately regulated.

The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn(2+) concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn(2+). This deficiency was not alleviated by compensation with either Mg(2+) or Fe(3+). Wild-type S. parasanguis could take up Mn(2+) and Fe(3+), while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn(2+) and Fe(3+). FimA expression itself was shown to be responsive to Mn(2+) concentration, but not to availability of Fe(3+) or Mg(2+).

Evidence that ORF3 at the Streptococcus parasanguis fimA locus encodes a thiol-specific antioxidant.

Streptococcus parasanguis is a primary colonizer of dental plaque and a major player in subacute bacterial endocarditis. In the present study, the authors report that an ORF (ORF3) located 77 bp downstream of the fimA operon on the S. parasanguis FW213 chromosome complements an Escherichia coli thiol peroxidase (tpx) mutation in glutamine synthetase (GS) protection assays and that GS is protected by the ORF3 gene product in S. parasanguis cell extracts. In addition, the putative streptococcal peroxidase (Tpx(Sp)) protects S. parasanguis from stress caused by H2O2 and is induced by oxygen, as revealed by Northern blot analysis. Taken collectively, these findings support a thiol-dependent antioxidant activity for Tpx in S. parasanguis.

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model.

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.

A recombinase A-deficient strain of Actinobacillus actinomycetemcomitans constructed by insertional mutagenesis using a mobilizable plasmid.

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.