Identification of a Streptococcus gordonii SspB domain that mediates adhesion to Porphyromonas gingivalis.

Porphyromonas gingivalis, a primary pathogen in adult periodontitis, may establish itself in the oral cavity by adhering to early plaque bacteria such as Streptococcus gordonii. Our previous studies (R. J. Lamont et al., Microbiology 140:867-872, 1994) suggested that this interaction is mediated by the SspB polypeptide, a member of the antigen I/II family of streptococcal surface proteins. S. gordonii was recently shown to express a second Ssp polypeptide (SspA) that resembles SspB and the structurally homologous antigen I/II polypeptide (Pac) of Streptococcus mutans. To determine if all of these related antigen I/II proteins interacted with P. gingivalis, SspA, SspB, and Pac were tested for adhesion to P. gingivalis cells. Both of the S. gordonii Ssp proteins bound labeled target cells, whereas the S. mutans Pac polypeptide did not, suggesting that antigen I/II-mediated binding of P. gingivalis by streptococci may be species specific. To investigate the molecular basis for this functional difference, the P. gingivalis binding domain of SspB was mapped. The binding properties of a family of truncated SspB polypeptides lacking C-terminal sequences were determined. In addition, the lack of binding activity exhibited by the Pac protein was exploited to construct and analyze chimeric SspB-Pac polypeptides. Both approaches revealed that the region defined by residues 1167 to 1250 of SspB was essential for P. gingivalis binding. This region of SspA and SspB is entirely conserved, consistent with the binding properties determined for these proteins. However, the corresponding region of Pac differs in both the primary sequence and predicted secondary structure, suggesting that the overall structure of this domain may define its functional activity.     

Identification of a molecule of Porphyromonas gingivalis that binds to Streptococcus gordonii

The molecules that mediate the adherence of Porphyromonas gingivalis, a periodontal pathogen, to Streptococcus gordonii, a commensal plaque organism, were investigated. Outer membrane proteins of P. gingivalis were labelled with biotin, extracted by EDTA and reacted with S. gordonii cells. Interactive porphyromonas components were identified by SDS-PAGE of the S. gordonii cells followed by electroblotting and visualization of the adsorbed porphyromonas molecules with streptavidin-alkaline phosphatase. A P. gingivalis molecule of 35 kDa bound to S. gordonii. Monospecific polyclonal antibodies to the 35 kDa protein inhibited binding of P. gingivalis to S. gordonii by 71%. The antibodies also reacted with the P. gingivalis fimbriae, indicating that the 35 kDa molecule is antigenically related to, or associated with, the fimbriae.     

Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.     

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.     

Identification of independent Streptococcus gordonii SspA and SspB functions in coaggregation with Actinomyces naeslundii.

The initial stages of dental plaque formation involve the adherence of early colonizing organisms such as Streptococcus gordonii and Actinomyces naeslundii to the saliva-coated tooth surface and to each other. The S. gordonii surface proteins SspA and SspB are known to play a role in adherence to salivary proteins and mediate coaggregation with other bacteria. Coaggregation is the adhesin receptor-mediated interaction between genetically distinct cell types and appears to be ubiquitous among oral isolates. To define the function of SspA and SspB separately on the surface of their natural host, we constructed and analyzed the coaggregation properties of an isogenic sspB mutant of S. gordonii DL1, an sspAB double mutant, and a previously described sspA mutant. A. naeslundii strains have been previously classified into six coaggregation groups based on the nature of their coaggregations with S. gordonii DL1 and other oral streptococci. Coaggregation assays with the sspA and sspB mutants showed that SspA and SspB are the streptococcal proteins primarily responsible for defining these coaggregation groups and, thus, are highly significant in the establishment of early dental plaque. SspA exhibited two coaggregation-specific functions. It participated in lactose-inhibitable and -noninhibitable interactions, while SspB mediated only lactose-noninhibitable coaggregations. Accordingly, the sspAB double mutant lacked these functions and allowed us to detect a third coaggregation interaction with one of these organisms. These proteins may play an important role in development of S. gordonii-A. naeslundii communities in early dental plaque. Understanding these adhesin proteins will aid investigations of complex microbial communities that characterize periodontal diseases.     

Structural dissection and in vivo effectiveness of a peptide inhibitor of Porphyromonas gingivalis adherence to Streptococcus gordonii

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro(1171) or Glu(1168) increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo, mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10(9) CFU of S. gordonii DL-1 or 10(7) CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10(9) CFU of S. gordonii followed by 10(7) CFU of P. gingivalis induced significantly greater bone loss (P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10(7) CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis.     

Secretion of Functional Salivary Peptide by Streptococcus gordonii Which Inhibits Fimbria-Mediated Adhesion of Porphyromonas gingivalis

Porphyromonas gingivalis, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of Streptococcus gordonii secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (prpC) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. prpC was connected downstream to a promoter and a gene encoding a signal peptide of Streptococcus downei glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant S. gordonii G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of P. gingivalis cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of P. gingivalis with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent P. gingivalis adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of P. gingivalis adherence to the oral cavity.     

Role of Streptococcus gordonii surface proteins SspA/SspB and Hsa in platelet function

Streptococcus gordonii colonization of damaged heart surfaces in infective endocarditis is dependent upon the recognition of host receptors by specific bacterial surface proteins. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial proteins required remains poorly understood. This study provides clear evidence that several S. gordonii surface proteins participate in the interaction with platelets to support platelet adhesion and induce platelet aggregation. S. gordonii strains were found to support strong (DL1-Challis, SK12, SK184, and Blackburn) or moderate (UB1545 Δhsa and CH1-Challis) adhesion or failed to support platelet adhesion (M5, M99, and Channon). In addition, under flow conditions, platelets rolled and subsequently adhered to immobilized S. gordonii at low shear (50 s⁻¹) in an Hsa-dependent manner but did not interact with S. gordonii DL1 at any shear rate of >50 s⁻¹. S. gordonii strains either induced (DL1-Challis, SK12, SK184, UB1545 Δhsa, and M99) or failed to induce (M5, CH1-Challis, Channon, and Blackburn) platelet aggregation. Using a proteomic approach to identify differential cell wall protein expression between aggregating (DL1) and nonaggregating (Blackburn) strains, we identified antigen I/antigen II family proteins SspA and SspB. The overexpression of SspA or SspB in platelet-nonreactive Lactococcus lactis induced GPIIb/GPIIIa-dependent platelet aggregation similar to that seen with S. gordonii DL1. However, they failed to support platelet adhesion. Thus, S. gordonii has distinct mechanisms for supporting platelet adhesion and inducing platelet aggregation. Differential protein expression between strains may be important for the pathogenesis of invasive diseases such as infective endocarditis.     

Oral immunization with recombinant Streptococcus gordonii expressing porphyromonas gingivalis FimA domains

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.     

Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin

Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology of pulpal and periapical diseases.     

Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii

Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity.     

Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

Coaggregation of Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77:5130–5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.     

Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva

Porphyromonas gingivalis causes periodontal diseases and its lipopolysaccharide (LPS) is considered as a major virulence factor responsible for pathogenesis. Since initial recognition of P. gingivalis LPS (Pg.LPS) in the oral cavity might be crucial for the host response, we identified Pg.LPS-binding proteins (Pg.LPS-BPs) using Pg.LPS-immobilized beads and a high-resolution mass spectrometry. LPS purified from P. gingivalis was conjugated onto N-hydroxysuccinimidyl-Sepharose^® 4 Fast Flow beads. Notably, Pg.LPS-conjugated beads could stimulate Toll-like receptor 2 (TLR2) as determined by a TLR2-depdendent reporter expression system using CHO/CD14/TLR2. In addition, the Pg.LPS-conjugated beads induced the production of inflammatory mediators such as nitric oxide and interferon-gamma-inducible protein-10 in the macrophage cell-line, RAW 264.7. These results imply that Pg.LPS retained its immunological properties during the conjugation process. Then, the Pg.LPS-conjugated beads were mixed with a pool of saliva obtained from nine human subjects to capture Pg.LPS-BPs and molecular identities were determined by LTQ-Orbitrap hybrid fourier transform mass spectrometry. Pg.LPS-BPs captured at high frequencies included alpha-amylase, cystatin, prolactin-inducible protein, lysozyme C, immunoglobulin components, serum albumin, lipocalin-1, and submaxillary gland androgen-regulated protein 3B. These proteins are known to be involved in bacterial adhesion and colonization, anti-microbial functions or modulation of immune responses.     

Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva

Porphyromonas gingivalis causes periodontal diseases and its lipopolysaccharide (LPS) is considered as a major virulence factor responsible for pathogenesis. Since initial recognition of P. gingivalis LPS (Pg.LPS) in the oral cavity might be crucial for the host response, we identified Pg.LPS-binding proteins (Pg.LPS-BPs) using Pg.LPS-immobilized beads and a high-resolution mass spectrometry. LPS purified from P. gingivalis was conjugated onto N-hydroxysuccinimidyl-Sepharose(?) 4 Fast Flow beads. Notably, Pg.LPS-conjugated beads could stimulate Toll-like receptor 2 (TLR2) as determined by a TLR2-depdendent reporter expression system using CHO/CD14/TLR2. In addition, the Pg.LPS-conjugated beads induced the production of inflammatory mediators such as nitric oxide and interferon-gamma-inducible protein-10 in the macrophage cell-line, RAW 264.7. These results imply that Pg.LPS retained its immunological properties during the conjugation process. Then, the Pg.LPS-conjugated beads were mixed with a pool of saliva obtained from nine human subjects to capture Pg.LPS-BPs and molecular identities were determined by LTQ-Orbitrap hybrid fourier transform mass spectrometry. Pg.LPS-BPs captured at high frequencies included alpha-amylase, cystatin, prolactin-inducible protein, lysozyme C, immunoglobulin components, serum albumin, lipocalin-1, and submaxillary gland androgen-regulated protein 3B. These proteins are known to be involved in bacterial adhesion and colonization, anti-microbial functions or modulation of immune responses.     

Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis functions as a coadhesin for Porphyromonas gingivalis major fimbriae

Cohesive interactions between Porphyromonas gingivalis and plaque-forming bacteria, such as Streptococcus oralis, are considered to play an important role in the colonization of P. gingivalis in periodontal sites. Although P. gingivalis fimbriae have been reported to mediate coaggregation with S. oralis, the S. oralis molecule involved has not been identified. We identified the coadhesin of S. oralis ATCC 9811 and purified it by affinity column chromatography. We found that the molecular mass of the purified protein was approximately 40 kDa. Dot blot and Western blot assays showed binding of the 40-kDa protein to P. gingivalis fimbriae. Further, turbidimetric assays showed that the coadhesin inhibited coaggregation between P. gingivalis and S. oralis in a dose-dependent manner. Analyses of the amino-terminal sequences of the protein and its lysyl endopeptidase-cleaved fragments revealed that the coadhesin was identical to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we cloned the gene that encodes S. oralis GAPDH and found that the sequence had a high degree of homology with the sequences of GAPDHs of various bacteria, including Streptococcus gordonii and Fusobacterium nucleatum. To confirm the contribution of S. oralis GAPDH to the interaction with P. gingivalis, a recombinant GAPDH protein was generated in Escherichia coli; this protein bound to P. gingivalis fimbriae and had an inhibitory effect on coaggregation. These results suggest that S. oralis GAPDH functions as a coadhesin for P. gingivalis fimbriae. In addition, considering the high degree of homology of the GAPDHs of various bacteria, those of other plaque-forming bacteria also may contribute to the colonization of P. gingivalis.     

Sequencing of the Ribosomal Intergenic Spacer Region for Strain Identification of Porphyromonas gingivalis

The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivalis were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalis ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison.     

Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Deltaivi10 and Deltaivi11, which indicated that mutant strains Deltaivi10 and Deltaivi11 are less able to survive in this model than the wild-type strain, while Deltaivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Deltaivi8, Deltaivi10, and Deltaivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Deltaivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.     

Interaction of Candida albicans Cell Wall Als3 Protein with Streptococcus gordonii SspB Adhesin Promotes Development of Mixed-Species Communities

Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3⁻ mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.Candida species are the fourth most common causative agents of nosocomial bloodstream infections (2, 47, 54). Crude mortality rates for Candida infections exceed 50% (10, 52), and attributable mortality rates vary between 5 and 48% (3, 10, 13). Candida albicans accounts for 62% of invasive candidiasis infections (46, 47) and is commonly isolated from the oral cavity, gastrointestinal tract, and vagina. The oral carriage rate of C. albicans in healthy subjects ranges from 25 to 60% (28, 42, 48). In the oral cavity, there are estimated to be approximately 700 different species of microorganisms present (45). C. albicans is able to interact physically, by coaggregation, or chemically, through small-molecule signaling, with some of these other microorganisms (1, 18, 20, 29, 33). Interactions of C. albicans with bacteria may be antagonistic, e.g., with Pseudomonas aeruginosa (20), or synergistic, e.g., with Streptococcus gordonii (1), resulting in the formation of diverse polymicrobial communities.Streptococcus gordonii is a primary colonizer of the oral cavity and may be isolated from mucosal or hard surfaces present there (17, 41). It has previously been shown that S. gordonii, and other viridans streptococci, can coaggregate with C. albicans cells both in vitro and in vivo (21, 29, 57). The interactions between oral streptococci and C. albicans are recognized as contributing to formation of enhanced biofilms (1), which may occur on dentures, leading to denture stomatitis (42). Oral streptococci express a range of cell surface polypeptides, many of which act as adhesins to promote colonization (31, 38). The antigen (Ag) I/II family of polypeptides are cell wall-anchored proteins produced by most indigenous species of oral streptococci (4). These adhesins have been shown to bind a wide range of host cell proteins, including fibronectin (49) and salivary agglutinin gp-340 (5, 12, 27). In addition, the Ag I/II family polypeptide SspB from S. gordonii has been shown to interact directly with other microorganisms, including Actinomyces naeslundii (27), Porphyromonas gingivalis (11), and C. albicans (1, 22). It is thus proposed that oral streptococci may promote colonization by these other microorganisms by providing alternative surfaces to adhere to (30) and possibly metabolic benefits (25).Candida albicans is a pleomorphic fungus, with the two most commonly identified morphologies being yeast cells and hyphae. Hyphal-filament formation may be induced by many factors, including pH, serum, temperature, nutrient availability, and diffusible cell signaling molecules (53). In a mixed-species biofilm model, S. gordonii enhances hyphal formation, and there is evidence that this may be mediated, at least in part, by soluble factors released by streptococci (1). Within mixed-species biofilms of S. gordonii and C. albicans, streptococci were found associated with yeast cells, pseudohyphae, and hyphae, but preferentially with hyphal filaments (1).The hyphal cell wall comprises a mixture of chitin, β-1,3 glucans, and β-1,6 glucans, as well as a vast array of proteins (7). One of the major families of C. albicans adhesins is the ALS (agglutinin-like sequence) group of cell wall glycoproteins (24). The family comprises 8 members, several of which have adhesive functions involved in host-pathogen interactions (24). One of these adhesins, Als3p, is a hypha-specific protein (9, 23) and has been shown to be required for mature-biofilm formation, binding extracellular matrix, adhesion to host cells, and internalization of C. albicans by endothelial cells (24, 50, 56). There is also evidence that the Als5 protein is involved in recognition of S. gordonii by C. albicans (32).In this study, we investigated the role of hypha-specific Als3p in early-stage biofilm formation and in intergeneric interactions of C. albicans with S. gordonii. The results suggest that Als3p interacts directly with SspB on the surface of S. gordonii, a binding event that may then enable additional concerted adhesin-receptor interactions to become established.