Yoonsuk Park

Role of the Streptococcus gordonii SspB protein in the development of Porphyromonas gingivalis biofilms on streptococcal substrates

Porphyromonas gingivalis is an aggressive periodontal pathogen that persists in the mixed-species plaque biofilm on tooth surfaces. P. gingivalis cells attach to the plaque commensal Streptococcus gordonii and this coadhesion event leads to the development of P. gingivalis biofilms. Binding of these organisms is multimodal, involving both the P. gingivalis major fimbrial FimA protein and the species-specific interaction of the minor fimbrial Mfa1 protein with the streptococcal SspB protein. This study examined the contribution of the Mfa1-SspB interaction to P. gingivalis biofilm formation. P. gingivalis biofilms readily formed on substrata of S. gordonii DL1 but not on Streptococcus mutans cells which lack a coadhesion-mediating homologue of SspB. An insertional inactivation of the mfa1 gene in P. gingivalis resulted in a phenotype deficient in S. gordonii binding and unable to form biofilms. Furthermore, analysis using recombinant streptococci and enterococci showed that P. gingivalis biofilms formed on Enterococcus faecalis strains expressing SspB or translational fusions of SspB with SpaP (the non-adherent SspB homologue in S. mutans) containing the P. gingivalis adherence domain (SspB adherence region, BAR) of SspB. In contrast, an isogenic Ssp null mutant of S. gordonii DL1 was unable to support biofilm growth, even though this strain bound to P. gingivalis FimA at levels similar to wild-type S. gordonii DL1. Finally, site-specific mutation of two functional amino acid residues in BAR resulted in SspB polypeptides that did not promote the development of P. gingivalis biofilms. These results suggest that the induction of P. gingivalis biofilms on a streptococcal substrate requires functional SspB-minor fimbriae interactions.

Signaling System in Porphyromonas gingivalis Based on a LuxS Protein

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

Short Fimbriae of Porphyromonas gingivalis and Their Role in Coadhesion with Streptococcus gordonii

ABSTRACT 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.

Intrinsic apoptotic pathways of gingival epithelial cells modulated by Porphyromonas gingivalis

Porphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase‐3. The anti‐apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriae‐deficient mutants and a naturally occurring non‐fimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real‐time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis‐infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP‐1 and cIAP‐2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro‐survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.

Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells

We have developed a fluorescence imaging technique using a DNA‐binding dye to visualize, over time, the physical interactions between Porphyromonas gingivalis and human gingival epithelial cells in vitro. The results extend previous observations of P. gingivalis invasion of gingival epithelial cells based on indirect measurements. An intracellular location for P. gingivalis was established by optical sectioning of images in the z‐plane. Kinetic analysis showed that P. gingivalis invasion of epithelial cells is a rapid and efficient process, reaching completion after 12 min. Imaging of infected monolayers revealed that over 90% of a population of gingival epithelial cells contained bacteria. Furthermore, only vital bacteria were capable of invasion, and intracellular bacteria congregated in the perinuclear region of the epithelial cells. P. gingivalis remained inside the epithelial cells over a 24 h period and induced rearrangement of the actin cytoskeleton along with alteration of the size and shape of the epithelial cells. These findings provide direct evidence that entry rates of P. gingivalis into gingival epithelial cells are high and rapid, and that internalized bacteria initially localize in a specific region of the epithelial cells.

Coinvasion of Dentinal Tubules by Porphyromonas gingivalis and Streptococcus gordonii Depends upon Binding Specificity of Streptococcal Antigen I/II Adhesin

ABSTRACT 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 toP. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant ofP. 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. gingivaliscoinvasion of dentin. The results demonstrate that collagen-binding andP. 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.

LuxS Involvement in the Regulation of Genes Coding for Hemin and Iron Acquisition Systems in Porphyromonas gingivalis

ABSTRACT The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe2+ ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.

Identification of Porphyromonas gingivalis genes specifically expressed in human gingival epithelial cells by using differential display reverse transcription-PCR.

ABSTRACT Porphyromonas gingivalis, one of the causative agents of adult periodontitis, can invade and survive within host epithelial cells. The molecular mechanisms by which P. gingivalis induces uptake and adapts to an intracellular environment are not fully understood. In this study, we have investigated the genetic responses of P. gingivalis internalized within human gingival epithelial cells (GECs) in order to identify factors involved in invasion and survival. We compared the differential display of arbitrarily PCR-amplified gene transcripts in P. gingivalis recovered from GECs with the display of transcripts in P. gingivalis control cultures. Over 20 potential differentially expressed transcripts were identified. Among these, pepO, encoding an endopeptidase, and genes encoding an ATP-binding cassette (ABC) transporter and a cation-transporting ATPase were upregulated in GECs. To investigate the functionality of these gene products, mutants were generated by insertional inactivation. Compared to the parental strain, mutants of each gene showed a significant reduction in their invasion capabilities. In addition, GEC cytoskeletal responses to the mutants were distinct from those induced by the parent. In contrast, adhesion of the mutant strains to GECs was not affected by lack of expression of the gene products. These results suggest that PepO, a cation-transporting ATPase, and an ABC transporter are required for the intracellular lifestyle of P. gingivalis.

Role of the Porphyromonas gingivalis InlJ protein in homotypic and heterotypic biofilm development.

ABSTRACT The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.

Regulation of the Porphyromonas gingivalis fimA (Fimbrillin) gene.

ABSTRACT In common with many bacterial virulence genes, the fimbrillin (fimA) gene of Porphyromonas gingivalis is modulated in response to environmental fluctuation. Thetrans-acting components that comprise the regulatory system for transcriptional activity of the fimA gene inP. gingivalis were investigated. Three major proteins were found to bind to the upstream region of the fimApromoter. One of these proteins was fimbrillin itself, and the other two were a major arginine protease (Rgp) and lysine protease (Kgp). Production of these proteins was necessary for maximal fimA transcription. An exogenousfimA promoter-lacZ reporter was inactive when introduced into a strain of P. gingivalis carrying a mutation in the indigenous fimA gene. Furthermore,fimA mRNA levels were significantly decreased inrgp and kgp mutant strains. These data indicate that P. gingivalis has evolved multiple levels of control of fimbrial gene expression to enhance its survival in hostile environments.