Yoshiaki Hasegawa

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.

Gingival Epithelial Cell Transcriptional Responses to Commensal and Opportunistic Oral Microbial Species

ABSTRACT Transcriptional profiling and ontology tools were utilized to define the biological pathways of gingival epithelial cells modulated by coculture with the oral commensal Streptococcus gordonii and the opportunistic commensal Fusobacterium nucleatum. Overall, F. nucleatum and S. gordonii perturbed the gingival epithelial cell transcriptome much less significantly than the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans perturbed the transcriptome, indicating that there was a greater degree of host adaptation by the commensal species (M. Handfield, J. J. Mans, G. Zheng, M. C. Lopez, S. Mao, A. Progulske-Fox, G. Narasimhan, H. V. Baker, and R. J. Lamont, Cell. Microbiol. 7:811-823, 2005). The biological pathways significantly impacted by F. nucleatum and S. gordonii included the mitogen-activated protein kinase (MAPK) and Toll-like receptor signaling pathways. Differential regulation of GADD45 and DUSP4, key components of the MAPK pathway, was confirmed at the protein level by Western blotting. Modulation of the MAPK pathway is likely to affect host cell proliferation and differentiation. In addition, both the MAPK and Toll-like receptor pathways ultimately converge on cytokine gene expression. An enzyme-linked immunosorbent assay of secreted interleukin-6 (IL-6) and IL-8 demonstrated that F. nucleatum induced production of these cytokines, whereas S. gordonii inhibited secretion from the epithelial cells. Stimulation of secretion of proinflammatory cytokines from epithelial cells may reflect the invasive phenotype of F. nucleatum and contribute to the greater pathogenic potential of F. nucleatum than of S. gordonii.

P. gingivalis accelerates gingival epithelial cell progression through the cell cycle

P. gingivalis, an opportunistic pathogen in periodontal disease, can reside within the epithelial cells that line the gingival crevice. A proteomic analysis revealed that infection of gingival epithelial cells with P. gingivalis induces broadly based changes in the level and phosphorylation status of proteins that exert multi-level control on the eukaryotic cell cycle. Pathways that were impacted by P. gingivalis included those involving cyclins, p53 and PI3K. The predicted infection-dependent phenotype was confirmed by cytofluorimetry that showed an enhanced proliferation rate of gingival epithelial cells infected with P. gingivalis associated with accelerated progression through the S-phase. Elevated cell proliferation was dependent on the presence of the long fimbriae of P. gingivalis. The ability of P. gingivalis, a common inhabitant of the subgingival crevice, to accelerate cell cycling could have biological consequences for barrier and signaling functions, and for physiological status, of the gingival epithelium.

Surface components of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE Research on Porphyromonas gingivalis, a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis. MATERIAL AND METHODS Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. RESULTS Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly. CONCLUSION Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.

A Novel Type of Two‐Component Regulatory System Affecting Gingipains in Porphyromonas gingivalis

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two‐component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene‐disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine‐ and lysine‐specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wild‐type strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly‐discovered two‐component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

FimB Regulates FimA Fimbriation in Porphyromonas gingivalis

The periodontitis-associated pathogen Porphyromonas gingivalis colonizes and forms a biofilm in gingival crevices through fimbriae. It is known that the often-used strains ATCC 33277 and 381 produce long FimA fimbriae. We found a possible nonsense mutation within fimB, immediately downstream from fimA, coding a major subunit of FimA fimbriae of the strains. Indeed, P. gingivalis strains, except for ATCC 33277 and 381, universally expressed FimB, the gene product of fimB. Electron micrographs revealed that a FimB-restored strain had short and dense, “toothbrush”-like, FimA fimbriae. FimA overexpression elongated the fimbriae, whereas FimB overexpression shortened them. FimB restoration increased production of FimA and its accessory proteins. Thus, FimB regulates the length and expression of FimA fimbriae. Additionally, FimB restoration significantly reduced the release of FimA fimbriae from the cell surface, suggesting that FimB functions as an anchor of the fimbriae. The restoration enhanced adherent activity as well.

Porphyromonas gingivalis FimA Fimbriae: Fimbrial Assembly by fimA Alone in the fim Gene Cluster and Differential Antigenicity among fimA Genotypes

The periodontal pathogen Porphyromonas gingivalis colonizes largely through FimA fimbriae, composed of polymerized FimA encoded by fimA. fimA exists as a single copy within the fim gene cluster (fim cluster), which consists of seven genes: fimX, pgmA and fimA-E. Using an expression vector, fimA alone was inserted into a mutant from which the whole fim cluster was deleted, and the resultant complement exhibited a fimbrial structure. Thus, the genes of the fim cluster other than fimA were not essential for the assembly of FimA fimbriae, although they were reported to influence FimA protein expression. It is known that there are various genotypes for fimA, and it was indicated that the genotype was related to the morphological features of FimA fimbriae, especially the length, and to the pathogenicity of the bacterium. We next complemented the fim cluster-deletion mutant with fimA genes cloned from P. gingivalis strains including genotypes I to V. All genotypes showed a long fimbrial structure, indicating that FimA itself had nothing to do with regulation of the fimbrial length. In FimA fimbriae purified from the complemented strains, types I, II, and III showed slightly higher thermostability than types IV and V. Antisera of mice immunized with each purified fimbria principally recognized the polymeric, structural conformation of the fimbriae, and showed low cross-reactivity among genotypes, indicating that FimA fimbriae of each genotype were antigenically different. Additionally, the activity of a macrophage cell line stimulated with the purified fimbriae was much lower than that induced by Escherichia coli lipopolysaccharide.

Identification and characterization of novel glycoproteins involved in growth and biofilm formation by Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated as a major pathogen associated with chronic periodontitis. To extend our knowledge of post-translational protein glycosylation in P. gingivalis, a proteomic analysis involving two-dimensional polyacrylamide gel electrophoresis combined with carbohydrate staining and mass spectrometry was performed. Four novel glycoproteins, PGN0743, PGN0876, PGN1513 and PGN0729, in P. gingivalis ATCC 33277 were identified. These four identified glycoproteins possess a range of biochemical activities and cellular localization. PGN0743 contains a sequence motif identifying it as a FKBP-type cis-trans isomerase, which has activity usually associated with chaperone functions. PGN0876 and PGN1513 contain tetratricopeptide repeat domains that mediate protein-protein interactions. PGN0729 encodes the outer membrane protein 41 precursor, which was previously identified as Pgm6, and is homologous to the OmpA protein in Escherichia coli. Several different types of glycoprotein were identified, suggesting that P. gingivalis possesses a general mechanism for protein glycosylation. PGN0743-deficient and PGN0876-deficient mutants were constructed to examine the role(s) of the two identified glycoproteins. Both mutants showed a decreased growth rate under nutrient-limited conditions and reduced biofilm formation activity. These results suggest that the novel glycoproteins PGN0743 and PGN0876 play an important role in the growth and colonization of P. gingivalis.

Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae.

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. Porphyromonas gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes, 40 and 43 kDa, in the cell. The 43-kDa and 40-kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40-kDa Mfa3 alone. Furthermore, the 40-kDa Mfa3 started with the Ala(44) residue of the deduced amino acid sequence, indicating that the N-terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno-electron microscopy revealed that Mfa3 localized at the tip of the fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in the absence of other accessory proteins, PGN0290 and PGN0291, in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto-aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1(-) , FimA(-) ). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis.