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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 Mfal 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. DOI 10.1099/mic.0.028928-0

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of [mu]-oxo haem dimer on the cell surface. Gingipain--adhesin complexes are responsible for production of [mu]-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43-48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain--adhesin complexes.

The FimA fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. In this study, the four open reading frames (ORF1, ORF2, ORF3 and ORF4) downstream of the fimbrilin gene (fimA) in strain ATCC 33277 were examined. ORF2, ORF3 and ORF4 were demonstrated to encode minor components of the fimbriae and were therefore renamed fimC, fimD and fimE, respectively. Immunoblotting analyses revealed that inactivation of either fimC or fimD by an ermF-ermAM insertion, but not inactivation of ORF1, was accompanied by concomitant loss of the products from the downstream genes, raising the possibility that fimC, fimD and time constitute a transcription unit. The fimE mutant produced FimC and FimD, but fimbriae purified from it contained neither protein, suggesting that FimE is required for the assembly of FimC and FimD onto the fimbrilin (FimA) fibre. The fimC, fimD and time mutants lost autoaggregation abilities. Fimbriae purified from these three mutants showed attenuated binding activities to glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis and to two extracellular matrix proteins, fibronectin and type I collagen. These results suggest that FimE, as well as FimC and FireD, play critical roles in the adhesive activities of the mature FimA fimbriae in P. gingivalis.

Tannerella forsythensis, one of the important pathogens in periodontal disease, has a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. The S-layer in T. forsythensis is suggested to be associated with haemagglutinating activity, adhesion and invasion of host cells; however, its precise functions have been unknown. ORFs encoding the major S-layer proteins (230 and 2,70 kDa) of T. forsythensis ATCC 43037, tfsA and tfsB, respectively, following the names in a recent report [Lee, S.-W., Sabet, M., Um, H. S., Yang, L., Kim, H. C. & Zhu, W. (2006). Gene 371, 102-111] were determined. To verify the function of the S-layer proteins, three mutants with tfsA, tfsB, or both deleted were successfully constructed by a PCR-based overlapping method. S-layer proteins were completely lost in the double mutant. The single-deletion mutants appeared to lose one of the 230 and 2,70 kDa proteins. Thin-section microscopy clearly revealed that the 230 and 2,70 kDa proteins composed the S-layer. Although the S-layer proteins may be weakly related to haemagglutinating activity, these proteins were highly responsible for adherence to human gingival epithelial cells (Ca9-22) and KB cells. These results suggest that the S-layer proteins in T. forsythensis play an important role in the initiation stage of oral infection including periodontal disease.

The effects of growth rate, medium and aeration on expression of major virulence factors and cellular proteins in Porphyromonas gingivalis ATCC 33277 in a chemostat are investigated. The results have shown P. gingivalis to actively control expression of several virulence factors to survive in the widely fluctuating oral environment.

The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded [beta]-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.

Research has been conducted on Porphyromonas gingivalis nonpigmented mutant. The construction of this mutant has been carried out via the use of transposon mutagenesis and the results indicate that the mutant possesses transposon DNA at the porR gene involved in the cell surface polysaccharide biosynthesis.

Arginine-specific cysteine proteinase (RGP) acts as a processing proteinase for maturation of fimbrilin in Porphyromonas gingivalis. RGP also appears to be involved in the maturation of some cell surface proteins. Double mutations in the rgpA and rgpB genes that code for RGP results in accumulation of a 75-kDa protein, one of the main outer membrane proteins of P. gingivalis. Immunoblot analysis of cell extracts shows that the double mutant produces two immunoreactive proteins with 45 kDA and 43 kDa molecular masses, representing the precursor and mature forms of fimbrilin.

Abstract Background Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. Methods Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. Results The results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. Conclusions In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non-opsonized Tannerella forsythia ATCC 43037 displaying a serum-resistant surface layer (S-layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of [ABSTRACT FROM AUTHOR]