Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

Characterization of a novel potential peptide import system in Treponema denticola.

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. METHODS: To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. RESULTS: Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation. CONCLUSION: These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth.

Csa2 is a member of both the Candida albicans Rbt5 protein family and the Common in Fungal Extracellular Membranes (CFEM) protein superfamily. CFEM proteins are characterized by an internal domain containing eight equally spaced cysteine residues. Csa2 is involved in iron uptake from hemoglobin and heme proteins; however, its precise role is unclear. Here, we provide quantitative evidence of the involvement of Csa2 in the utilization of iron from human hemoglobin during C. albicans hyphal growth. The ability of the hyphal form of the wild-type (wt), a homozygote csa2Δ mutant, and a complemented strain of C. albicans to utilize hemoglobin as an iron source under iron-restricted conditions was examined through growth studies and a crystal violet-staining assay. Hemoglobin-binding activity was assessed indirectly using a hemoglobin-sensitized tube method. Although hyphal growth of the wt and csa2Δ/Δ::CSA2 strains was completely recovered when a high concentration of human hemoglobin was added to the iron-restricted culture medium, the recovery of the csa2Δ/Δ mutant was significantly diminished. Furthermore, hemoglobin binding was impaired in the csa2Δ/Δ mutant compared with the wt and csa2Δ/Δ::CSA2 strains, revealing that Csa2 is involved in the utilization of hemoglobin as an iron source by the hyphal form of C. albicans.

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis.

The signaling molecule Wnt regulates bone homeostasis through ß-catenin-dependent canonical and ß-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.

Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis.

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.