Hemin-binding protein 35 (HBP35) plays an important role in bacteria-mammalian cells interactions in Porphyromonas gingivalis.

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.

Role of the hemin-binding protein 35 (HBP35) of Porphyromonas gingivalis in coaggregation.

Hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis is one of the outer membrane proteins and has been reported to be a non-fimbrial coaggregation factor. In this study, a P. gingivalis HBP35-deficient mutant (MD774) was constructed from wild-type strain FDC381 by insertion mutagenesis in order to provide a better understanding of this protein's role in coaggregation. The intact cells and vesicles in FDC381 were found to have strong aggregation activities with Gram-positive bacteria. But neither the vesicles nor the intact cells showed aggregation activity in MD774. In addition, MD774 reduced autoaggregation activity. Immunoblot analysis of MD774 showed the presence of a non-maturated 45-kDa fimbrillin protein. Electron microscopy showed that the MD774 had no long fimbriae on the cell surface. Arg- and Lys-gingipain activity in MD774 was significantly decreased, compared with FDC381. Real-time RT-PCR demonstrated a significant reduction in the expression of gingipain-associated genes rgpA, rgpB, and kgp. In conclusion, we suggest that the reduction in coaggregation was caused by the combined reduction of a variety of molecules, including HBP35, gingipains, and fimbriae. Our results suggest that the HBP35 protein directly influences not only coaggregation as an adhesion molecule but also indirectly influences the expression of other coaggregation factors.

Gene expression profiling and characterization under hemin limitation in Porphyromonas gingivalis.

Hemin is an important nutrient for Porphyromonas gingivalis growth and pathogenicity. We examined the gene expression profile of P. gingivalis, including genes involved in its pathogenicity, at various growth stages under hemin-standard and limited conditions by using a custom-made microarray. The transcription of many genes decreased after late-log and mid-log phases under hemin-standard and limited conditions, respectively. We focused on two groups of genes while comparing gene expression profiles under hemin-standard and limited conditions by gene tree analysis. Genes belonging to group A maintained high transcriptional levels, whereas genes in group B were expressed at low levels under standard hemin conditions. However, group B genes increased remarkably under hemin-limited conditions. Groups A and B contained genes involved in regulatory functions and protein fate, respectively. Genes related to energy metabolism, transport, and protein binding were present in both groups. Our results suggest that P. gingivalis experienced severe stress under hemin-limited conditions, and growth phase-dependent changes in transcription levels were observed for many genes. Moreover, increased expression of genes involved in energy metabolism suggests that hemin is related not only to pathogenicity, but also energy metabolism.

Inhibition of hemagglutinating activity by monoclonal antibody against Porphyromonas gingivalis 40-kDa outer membrane protein.

Periodontitis is a chronic inflammatory disease of periodontal tissues that results in alveolar bone loss, and Porphyromonas gingivalis, which has a high hemagglutinating activity, has been implicated as an important pathogen in the development of periodontitis. This bacterium has a high hemagglutinating activity. We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P. gingivalis 381. Although recombinant (r) 40-kDa OMP itself did not show hemagglutinating activity, its polymeric form, constructed with a cross-linking reagent, significantly expressed that activity. Furthermore, an affinity-purified antibody against r40-kDa OMP inhibited the hemagglutinating activity of P. gingivalis vesicles. In the present study, in order to clarify the pathological role of 40-kDa OMP and develop passive immunotherapy, we examined the inhibitory effect of monoclonal antibodies (MAbs) against r40-kDa OMP on the hemagglutinating activity of P. gingivalis vesicles. The MAbs reacted with r40-kDa OMP, the outer membrane fraction, vesicles, and P. gingivalis cell extracts, and significantly inhibited the hemagglutinating activities of the polymeric r40-kDa OMP as well as of P. gingivalis vesicles. These findings suggest that MAbs against 40-kDa OMP may be useful for the development of passive immunotherapy and for assessing treatment for periodontal diseases caused by P. gingivalis infection.

Monitoring of dnaK gene expression in Porphyromonas gingivalis by oxygen stress using DNA microarray.

Porphyromonas gingivalis, a Gram-negative anaerobe associated with adult periodontitis, expresses numerous potential virulence factors. dnaK, a member of the heat shock protein family, functions as a molecular chaperone and plays a role in microbial pathogenicity. However, little is known regarding its gene expression caused by oxygen stress in P. gingivalis. In the present study, a custom-made DNA microarray was designed and used to monitor dnaK gene expression in P. gingivalis caused by oxygen stress. The results demonstrated that dnaK mRNA was up-regulated in a short time, and the DNA microarray results were confirmed by real-time polymerase chain reaction analysis. These findings suggest that oxygen stress stimulates gene expression of dnaK and may have a relationship to the aerotolerance activity of this organism as well as its expression of pathogenesis.

Age-related changes in IGF-1 expression in submandibular glands of senescence-accelerated mice.

Saliva is known to play important roles in such functions as swallowing, mastication, speech, and taste. Furthermore, salivary glands synthesize and secrete a number of growth factors involved in cell/tissue homeostasis. It has been demonstrated that IGF-1, which is structurally analogous to insulin, has been shown to be expressed in mouse submandibular glands, and that IGF-1 stimulates DNA synthesis, amino acid uptake, protein synthesis, and glucose transport in various cells. Diminished function of the salivary glands is thought to lead to increased dental caries and periodontal diseases, which are commonly associated with aging. However, very little is known regarding the effects of age on IGF-1 expression in submandibular glands. The senescence-accelerated mouse (SAM), an experimental murine model of accelerated aging, has been extensively used to examine the mechanisms responsible for aging. In the present study, IGF-1 production and mRNA levels in the submandibular glands of SAM-P1 mice were examined. IGF-1 levels were determined by radioimmunoassay and IGF-1 mRNA levels by semi-quantitative RT-PCR. We found that IGF-1 protein levels in homogenates and IGF-1 mRNA levels decreased with age in SAMP1 mice. These findings suggest that IGF-1 synthesis in submandibular glands decreases with aging, and this may result in lower levels of cellular proliferation, regeneration and wound healing in aged oral tissues.

Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.