Molecular and biological characterization of histidine triad protein in group A streptococci.

Four Streptococcus pneumoniae genes, phtA, phtB, phtD, and phtE, as well as the slr gene of group A streptococci (GAS), encode proteins with a histidine triad motif (HxxHxH). Pht proteins function as protective antigens against S. pneumoniae infection. A search of the GAS genome database identified a novel protein, HtpA, possessing five histidine triad motifs. The htpA gene was shown to encode a 92.5-kDa protein located downstream of the fbaA and lbp genes, while Western blot analyses revealed that HtpA protein was expressed on the cell surfaces of all group A, B, C, and G streptococcal isolates tested. Immunization of mice with rHtpA induced antigen-specific antibody production and was effective after a single immunization, with antibody titers remaining constant for at least 84days. In addition, HtpA-immunized mice survived after challenge with GAS strains isolated from patients with streptococcal toxic shock syndrome for significantly longer periods than sham-immunized mice. In that experiment, the HtpA-specific antibody was effectively induced by a single immunization and the specific antibody titer remained constant for at least 84days. These results indicate that the novel histidine triad protein HtpA is a candidate vaccine for GAS infection.

Site-specific development of periodontal disease is associated with increased levels of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in subgingival plaque.

BACKGROUND: Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (previously T. forsythensis), which are regarded as the principal periodontopathogenic bacteria, exist as a consortium in subgingival biofilms. We aimed to examine quantitative relationships between P. gingivalis, T. denticola, and T. forsythia in subgingival biofilms and the relationship between the quantity and prevalence of these three bacteria and site-specific periodontal health. METHODS: This study was cross-sectional. The study population consisted of 35 adult subjects who visited the Kyushu Dental College Hospital. Plaque samples were collected from 105 periodontal pocket sites. Quantitative analyses of each of the three periodontopathogenic bacteria were performed using real-time polymerase chain reaction with species-specific primers and hybridization probes. RESULTS: The plaque samples were divided into four groups based on the presence or absence of a periodontal pocket (probing depth [PD] > or =4 mm) and bleeding on probing (BOP), regardless of attachment loss. The proportions of all three target bacteria detected in samples from sites of periodontal disease (with PD and BOP) were markedly higher than those in the other sample groups. Cell numbers of P. gingivalis, T. denticola, and T. forsythia in the subgingival plaque of each sampling site were significantly mutually correlated and were increased in the plaque of sites of periodontal disease with PD > or =4 mm and BOP. CONCLUSION: The symbiotic effects of P. gingivalis, T. denticola, and T. forsythia, which coaggregate and exist concomitantly in subgingival biofilms, may be associated with the local development of periodontitis.

Preparation of rat gingival mitochondria with an improved isolation method.

In order to establish a method of obtaining rat gingival mitochondria (Mt), Mt fractions were prepared in various combinations of homogenizing time with collagenase concentration. Rat gingival tissues were excised, minced, treated with collagenase, homogenized, and subjected to differential centrifugation rates. Both the respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio of the Mt fraction prepared in a combination of 40, 50, or 60 sec homogenization with collagenase in a concentration range of 0.115%-0.130% (w/v) were measured. The values for the RCR and ADP/O ratio of the Mt fraction obtained in an optimal condition was 1.80 ± 0.05 and 1.65 ± 0.03, respectively. These results suggest that Mt of fairly high quality can be obtained through this refined combination of the homogenizing time and collagenase concentration.

Relationship between endothelin-1 and interleukin-1beta in inflamed periodontal tissues.

We recently demonstrated that endothelin-1 (ET-1) was strongly expressed in inflamed gingival tissues, but the biological role of ET-1 in gingival tissue remains unknown. This study focused on the relationship between ET-1 and interleukin-1beta (IL-1beta), an important cytokine during the periodontal inflammatory process. We determined the protein levels of ET-1 and IL-1beta in gingival tissues from patients and examined whether ET-1 could regulate the expression of the IL-1beta gene and protein in oral epithelial cells and fibroblasts in vitro. There was a significant correlation between the levels of ET-1 and IL-1beta in 26 gingival tissues, as determined by ELISA. Following the confirmation of two specific ET-1 receptors (ETA and ETB receptors) on the cultured cells, the effects of ET-1 stimulation on IL-1beta mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. The IL-1beta mRNA and protein levels were enhanced by ET-1 stimulation in a dose-dependent manner, and the enhancement of IL-1beta was inhibited by ETA or ETB receptor antagonists. These findings indicate that ET-1 is involved in the regulation of IL-1beta expression in gingival tissues and suggest that ET-1 signaling to the cells may be a therapeutic target for treating IL-1beta-dependent inflammatory responses.