Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

Effectiveness of customized master cone on apical sealing in various apical size of prepared root canals / 대한치과보존학회지

The purpose of this study is to evaluate the effectiveness of customized master cone on apical sealing in various apical size of prepared root canals, that is MAF(Master Apical File) and to know at which apical size the apical leakage is to be significantly reduced using customized master cone. 120 extracted single rooted premolars were divided into four groups according to their apical size(MAF), #30, 40, 50 and 60. And then, each group was subdivided into three in accordance with three obturation methods, lateral condensation with standardized master cone, lateral condensation with chloroform-dipped customized master cone, and continuous wave of obturation technique. Resorcinol-formaldehyde resin was used for the microleakage test of this study. Teeth were sectioned horizontally at 1.5mm(Level 1), 2.5mm(Level 2), and 3.5mm(Level 3) from the anatomical root apex using low speed microtome. All sections were examined under x40 magnification with a stereomicroscope, photographed, and then scanned. With the scanned images, resin-infiltrated area presenting the microleakage was calculated using SigmaScan/Image, and the ratio of leakage to the total root canal area of each group was analyzed statistically(one way ANOVA). The results were as follows; 1. In groups of MAF #30, there was no significant difference of mean leakage ratio among three obturation methods at all three levels. 2. In groups of MAF #40, the group using lateral condensation with customized master cone had the lowest mean leakage ratio at all three levels, but there was no significant difference among three obturation techniques. 3. In groups of MAF #50, the mean leakage ratio of the group using lateral condensation with standard master cone was the highest among those of three obturation techniques at level 1, and this difference was statistically significant(p<0.05). 4. In groups of MAF #60, the groups using lateral condensation with standard master cone had also the highest mean leakage ratio at all levels, but there was no significant difference at level 1 and 2. At level 3, the leakage of the group using lateral condensation with standard master cone was significantly higher than that of the group using continuous wave of obturation(p<0.05). The results of this study suggested that the obturation method using customized master cone or the continuous wave of obturation is more effective for apical sealing than that using standardized master cone when MAF is larger than #50.