Bhatti M, MacRobert A, Meghji S, Henderson B, Wilson M
Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, University of London, UK.
Photochem Photobiol. 1998 Sep;68(3):370-6.
The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a heliumneon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and signlet oxygen scavenger L-tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.
本研究的目的是确定光敏剂甲苯胺蓝O（TBO）在牙龈卟啉单胞菌内的分布情况以及该生物体致死性光致敏作用的可能机制。通过用氚标记的TBO（3H-TBO）孵育牙龈卟啉单胞菌，并将细胞分离为外膜（OM）、质膜（PM）、细胞质蛋白、其他细胞质成分和DNA来确定TBO的分布。发现各组分中TBO的百分比分别为86.7%、5.4%、1.9%、5.7%和0.3%。通过使用重水（D2O）（可延长单线态氧的寿命）以及自由基和单线态氧清除剂L-色氨酸，研究了细胞毒性物质在氦氖（HeNe）激光和TBO诱导的致死性光致敏作用中的参与情况。在光剂量为0.44 J（能量密度 = 0.22 J/cm2）时，分别在D2O和H2O（盐溶液）存在的情况下，细胞数量减少了9.0个对数10和2个对数10，这表明单线态氧参与其中。随着L-色氨酸浓度的增加，杀菌效果降低。通过测量色氨酸荧光来确定致死性光致敏对全细胞蛋白的影响，使用4.3 J（能量密度 = 4.3 J/cm2）的光时色氨酸荧光降低了30%。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳确定对OM和PM蛋白的影响。与对照组相比，有证据表明几种PM蛋白和OM蛋白的分子量发生了变化。有证据表明从辐照细胞中获得的DNA受到了损伤。扫描电子显微镜研究表明，致敏后再暴露于激光的牙龈卟啉单胞菌细胞会发生共聚集。这些结果表明，牙龈卟啉单胞菌的致死性光致敏作用可能涉及OM和/或PM蛋白的变化以及由单线态氧介导的DNA损伤。
