Study On Localization And Enzyme Activity Of GlpK And GlpO In Mycoplasma Genitalium And Cytotoxicity To Host Cell

BackgroundMycoplasma genitalium is the smallest prokaryotic microbial cells which is able to grow in non-life conditions. M. genitalium cause acute and chronic urethritis, vaginitis, endometritis, pelvic inflammatory disease, tubal infertility, and closely related to the death of patients as AIDS associated mycoplasma. Therefore, the research on the pathogenesis of M. genitalium will help to improve the understanding and treatment of M. genitalium related diseases.So far, mycoplasma has not found the typical cytolysin virulence such as toxins and invasion factors. This is mainly due to the small mycoplasma genome which only have evolved genetic material of maintain basic life functions. Thus, mycoplasma may play a pathogenic role through its structure and endogenous metabolites as toxic effectors. Recent studies showed that the pathogenicity of mycoplasma was closely related to their carbon metabolism and different mycoplasma used different carbon sources. More and more studies showed that many microorganisms have a complete set of glycerol utilization system and glycerin may involved a variety of pathophysiology and pathogenesis. Therefore, this study intends to choose two primary enzyme GlpK and GlpO of glycerol metabolism from M. genitalium, and to study their localization and enzyme activity, and to involve in the cytotoxicity of M. genitalium on host cell.ObjectiveGlpK protein and anti GST-GlpK antibody were purified which are to be applied to the enzyme activity of GlpK and GlpK involvement in the cytotoxicity of M. genitalium on host cell. GlpO protein and anti GST-GlpO antibody were purified which are to be applied to their localization and enzyme activity and GlpO involvement in the cytotoxicity of M. genitalium on host cell. Methods1、Glycerol kinase activity of M. genitalium GlpKProkaryotic expression vector pGEX6p-1/GlpK were structured and GST-GlpK fusion protein were expressed. BALB/c mice were immuned by purified protein and purified antiserum titers were detected. Enzyme activity of purified GlpK protein and the effects of temperature and pH on enzyme activity were detected.2、Glycerol-3-phosphate oxidese activity of M. genitalium GlpO Prokaryotic expression vector pGEX6p-1/GlpO were structured and GST-GlpO fusion protein expressed. BALB/c mice were immuned by purified protein and purified antiserum titers were detected. Enzyme activity of purified GlpO protein and the effects of temperature and pH on enzyme activity were detected. Cellular localization of GlpO were analyzed by the separation of membrane proteins and cytoplasmic proteins.3、Regulation of GlpK and GlpO on the cytotoxicity of host cellFunctional defect strains of GlpK and GlpO were electroporated and identified. Carbon growth curve of M. geniialium were drawed by wet weight method. GlpK and GlpO expression in glucose and glycerol were detected by Western blotting. M. genitalium produce H2O2, cytotoxicity of HeLa cells and oxidative stress in HeLa cells with glycerol were observed by combination of antibody inhibition assay. Results1、1524bp fragment of GlpK were amplified by PCR. pGEX6p-1/GlpK recombinant plasmid were constructed successfully and the soluble protein of83kDa were expressed successfully. The pAb titer was1:16000after purified protein immunized BALB/c mice. GST-GlpK fusion protein were cut and purified to GlpK protein of57kDa. Gycerol kinase activity of GlpK protein was1.73U/mL, and activity rised with the rise of temperature and pH..2、1152bp fragment of GlpO were amplified by PCR. pGEX6p-1/GlpO recombinant plasmid were constructed successfully and the soluble protein of68kDa were expressed successfully. The pAb titer was1:32000after purified protein immunized BALB/c mice. GST-GlpO fusion protein were cut and purified to GlpO protein of42kDa. Glycerol-3-phosphate oxidese activity of GlpO protein was4.26U/mL, and the activity was highest in40℃and pH7.3、GlpK and GlpO mutant strains were not screened. M. genitalium grew rapidly in the glucose while grew slow in the glycerol or without adding carbon. H2O2of glycerol groups and anti GST-GlpK antibody group were significantly higher than anti GST-GlpO antibody group and without glycerol groups. HeLa cell survival rate of glycerol groups and anti-GST-GlpK antibody group were significantly lower than anti-GST-GlpO antibody group and without glycerol groups. Plenty of H2O2and ROS in intracellular of HeLa cells were detected in glycerol group while were not detected in anti GST-GlpO antibody group and without glycerol groups. Conclusions1、Recombinant plasmid pGEX6p-1/GlpK and pGEX6p-1/GlpO were constructed successfully. GST-GlpK and GST-GlpO recombinant protein were expressed successfully.2、GlpK has glycerol kinase activity. GlpO is transmembrane protein and has Glycerol-3-phosphate Oxidese activity.3、GlpO involved in the cytotoxicity of Mg on host cell in glycerol metabolic.