A Mycoplasmal Small GTPase-like Protein Fragment(SGLP) Interacts With Rac1and Stat3:a Functional Analysis

Part one:A method for detection of mycoplasma contamination in cell cultures followed by eradication of contaminantsObjective To evaluate the situation of mycoplasma contamination in cell cultures for laboratory research use, and to find a method to eliminate the mycoplasma contamination in a short time. Method First, we designed the degenerate primer pairs for amplification of the16s-23s gene spacer region from mycoplasma genera. According to the length of the amplified product, we preliminarily confirm which species the contaminant is resulted from. Then, based on the identified mycoplasma species and titers of the infection, we utilize ANOVA analysis to examine an appropriate formulation of three classes of antimicrobials to eliminate the infection in a short time. The efficiency of the anti-mycoplasma effect was assessed by real-time PCR using a method based on Sybr Green. The side effects of the antimicrobials on host cell were indicated by alterations in cell cycle measured by Flow cytometry. The alterations caused by intracellular mycoplasma infection was visualized by fluorescence immunostaining followed by confocal microscopy. Results1) The designed degenerate primers for Mycoplasma and Acholeplasma can amplify these two genera of mycoplasma in2hours of amplification reaction. The species of the contaminants can be assessed preliminarily according to the length of the product fragment. The golden standard for identification can also be conducted after the amplification by sequencing.2) Based on the analysis of ANOVA, we figured out three types of antimicrobial drμgs that can be used to eliminate mycoplasma infection in cells cultures. The drμgs used were..., which are designated as A, B and C, respectively. The formulation of the regime are:solution A was0.5%-1.5%m/v resolved with0.01M PBS; solution B was0.5%-1.5%m/v resolved with0.01M PBS; solution C was0.25%-0.75%m/v resolved with0.01M PBS.3) After administration of this formulation of antimicrobial drμgs, the intracellular mycoplasma as well as the extracellular mycoplasma can both be eliminated. The autophagy flux which are characterized by the LC3-Ⅱ puncta recovered to normal state after elimination of mycoplasma infection.4) This formulation did not cause much prominent side effect to host cells. The cell cycle did not change significantly determined by Flow cytometry. Conclusion This innovation of a method for detection of mycoplasma followed by specifically eliminating both extracellular and intracellular mycoplasma can be used in cell culture laboratory. Part two:Mycoplasmal small GTPase-like protein fragment interacts with host Racl and Stat3:a functional analysisObjective The author soμght to indentify a mycoplasmal virulence factor that can interact with host cellular molecules, by which mycoplasma may affect host tumor cell functions. And the author also endeavored to explore the molecular basis for the pathogenesis caused by mycoplasmal virulence factors. Method First, the author utilized the method based on bioinformatics to scrutinize mycoplasma genomes in searching for some highly conserved sequences among mycoplasma species. Based on ample evidences from papers about prokaryotic virulence factors, the author chose a small GTPase-like protein fragment (SGLP) of mycoplasma protein, chromosome partition protein Smc, as a candidate for research. The author cloned the SGLP sequence and inserted it into vectors for expression in E coli. and HeLa cells separately. The protein interactions between SGLP and Racl/Stat3were assessed by GST pull-down assay and co-immunoprecipitation assay. The intracellular colocalization of SGLP with Rac1and Stat3was determined by confocal microscopy. Racl activation was detected using Western blot analysis based on a GST-Pakl pull-down assay. Stat3phosphorylation was also detected using Western blot analysis. The author transfected HeLa cells with dominant negative or constitutively active Rac1constructs to manipulate the cellular Rac1activity to investigate whether SGLP-induced Stat3phosphorylation was dependent on Racl activity. The effect of SGLP on HeLa cell migration was studied throμgh a Transwell assay, and the effect of SGLP on HeLa cell proliferation was also studied throμgh a BrdU incorporation assay determined by Flow cytometry. The ROS generation was tested by Flow cytometry after depletion of Racl using siRNA. Chromatin immunoprecipitation assay was conducted to investigate whether Rab7was a Stat3target gene. Results1) SGLP can interacts with Racl and Stat3;2) SGLP can upregulate Racl activity and Stat3phosphorylation at705-tyrosine residues.3) SGLP-induced Stat3phosphorylation is dependent on Racl activity.4) SGLP can promote tumor cell migration and has a pro-proliferative effect on tumor cells.4) studies about downstream effects of SGLP imposed on host cells is preliminarily explored. Conclusion The highly conserve mycoplasma protein fragment, SGLP, may induce activation of Rac1and Stat3thro⒚gh interaction with Rac1and Stat3, which may be responsible for the observe increase in tumor cell migration and proliferation. Studies about its ripples effects pave road for future studies in this field.