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

Objective To evaluate the situation of mycoplasma contamination in cellcultures for laboratory research use, and to find a method to eliminate the mycoplasmacontamination in a short time. Method First, we designed the degenerate primer pairs foramplification of the16s-23s gene spacer region from mycoplasma genera. According to thelength of the amplified product, we preliminarily confirm which species the contaminant isresulted 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 ofantimicrobials to eliminate the infection in a short time. The efficiency of theanti-mycoplasma effect was assessed by real-time PCR using a method based on SybrGreen. The side effects of the antimicrobials on host cell were indicated by alterations incell cycle measured by Flow cytometry. The alterations caused by intracellular mycoplasmainfection was visualized by fluorescence immunostaining followed by confocal microscopy.Results1) The designed degenerate primers for Mycoplasma and Acholeplasma canamplify these two genera of mycoplasma in2hours of amplification reaction. The speciesof the contaminants can be assessed preliminarily according to the length of the productfragment. The golden standard for identification can also be conducted after theamplification by sequencing.2) Based on the analysis of ANOVA, we figured out threetypes of antimicrobial dr gs that can be used to eliminate mycoplasma infection in cellscultures. The dr gs used were…, which are designated as A, B and C, respectively. Theformulation 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 antimicrobialdr gs, 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 tonormal state after elimination of mycoplasma infection.4) This formulation did not causemuch prominent side effect to host cells. The cell cycle did not change significantlydetermined by Flow cytometry. Conclusion This innovation of a method for detection ofmycoplasma followed by specifically eliminating both extracellular and intracellularmycoplasma can be used in cell culture laboratory. Objective The author so ght to indentify a mycoplasmal virulence factor thatcan interact with host cellular molecules, by which mycoplasma may affect host tumor cellfunctions. And the author also endeavored to explore the molecular basis for thepathogenesis caused by mycoplasmal virulence factors. Method First, the author utilizedthe method based on bioinformatics to scrutinize mycoplasma genomes in searching forsome highly conserved sequences among mycoplasma species. Based on ample evidencesfrom papers about prokaryotic virulence factors, the author chose a small GTPase-likeprotein fragment (SGLP) of mycoplasma protein, chromosome partition protein Smc, as acandidate for research. The author cloned the SGLP sequence and inserted it into vectorsfor expression in E coli. and HeLa cells separately. The protein interactions between SGLPand Rac1/Stat3were assessed by GST pull-down assay and co-immunoprecipitation assay.The intracellular colocalization of SGLP with Rac1and Stat3was determined by confocalmicroscopy. Rac1activation was detected using Western blot analysis based on aGST-Pak1pull-down assay. Stat3phosphorylation was also detected using Western blotanalysis. The author transfected HeLa cells with dominant negative or constitutively activeRac1constructs to manipulate the cellular Rac1activity to investigate whetherSGLP-induced Stat3phosphorylation was dependent on Rac1activity. The effect of SGLPon HeLa cell migration was studied thro gh a Transwell assay, and the effect of SGLP onHeLa cell proliferation was also studied thro gh a BrdU incorporation assay determined byFlow cytometry. The ROS generation was tested by Flow cytometry after depletion of Rac1using siRNA. Chromatin immunoprecipitation assay was conducted to investigate whetherRab7was a Stat3target gene. Results1) SGLP can interacts with Rac1and Stat3;2)SGLP can upregulate Rac1activity and Stat3phosphorylation at705-tyrosine residues.3)SGLP-induced Stat3phosphorylation is dependent on Rac1activity.4) SGLP can promote tumor cell migration and has a pro-proliferative effect on tumor cells.4) studies aboutdownstream effects of SGLP imposed on host cells is preliminarily explored. ConclusionThe highly conserve mycoplasma protein fragment, SGLP, may induce activation of Rac1and Stat3thro gh interaction with Rac1and Stat3, which may be responsible for theobserve increase in tumor cell migration and proliferation. Studies about its ripples effectspave road for future studies in this field.