Chlamydia Trachomatis CT225 Interacting With Vimentin From HeLa Cell

Chlamydiae is an obligate intracellular pathogen whose hosts include humans and animals.chlamydia trachomatis is one of the important human pathogens that can cause eye and genital infections.Chlamydia trachomatis strain（serum A-C）is the main cause of infectious blindness.The genitourinary system（serum D-K）can cause the most common sexually transmitted diseases in the world.However,STD lymphogranuloma（LGV）strain（serum L1-L3）can cause invasive urogenital or anorectal infections.Inclusion membrane protein（Incs）exist at the interface between the Inclusion and the cytoplasm of the host cell.Many Incs have been shown to play an important role in the chlamydia-host interaction.In this study,CT225 was found to belong to the CT223-CT229 gene cluster and has a transport function using bioinformatics analysis.Affinity purification mass spectrometry was used to screen CT225 interacting molecules from HeLa cells,and identification between interacting molecules was carried out with different methods.In the first part of the study,The CT225 interacting protein was screened.The GST-CT225 protein was expressed,purified by affinity chromatography,and fixed on beads as a bait protein.HeLa cells were cultured to a sufficient number,collected and lysed,and the lysate supernatant was used as a prey protein.They were mixed and incubated to enrich for proteins that may interact with GST-CT225.Proteins were initially searched by SDS-PAGE combined with silver staining.The components of the enriched protein were identified by mass spectrometry.As a result of mass spectrometry,the ligand molecules with higher scores included Alpha-actinin,Keratin,Vimentin,and Tubulin.According to bioinformatics and documents,it was suggested that Vimentin was choosed as candidate ligand interacting with CT225.In the second part of the study,candidate protein was verified.Firstly,the recombinant plasmid containing geneen coding the candidate protein（Vimentin）was constructed,and co-immuno precipitation was used to detect whether CT225 can ectopically interact with Vimentin in HeLa cells.HeLa cells were resuscitated,cultured,and transferred to 6-well plates.After growing to a certain number,the cells were transfected with the constructed recombinant plasmid with transfection reagent.After transfected,the cells were continue to be incubated in a cell incubator at37℃and 5%CO₂for 24 h.The cells were collected via trypsin digestion,washed with phosphate buffer solution（PBS）,centrifuged and lysed on ice,and the lysate supernatant was detected by Western Blot.There were two bands at 13kDa and 53kDa,corresponding to Myc-CT225 and Flag-VIM.Then,GST pull-down technology was used to verify exogenous interactions.The GST-CT225 prepared in advance was used as the bait protein.A Flag-tag-ligand plasmid was constructed and transfected into HeLa cell to over-expressthe prey protein.By Westen Blot,there were two bands at 39kDa and 53kDa,corresponding to GST-CT225 and Flag-VIM as expected.Finally,subcellular colocalization techniques were used to verify spatial interactions.HeLa cells were cultured on sterile slides in a 24-well plate to a sufficient number and transfected with plasmid.Cells were fixed with acetone at room temperature,blocked with serum,incubated with the primary antibody for 1 h,washed with PBS and incubated with the appropriate secondary antibody for 1 h.Nuclei were stained with DAPI.The slides were washed with PBS and placed on glass slides,and the samples were analyzed using a laser scanning confocal microscope.It was abserved that Myc-CT225 protein was green fluorescence and Flag-VIM protein was red fluorescence,and overlap of them showed yellow fluorescence at three levels.This experiment confirmed the interaction between CT225 and vimentin.This study provides a basis for the biological function study of CT225,and may be helpful for further research on the molecular mechanism of Chlamydia trachomatis growth and development.