Preliminary Study On The Regulation Mechanism Of RovA Protein Of Yersinia Enterocolitica Targeting To The Nucleus

The nucleus is the control center of cell genetics and metabolism,regulating the cell’s response to the outside environment,metabolism,growth and differentiation and other cellular activities.Studies have found that in the process of bacterial infection of host cells,individual bacterial-derived effector proteins can target the host cell nucleus and affect life activities such as gene transcription,RNA shearing,DNA repair,and chromatin recombination in the nucleus.This study aims to screen bacterial proteins that can be targeted to enter the host cell nucleus through nuclear localization signal peptides,and to make a preliminary exploration of its regulatory mechanism,providing a theoretical basis for further exploration of the pathogenic mechanism of pathogenic bacteria on the host cell infection.Firstly,seven potential nuclear proteins were screened by nuclear localization signal.These seven gene sequences were constructed into p EGFP-N1 vector with green fluorescent protein and transfected into 293T cells.The cell subposition of green fluorescent protein was observed under fluorescence inverted microscope.It was found that RovA protein from Yersinia enterocolitica could target the host cell nucleus.Subsequently,scanning laser confocal microscopy confirmed that RovA protein can target the host nucleus,and RovA_87-92AKRIKL is crucial for its nuclear.After confirming that RovA targets the host cell nucleus,Ch IP-seq（Chromatin Immunoprecipitation sequence）technology was used to initially explore which DNA the RovA protein binds to after entering the host cell nucleus.First,we constructed the p ECMV-3×Flag-rov A fusion protein expression vector,and used Ch IP-seq technology to screen 21 DNA fragments to bind to RovA protein,and further constructed the p ET-28a-rov A vector with His tag to purify RovA protein.Electrophoretic Mobility Shift Assay（EMSA）method was used to verify in vitro that three DNA sequences are bound to RovA protein.The genes corresponding to the start transcription sites closest to the summit of the three DNA fragment peak positions were ENSG00000201178.1,respectively.ENSG00000238129.1 and ENSG00000264970.1In addition,Useing Co-Immunoprecipitation（Co-IP）and mass spectrometry identification（MST）technology explored the protein that interacts with RovA in the cell,and preliminarily analyzed the mechanism of RovA entering the nucleus.The results of mass spectrometry identified seven nuclear transport proteins.Functional analysis and Blast comparison of the seven proteins were performed,and the four proteins P52292,Q14974,O00629 and Q5TFJ7 were purified after screening.Further,using surface plasmon resonance（SPR）and microscale thermophoresis（MST）technologies to verify that RovA protein interacts with four proteins,and their affinities were 2.5836×10^-6,5.2276×10^-7,3.4955×10^-6 and 1.218×10^-5.According to the affinity results,RovA protein binded to Q14974（Importinβ1）the strongest.It was preliminarily speculated that RovA might be combined with the nuclear localization signal peptide（AKRIKL）of the four transporters and targeted to enter the host cell nucleus.