Construction And Application Of TEM-1?-lactamase Based Protein Translocation Assay For Identification Of Anaplasma Phagocytophilum Type ? Secretion System Effector Proteins

Research background and objective:Anaplasma phagocytophilum(Ap)is a Gram-negative obligate intracellular bacteria,it causes human granulocytic anaplasmosis(HGA)by invading human peripheral blood neutrophils.The Type ? secretion system(T4SS)is an important virulence factor,which causes physiological dysfunction of host cell by transporting various effector proteins into the host cell,leading to disease.This study attempts to identify new effector molecules by constructing the Ap T4SS effector molecule translocation reporting system and to elucidate the pathogenic mechanism of this pathogen by biological analysis of the identified effector proteins.Research content and methods:1.Construction of TEM-1 ?-lactamase based protein translocation assay for identification of Ap T4SS effector proteins.This reporter system consists of a donor bacterium(E.coli SM10 ?pir strain)and a recipient eukaryotic cell(Chinese hamster oocyte,CHO K1)and is divided into three functional parts,including transport channels,coupling proteins and a reporter gene.Transport channels are provided by E.coli SM10?pir strain(this strain carries RP4 conjugation plasmid);the coupling protein is a chimeric protein(TraG-VirD4)formed by fusion of the transmembrane end of the couple protein TraG amino acid encoded by the RP4 plasmid and the Ap VirD4 cytoplasmic region;the reporter gene is one encoding TEM-1 ?-lactamase-AP effector protein.If the reporter gene fused with a gene encoding an Ap effector protein,it will be recognized by the TraG-VirD4 coupling protein and transported from the E.coli SM10 Xpir to the CHO K1 cell via the transport channel.In the recipient cells,the reporter gene-encoded TEM-1 ?-lactamase cleaves the preloaded CCF2-AM,which converts from green fluorescence to blue fluorescence.The emitting light indicates that the Ap protein fused to the reporter gene is a T4SS effector protein.2.Application of TEM-1 ?-lactamase based protein translocation assay for identification of Ap T4SS effector proteins.Ats-1,a known AP T4SS effector protein and the potential effector proteins predicted by bioinformatics,which their genes were fused with TEM-1 ?-lactamase gene,and verified by the constructed reporter system.3.Localization of APH0215 in transfected cells.HeLa cells were transfected with the APH0215-GFP expressing plasmid,and the organelles were immunolabeled with antibodies to observe the location of APH0215 in the transfected cells.Research results:1.We developed a TEM-1 ?-lactamase based translocation assay for identification of Ap T4SS effector proteins.2.This reporter system can transport Ats-1 and the potential effector protein APH0215 to CHO K1,and fail to transport the potential effector protein APH0756 and the carboxy-terminal knockout APH0215 to CHO K1.It indicated that APH0215 is an Ap T4SS effector protein,and the carboxy-terminus of APH0215 plays an important role in its transport.3.APH0215 colocalizes with the trans-Golgi network marker protein TGN38,indicating that APH0215 is localized to the trans-Golgi tube network of the host cell.Conclusion:1.This study successfully developed a TEM-1 ?-lactamase based translocation assay for identification of Ap T4SS effector proteins,which provides a research tool for the identification of other Ap T4SS effector proteins in the future.2.In this study,APH0215 was identified as an Ap T4SS effector protein by the developed reporter system.3.Further investigation of the APH0215 protein revealed that the APH0215 C-terminal sequence is required for its transport,and APH0215 is localized to the trans-Golgi network in transfected cells.It provides hints for exploring the biological function of APH0215 in infected cells.