iProXProteomicsXiaolu XiongHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0004207000xiongxiaolu624@163.comBeijing Institute of Microbiology and EpidemiologyiProXCoxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91-120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.A protein-protein interaction map reveals that the Coxiella burnetii effector CirB inhibits host proteasome activity.Fu Mengjiao M, Liu Yuchen Y, Wang Guannan G, Wang Peng P, Zhang Jianing J, Chen Chen C, Zhao Mingliang M, Zhang Shan S, Jiao Jun J, Ouyang Xuan X, Yu Yonghui Y, Wen Bohai B, He Chengzhi C, Wang Jian J, Zhou Dongsheng D, Xiong Xiaolu XfalseAP-MS Raw data of C. burnetii-host PPIsFifty-three Coxiella effectors were expressed in HEK-293T cells, and the Strep-tagged Coxiella effectors and the corresponding interacting complex were purified for MS analysis. The LC-MS/MS analyses were performed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled online to a nanoflow LC system (EASY-nLC 1000 or 1200, Thermo Fisher Scientific). The MS data acquired were searched against the Uniprot Swiss-Prot Human database with the addition of C. burnetii proteins by Proteome Discoverer software (version 2.4, Thermo Fisher Scientific, Bremen, Germany). Only the two biological replicates with the closest protein numbers and the highest PCC were retained for interaction identification. The negative controls, which consisted of only 200-600 proteins, were discarded altogether. Instead, we downloaded the contaminant repository of all 28 groups of HEK293T cells from the CRAPome, and took the two with the highest spectral count as negative controls, to obtain the high-confidence interactions. Finally, 53 effectors and 2 control was used for SAINTexpress analysis with the cut-off BFDR<0.01. The top 5th percentiles proteins ranked by occurrence rate in CRAPome all human data (443 genes) were removed from pathogen-host interactions identified by SAINTexpress.Fri Mar 18 00:00:00 GMT 2022PXD032380960635816513