Brucella effector protein BspJ inhibits the NF-κB signaling pathway by targeting the Zfp740 promoter region to promote bacterial survival within macrophages

Abstract

Brucella species are responsible for the zoonotic disease brucellosis and utilize the type IV secretion system (T4SS) to introduce effector proteins into host cells, thereby modulating immune responses and sustaining intracellular infection. Notably, BspJ was identified as the first Brucella nucleomodulin. Although our previous studies demonstrated its influence on host inflammatory responses and bacterial colonization, the precise molecular mechanism remains elusive. In this study, we report novel findings that BspJ directly targets the host transcription factor Zfp740 promoter region through three critical amino acid residues (His66, Arg87, and Arg96), leading to the upregulation of Zfp740 expression. This upregulation subsequently blocks IκBα degradation and inhibits P65 phosphorylation and nuclear translocation, thereby obstructing the activation of the NF-κB signaling pathway and ultimately diminishing the production of proinflammatory cytokines. Moreover, both the BspJ deletion strain and strains with mutations in key amino acids significantly enhanced the inflammatory response and compromised the intracellular survival capacity of Brucella. In conclusion, our study systematically elucidates a novel regulatory axis, BspJ-Zfp740-P65/NF-κB, and reveals a new mechanism by which Brucella directly interferes with host transcriptional programs through a nucleus-targeting effector protein to suppress inflammation and establish persistent infection. These findings provide a significant theoretical foundation for understanding bacteria-mediated immune evasion and chronic infection and introduce new research avenues for the development of host-directed anti-Brucella strategies.

Keywords

Brucella / Inflammatory response / BspJ / Zinc finger protein / NF-κB

References

[1] Benjamin R, Banerjee A, Balakrishnan K, Sivangala R, Gaddam S, Banerjee S. Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation. PLoS ONE, 2014, 9(8): 11

[2] Bondar A. Graphs of Hydrogen-Bond Networks to Dissect Protein Conformational Dynamics. The Journal of Physical Chemistry B, 2022, 126(22): 3973-3984

[3] Bourg G, Sube R, O'callaghan D, Patey G. Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems. Journal of Bacteriology, 2009, 191(9): 2985-2992

[4] Byndloss M, Tsolis R. Lewin HA, Roberts RM. "Brucella spp. virulence factors and immunity,". Annual review of animal biosciences, 2016, Palo Alto, Annual Reviews: 111-127Vol 4

[5] Catrysse L, Vereecke L, Beyaert R, Van Loo G. A20 in inflammation and autoimmunity. Trends in Immunology, 2014, 35(1): 22-31

[6] Celli J. The Intracellular Life Cycle of Brucella spp. Microbiol. Spectr., 2019, 7(2): 11

[7] Celli J, De Chastellier C, Franchini D, Pizarro-Cerda J, Moreno E, Gorvel A. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. Journal of Experimental Medicine, 2003, 198(4): 545-556

[8] Comerci D, Martínez-Lorenzo M, Sieira R, Gorvel J, Ugalde R. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cellular Microbiology, 2001, 3(3): 159-168

[9] Degos C, Hysenaj L, Gonzalez-Espinoza G, Arce-Gorvel V, Gagnaire A, Papadopoulos A, Pasquevich K, Méresse S, Cassataro J, Mémet S, et al. . Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favors bacterial persistence in vivo. Cellular Microbiology, 2020, 22(4): 16

[10] Djokic V, Freddi L, De Massis F, Lahti E, Van Den Esker M, Whatmore A, Haughey A, Ferreira A, Garofolo G, Melzer F, et al. . The emergence of Brucella canis as a public health threat in Europe: What we know and what we need to learn. Emerg. Microbes Infect., 2023, 12(2): 27

[11] Ecco G, Imbeault M, Trono D. KRAB Zinc Finger Proteins. Development, 2017, 144(15): 2719-2729

[12] Escoll P, Buchrieser C. Metabolic reprogramming of host cells upon bacterial infection: Why shift to a Warburg-like metabolism?. FEBS Journal, 2018, 285(12): 2146-2160

[13] Guo, C.Y., X.J. Zhu, Y.Q. Zhang, X.W. Peng, W.F. Sun, K.X. Guo, J.D. Zhang, X.Q. Zhang, J.P. Li, Z.F. Liu. 2025. Evaluation of the genetic profiles of Brucella with different biotypes using MLVA and MLST techniques Abstract Animal Diseases 5(1). https://doi.org/10.1186/s44149-025-00181-y.

[14] Guo X, Zeng H, Li M, Xiao Y, Gu G, Song Z, Shuai X, Guo J, Huang Q, Zhou B, et al. . The mechanism of chronic intracellular infection with Brucella spp. Frontiers in Cellular and Infection Microbiology, 2023, 13: 11

[15] Hansen G, Markesich D, Burnett M, Zhu Q, Dionne K, Richter L, Finnell R, Sands A, Zambrowicz B, Abuin A. Large-scale gene trapping in C57BL/6 N mouse embryonic stem cells. Genome Research, 2008, 18(10): 1670-1679

[16] Hayden M, Ghosh S. NF-κB in immunobiology. Cell Research, 2011, 21(2): 223-244

[17] Janczarek M. The Ros/MucR Zinc-Finger Protein Family in Bacteria: Structure and Functions. International Journal of Molecular Sciences, 2022, 23(24): 21

[18] Lawrence T. The Nuclear Factor NF-κB Pathway in Inflammation. Cold Spring Harbor Perspect. Biol., 2009, 1(6): 10

[19] Li C, Wang J, Sun W, Liu X, Wang J, Peng Q. The Brucella Effector BspI Suppresses Inflammation via Inhibition of IRE1 Kinase Activity during Brucella Infection. The Journal of Immunology, 2022, 209(3): 488-497

[20] López-Santiago R, Sánchez-Argáez A, De Alba-Núñez L, Baltierra-Uribe S, Moreno-Lafont M. Immune Response to Mucosal Brucella Infection. Frontiers in Immunology, 2019, 10: 21

[21] Luizet J, Raymond J, Lacerda T, Barbieux E, Kambarev S, Bonici M, Lembo F, Willemart K, Borg J, Celli J, et al. . The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells. Proc. Natl. Acad. Sci. u. s. a., 2021, 118(32): 10

[22] Ma Z, Li R, Hu R, Deng X, Xu Y, Zheng W, Yi J, Wang Y, Chen C. Brucella abortus BspJ Is a Nucleomodulin That Inhibits Macrophage Apoptosis and Promotes Intracellular Survival of Brucella. Frontiers in Microbiology, 2020, 11: 16

[23] Ma Z, Yu S, Cheng K, Miao Y, Xu Y, Hu R, Zheng W, Yi J, Zhang H, Li R, et al. . Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host. Journal of Veterinary Science, 2022, 23(1): 15

[24] Malik F, Guo J. Insights into protein-DNA interactions from hydrogen bond energy-based comparative protein-ligand analyses. Proteins, 2022, 90(6): 1303-1314

[25] May M, D'acquisto F, Madge L, Glöckner J, Pober J, Ghosh S. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science, 2000, 289(5484): 1550-1554

[26] Miller C, Smith E, Cundiff J, Knodler L, Blackburn J, Lupashin V, Celli J. A Brucella type IV effector targets the COG tethering complex to remodel host secretory traffic and promote intracellular replication. Cell Host & Microbe, 2017, 22(3): 317

[27] Mittal R, Peak-Chew S, Sade R, Vallis Y, Mcmahon H. The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate. Journal of Biological Chemistry, 2010, 285(26): 19927-19934

[28] Mussbacher M, Derler M, Basílio J, Schmid J. NF-κB in monocytes and macrophages - an inflammatory master regulator in multitalented immune cells. Frontiers in Immunology, 2023, 14: 13

[29] Myeni S, Child R, Ng T, Kupko J, Wehrly T, Porcella S, Knodler L, Celli J. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins. PLoS Pathogens, 2013, 9(8): 18

[30] Niebuhr K, Giuriato S, Pedron T, Philpott D, Gaits F, Sable J, Sheetz M, Parsot C, Sansonetti P, Payrastre B. Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology. The EMBO Journal, 2002, 21(19): 5069-5078

[31] Poladian N, Orujyan D, Narinyan W, Oganyan AK, Navasardyan I, Velpuri P, Chorbajian A, Venketaraman V. Role of NF-κB during Mycobacterium tuberculosis infection. International Journal of Molecular Sciences, 2023

[32] Seleem M, Boyle S, Sriranganathan N. Brucella: A pathogen without classic virulence genes. Veterinary Microbiology, 2008, 129(1–2): 1-14

[33] Sivanesan D, Hancock M, Giraldo A, Baron C. Quantitative Analysis of VirB8-VirB9-VirB10 Interactions Provides a Dynamic Model of Type IV Secretion System Core Complex Assembly. Biochemistry, 2010, 49(21): 4483-4493

[34] Sun M, Ju J, Ding Y, Zhao C, Tian C. The signaling pathways regulated by KRAB zinc-finger proteins in cancer. Biochim. Biophys. Acta-Rev. Cancer, 2022, 1877(3): 12

[35] Tsai R, Meng L. Nucleostemin: A latecomer with new tricks. International Journal of Biochemistry & Cell Biology, 2009, 41(11): 2122-2124

[36] Wang Y, Zhan Q, Wang X, Li P, Liu S, Gao G, Gao P. Insights into catalysis and regulation of noncanonical ubiquitination and deubiquitination by bacterial deamidase effectors. Nature Communications, 2020

[37] Wang Z, Wang G, Wang Y, Liu Q, Li H, Xie P, Wang Z. Omp31 of Brucella Inhibits NF-κB p65 Signaling Pathway by Inducing Autophagy in BV-2 Microglia. Neurochemical Research, 2021, 46(12): 3264-3272

[38] Watarai M, Makino S, Fujii Y, Okamoto K, Shirahata T. Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cellular Microbiology, 2002, 4(6): 341-355

[39] Yang J, Liu M, Liu J, Liu B, He C, Chen Z. Proteomic Analysis of Stationary Growth Stage Adaptation and Nutritional Deficiency Response of Brucella abortus. Frontiers in Microbiology, 2020, 11: 12

[40] Yokoyama S, Ito Y, Ueno-Kudoh H, Shimizu H, Uchibe K, Albini S, Mitsuoka K, Miyaki S, Kiso M, Nagai A, et al. . A Systems Approach Reveals that the Myogenesis Genome Network Is Regulated by the Transcriptional Repressor RP58. Developmental Cell, 2009, 17(6): 836-848

[41] Yuan Q, Carle A, Gao C, Sivanesan D, Aly K, Höppner C, Krall L, Domke N, Baron C. Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems. Journal of Biological Chemistry, 2005, 280(28): 26349-26359

[42] Zai X, Yang Q, Yin Y, Li R, Qian M, Zhao T, Li Y, Zhang J, Fu L, Xu J, et al. . Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses. Frontiers in Microbiology, 2017, 8: 15

[43] Zhang H, Xing Y, Zhu J, Wu S, Gao J, Wang Y, Yu Z, Li A, Li Y, Chen X, et al. . The Brucella Effector Protein BspF Crotonylates TRIM38 to Inhibit NF-κB and MAPK Signaling Pathway. International Journal of Molecular Sciences, 2025, 26(8): 16

[44] Zheng C, Wu H, Jin S, Li D, Tan S, Zhu X. Roles of Myc-associated zinc finger protein in malignant tumors. Asia-Pac. J. Clin. Oncol., 2022, 18(6): 506-514

[45] Zhou Y, Bu Z, Qian J, Cheng Y, Qiao L, Yang S, Cheng S, Wang X, Ren L, Yang Y. Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO. BMC Veterinary Research, 2021

Funding

National Natural Science Foundation of China(Grant No. 32060789)

Xinjiang Uygur Autonomous Region "Tianchi Talents (Young Doctor) Program(Grant No. BT-2025-TCYC-0067)

RIGHTS & PERMISSIONS

The Author(s)