The Brucella type IV secretion system (T4SS) can promote the intracellular survival and reproduction of Brucella. T4SS secretes effector proteins to act on cellular signaling pathways to inhibit the host’s innate immune response and cause a chronic, persistent Brucella infection. Brucella can survive in host cells for a long time by inhibiting macrophage apoptosis and avoiding immune recognition. The effector protein, BspF, secreted by T4SS, can regulate host secretory transport and accelerate the intracellular replication of Brucella. BspF has an acetyltransferase domain of the GNAT (GCN5-related N-acetyltransferases) family, and in our previous crotonylation proteomics data, we have found that BspF has crotonyl transferase activity and crotonylation regulation of host cell protein in the proteomics data. Here, we found that BspF attenuates the crotonylation modification of the interacting protein p53, which reduces the p53 expression through the GNAT domain. BspF can inhibit the transcription and protein expression of downstream apoptotic genes, thereby inhibiting host cell apoptosis. Additionally, the Brucella ΔbspF mutant stain promotes apoptosis and reduces the survival rate of Brucella in the cells. In conclusion, we identified that the T4SS effector protein BspF can regulate host cell apoptosis to assist Brucella in its long-term survival by attenuating crotonylation modification of p53 and decreasing p53 expression. Our findings reveal a unique mechanism of elucidating how Brucella regulates host cell apoptosis and promotes its proliferation through the secretion of effector proteins.

The type IV secretion system (T4SS) is an important virulence factor of Brucella. T4SS secretes 16 effector proteins, which affect the intracellular transport of Brucella-containing vacuoles and regulate the host immune response, helping Brucella survive and replicate in host cells. In our previous crotonylation proteomics data of HEK-293T cell proteins triggered by BspF, we found BspF crotonylated on TRIM38, which is an important modulator in the pathways of inflammation, and the crotonylation site is K142. Therefore, it is speculated that BspF may be involved in the regulation of host inflammatory response during Brucella infection. In this study, we found that BspF-mediated TRIM38K142 crotonylation promotes the ubiquitination of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), leading to the degradation of TRAF6 and thereby inhibiting the transduction of Nuclear factor-kappaB (NF-κB), p38 Mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinases (JNK) MAPK signaling pathways and the secretion of pro-inflammatory factors IL-6 and IL-8, which finally helps Brucella promote intracellular survival. This study provides a new theoretical basis for the intracellular survival of host innate immunity through the T4SS, provides new insights into the pathogenic mechanism and treatment of Brucella, and provides an important reference for the study of non-histone crotonylation function.