The Brucella Effector Protein BspF Crotonylates TRIM38 to Inhibit NF-κB and MAPK Signaling Pathway

Abstract

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.

Keywords: Brucella; BspF; MAPK; NF-κB; crotonylation.

Figure 1

BspF enhanced the crotonylation of TRIM38. (A) HA-BspF and FLAG-TRIM38 plasmids were co-transfected into HEK-293T cells and the effect of BspF on TRIM38 crotonylation was detected by Co-immunoprecipitation (Co-IP) with Lysine crotonylation (Kcr), HA-tagged, and FLAG-tagged antibodies. WCL represents the whole cell lysate. (B) Densitometric analysis of Western blot bands was performed, and statistical comparisons between the BspF+ and BspF− groups were conducted to evaluate protein expression levels. Data are means ± standard deviation (SD) from three independent experiments. **** indicates p < 0.0001.

Figure 2

BspF inhibits the transcription of pro-inflammatory factors. (A) RAW264.7 cells were infected with 2308WT and 2308bspF strains. After RNA extraction and reverse transcription, IL-6, IL-8, and TNF-α were detected using RT-PCR. (B) HeLa cells were transfected with HA, HA-BspF, and HA-BspFΔGNAT plasmids, and RNA was extracted and reversely transcribed. The mRNA expression of IL-6 and IL-8 genes was detected by RT-PCR. (C) HeLa cells were transfected with HA, HA-BspF, and HA-BspFΔGNAT plasmids after being treated with TNF-α. The mRNA expression of IL-6 and IL-8 genes were detected by RT-PCR. Data are means ± SD from three independent experiments. ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001.

Figure 3

BspF inhibits the activation of NF-κB, p38 MAPK, and JNK MAPK signaling pathways. (A) RAW264.7 cells were infected with 2308WT and 2308ΔbspF strains, and the phosphorylation of p65, p38, JNK, and ERK was detected through Western blot. NI indicates uninfected cells. (B) HeLa cells were transfected with HA, HA-BspF, and HA-BspFΔGNAT plasmids and the phosphorylation of p65, p38, JNK, and ERK was detected through Western blot analysis. EV indicates empty vector group. (C) HeLa cells were transfected with HA, HA-BspF, and HA-BspFΔGNAT plasmids after being treated with TNF-α, and the phosphorylation of p65, p38, JNK, and ERK was detected through Western blot analysis. EV indicates empty vector group. Data are means ± SD from three independent experiments. p-p65, p-p38, p-JNK, p-ERK: phosphorylated forms of the proteins.

Figure 4

BspF modulates TRAF6 expression via TRIM38. (A) RAW264.7 cells were infected with 2308WT and 2308ΔbspF strains, and expression of TRAF6 was detected through Western blot analysis. NI indicates uninfected cells. (B) HeLa cells were transfected with different doses of HA-BspF plasmids and TRAF6 expression was detected by Western blot. (C) HeLa cells were transfected with HA, HA-BspF, and HA-BspFΔGNAT plasmids and TRAF6 expression was detected by Western blot. EV indicates empty vector group. (D) Knockdown effect of the siRNAs at the protein level. HeLa cells were transfected with TRIM38 specific siRNAs and the endogenous TRIM38 protein was assayed through Western blot analysis. NC indicates negative control group. (E) HA-BspF plasmid was co-transfected with NC or si-TRIM38 in HeLa cells, and TRAF6 expression was detected through Western blot analysis. Data are means ± SD from three independent experiments.

Figure 5

BspF does not affect the TRIM38 expression level through the GNAT domain, and TRIM38K142Cr has no effect on the TRIM38 expression level. (A,B) Different doses of HA-BspF/BspFΔGNAT and FLAG-TRIM38 plasmids were co-transfected into HEK-293T cells, and the expression of BspF/BspFΔGNAT and TRIM38 was detected through Western blot analysis. (C) HA-BspF was co-transfected with the TRIM38 mutant (TRIM38K142R and TRIM38K142Q) into HEK-293T cells and the expression of the TRIM38 mutant (TRIM38K142R and TRIM38K142Q) was detected through Western blot analysis. WCL represents the whole cell lysate. IB represents immunoblotting. Data are means ± SD from three independent experiments.

Figure 6

BspF regulates the ubiquitination of TRAF6 through TRIM38K142Cr to affect the degradation of TRAF6. (A) HA-BspF was co-transfected with TRIM38 mutant (TRIM38K142R and TRIM38K142Q) plasmids, and the interaction between BspF and the mutant (TRIM38K142R and TRIM38K142Q) was detected with co-immunoprecipitation with FLAG-tagged and HA-tagged antibodies. (B) HA-BspF was co-transfected with the TRIM38 mutant (TRIM38K142R and TRIM38K142Q) and MYC-TRAF6 plasmids, performing immunoprecipitation by the MYC antibody, and then the expression and ubiquitination level of TRAF6 was detected through Western blot analysis. (C) MYC-TRAF6 was co-transfected with the TRIM38 mutant (TRIM38K142R and TRIM38K142Q) into HEK-293T cells and the expression of the TRIM38 mutant (TRIM38K142R and TRIM38K142Q) was detected through Western blot analysis. Data are means ± SD from three independent experiments.