Rv3722c Promotes Mycobacterium tuberculosis Survival in Macrophages by Interacting With TRAF3

Abstract

Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-β expression and decrease of IL-1β, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.

Keywords: Mycobacterium tuberculosis; Rv3722c; TRAF3; cytokines; intracellular survival.

Figure 1

Rv3722c interacts with TRAF3. (A) Delineation of the filtered PPI sub-network between Rv3722c and host proteins. The orange node represented Rv3722c and other colors represented host different immune pathway proteins. (B) Verification of the protein-protein interaction by Y2H assay. Rv3722c in the pmBD destination vector was used as bait. TRAF3 in the pmAD destination vector was used as prey. Co-transformation of pmBD empty vector with pmAD vector containing TRAF3 and co-transformation of the pmAD empty vector with pmBD vector containing Rv3722c were used as negative controls. Yeast cells grown on SD/Leu⁻Trp⁻His⁻Ade⁻ selective plates indicated the interaction between Rv3722c and TRAF3. (C) Verification of the protein-protein interaction by BiFC assay. ΔJun-Rv3722c was co-transfected with ΔFos-TRAF3 and the negative controls into HEK 293T cells, respectively. Cells were harvested 36 h after transfection for fluorescence microscopy-based image analysis. The interaction between Rv3722c and TRAF3 allowed the re-formation of a bimolecular fluorescent complex. (D) Verification of the protein-protein interaction by Co-IP assay. V5-tagged Rv3722c and HA-tagged TRAF3 were co-transfected into HEK 293T cells. Cells were harvested 36 h after transfection for Co-IP assay. Co-transfection of V5-tagged Rv3722c with HA-tagged empty vector and HA-tagged TRAF3 with V5-tagged empty vector were used as negative controls. (E) Protein-protein docking simulated the interaction of Rv3722c and TRAF3. The purple structure represented Rv3722c and the blue-green structure represented TRAF3. (F) Verification of the protein-protein co-localized by immunofluorescence confocal microscopy assay. V5-tagged Rv3722c and HA-tagged TRAF3 were co-transfected into HEK 293T cells. Cells were harvested at 36 h after transfection for immunofluorescence confocal microscopy assay. Co-transfection of V5-tagged Rv3722c with HA-tagged empty vector and HA-tagged TRAF3 with V5-tagged empty vector were used as negative controls. V5-tagged Rv3722c and HA-tagged TRAF3 signals were co-localized in the cytoplasm of HEK 293T cells. All experiments were performed in triplicate.

Figure 2

Knock-down of TRAF3 attenuates the effect of Rv3722c on M.tb proliferation. (A) pcDNA3.1-V5-Rv3722c or pcDNA3.1-V5 vector control and pAAV-eGFP were transiently co-transfected into RAW264.7 cells. Cells were infected with RFP-H37Ra at an MOI of 10 after transfected for 6 h. Cells were harvested for confocal microscopy-based image analysis at the indicated times. (B) Quantification of the average number of intracellular bacilli per cell in a total of 100 cells. (C) Flowchart of RAW-Rv3722c cell line construction. (D) RAW-Vector and RAW-Rv3722c cell lines were infected with RFP-H37Ra at an MOI of 10. Cells were harvested for confocal fluorescence microscopy-based image analysis at the indicated times. (E) Quantification of the average number of intracellular bacilli per cell in a total of 100 cells. (F) Identification of the mRNA expression of endogenous TRFA3 by qRT-PCR. (G) After infection of RAW-Rv3722c cells with lentivirus containing shTRAF3-1 gene for 48 h, the cells were infected with H37Ra at an MOI of 10. Cells were lysed and the CFUs of intracellular M.tb were detected by plating on 7H11 plates at the indicated times. Data shown in (F, G) are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant; by two-way ANOVA or student t-test. All experiments were performed in triplicate.

Figure 3

Genes and proteins in different signal pathways are potentially regulated by Rv3722c. (A) Volcano plot indicating significant up-regulation (red) and down-regulation (blue) genes after transmission of Rv3722c. (B) KEGG enrichment of genes that had significant change overexpression level comparing between RAW-Vector and RAW-Rv3722c cell lines. (C) Polarized heatmap showing the normalized gene expression level of TRAF3 related pathways. (D) Heatmap showing the normalized count of the expression level of cytokines.

Figure 4

M.tb Rv3722c modulates MAPK and NF-κB pathways via TRAF3. (A–F) qRT-PCR analysis of TRAF3, IFN-β, IL-1β, IL-6, IL-12p40, and TNF-α mRNA expression in RAW-Vector or RAW-Rv3722c cells with or without lentivirus containing shTRAF3-1 gene infection for 48 h. (G, H) Western blot analysis of TRAF3, p100/p52, p65, p-p65, p38, p-p38, JNK, p-JNK, Erk, p-Erk expression in RAW-Vector and RAW-Rv3722c cells with or without lentivirus containing shTRAF3 gene infection for 48 h. (I) Model of Rv3722c interacted with TRAF3 to regulate host immunity and promote M.tb survival. The PAMPs of M.tb was recognized by the host PRRs and then parasitized in the host cells. Rv3722c secreted by M.tb interacted with TRAF3 in the host cell to inhibit the processing of p100/p52, the phosphorylation of p65, p38, and JNK, promote the secretion of IFN-β, and inhibit the production of IL-1β, IL-6, IL-12p40, and TNF-α, which in turn promoted the proliferation of M.tb in host cells. Data shown in (A–F) are mean ± SD of three independent experiments. *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant; both by student t-test. All experiments were performed in triplicate.