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. 2019 Sep;20(5):e54.
doi: 10.4142/jvs.2019.20.e54.

Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages

Affiliations

Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages

Jihai Yi et al. J Vet Sci. 2019 Sep.

Abstract

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M (p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.

Keywords: Brucella; SUMO; coupling enzyme Ubc9; interaction; intracellular.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1. Western blot results of SUMO1 and Ubc9 in B. melitensis 16M-infected normal RAW264.7 cells. Cells were lysed at 0, 4, 8, 12, 24, and 48 h post-infection and proteins extracted. β-actin was the reference gene.
SUMO1, small ubiquitin-related modifier 1; Ubc9, E2 conjugating enzyme 9.
Fig. 2
Fig. 2. Cell viability is inhibited by knockdown and overexpression plasmids in a time-dependent manner. Statistical significance is indicated as *p < 0.05 or **p < 0.01.
SUMO1, small ubiquitin-related modifier 1; Ubc9, E2 conjugating enzyme 9; DMSO, dimethyl sulfoxide.
Fig. 3
Fig. 3. Quantitative real-time polymerase chain reaction was performed to analyze the mRNA levels of SUMO1 and Ubc9 in lentivirus-infected RAW264.7 macrophages. Statistical significance is indicated as *p < 0.05 or **p < 0.01.
mRNA, messenger RNA; SUMO1, small ubiquitin-related modifier 1; Ubc9, E2 conjugating enzyme 9; NC, no-treatment cell lines; K-S group, stable SUMO1 knockdown cells; O-S group, cells stably overexpressing SUMO1; OS-KS group, SUMO1 overexpression-knockdown cells; K-U group, stable Ubc9 knockdown cells; O-U group, cells stably overexpressing Ubc9; OU-KU group, Ubc9 overexpression-knockdown cells.
Fig. 4
Fig. 4. Enzyme-linked immunosorbent assay detection of IFNγ (A) and TNFα (B) in the supernatants of different treatment groups of RAW264.7 cells after B. melitensis 16M infection. Brucella-infected-untreated cells acted as the control group. Statistical significance is indicated as *p < 0.05 or **p < 0.01.
IFN, interferon; TNF, tumor necrosis factor; K-S group, stable SUMO1 knockdown cells; O-S group, cells stably overexpressing SUMO1; OS-KS group, SUMO1 overexpression-knockdown cells; K-U group, stable Ubc9 knockdown cells; O-U group, cells stably overexpressing Ubc9; OU-KU group, Ubc9 overexpression-knockdown cells.
Fig. 5
Fig. 5. Intracellular survival of post-infection B. melitensis 16M in different RAW264.7 cell treatment groups. RAW264.7 cells were lysed, and Brucella was coated with tryptic soy agar culture medium and cultured at 37°C for 72 h. B. melitensis 16M numbers were then counted. Brucella-infected untreated cells served as the control group. Statistical significance is indicated as *p < 0.05 or **p < 0.01.
CFU, colony-forming unit; K-S group, stable SUMO1 knockdown cells; O-S group, cells stably overexpressing SUMO1; OS-KS group, SUMO1 overexpression-knockdown cells; K-U group, stable Ubc9 knockdown cells; O-U group, cells stably overexpressing Ubc9; OU-KU group, Ubc9 overexpression-knockdown cells.
Fig. 6
Fig. 6. Effect of T4SS-VirB2 on expression of SUMO1 and Ubc9 after infection of RAW264.7 cells with B. melitensis 16M or B. melitensis 16M△VirB mutant. Western blotting was used to detect the protein levels of SUMO1 and Ubc9 (A). Quantitative real-time polymerase chain reaction was conducted to detect the levels of SUMO1 and Ubc9 mRNAs (B). Statistical significance is indicated as *p < 0.05 or **p < 0.01.
NC, no-treatment cell lines; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; SUMO1, small ubiquitin-related modifier 1; Ubc9, E2 conjugating enzyme 9.
Fig. 7
Fig. 7. VirB2 affects intracellular survival of Brucella 16M by inhibiting Ubc9 expression. Levels of Ubc9 after pre-stimulation with VirB2 for 0, 4, 8, and 24 h were assessed by western blotting (A). Brucella 16M and Brucella 16M△VirB2 cells pre-stimulated or non-prestimulated with VirB2. At 4, 8, 12, 24, and 48 h post infection, the number of bacteria in cells was counted (B). Statistical significance is indicated as *p < 0.05 or **p < 0.01.
CFU, colony-forming unit; Ubc9, E2 conjugating enzyme 9.

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