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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in
Jihai Yi1, Yueli Wang1, Qifeng Li1
1Department of Veterinary Medicine, College of Animal Science and Technology, Shihezi University, Shihezi 832000, China.
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.
Insights
Brucella melitensis 16M manipulates SUMOylation via Ubc9 and the VirB system to survive within host immune cells. Manipulating SUMO1/Ubc9 levels impacts Brucella survival, apoptosis, and immune factor production.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Brucella is an intracellular pathogen that establishes infection within host immune cells.
- The precise mechanisms underlying Brucella's intracellular survival remain incompletely understood.
- Small ubiquitin-related modifier (SUMO) conjugation, mediated by E2 conjugating enzyme 9 (Ubc9), plays diverse cellular roles.
Purpose of the Study:
- To investigate the role of SUMOylation, specifically SUMO1 and Ubc9, in the intracellular survival of Brucella melitensis 16M.
- To elucidate the interaction between Brucella 16M and the host SUMOylation machinery.
- To determine the contribution of the VirB secretion system to Brucella's intracellular persistence.
Main Methods:
- Construction of plasmids for SUMO1 and Ubc9 gene overexpression and knockdown.
- Time-dependent analysis of SUMO1/Ubc9 expression during Brucella 16M infection.
- Assessment of Brucella 16M intracellular survival, apoptosis, and immune factor production in manipulated host cells.
- Evaluation of a VirB2 secretion system mutant (16M△VirB2) for intracellular survival.
Main Results:
- Brucella 16M infection upregulated SUMO1/Ubc9 expression in a time-dependent manner.
- Overexpression of SUMO1/Ubc9 inhibited Brucella 16M intracellular survival, while depletion promoted it.
- SUMO1/Ubc9 manipulation influenced Brucella-induced apoptosis and immune factor production in macrophages.
- The 16M△VirB2 mutant exhibited reduced intracellular survival compared to wild-type Brucella 16M.
- VirB2 was found to affect Ubc9 expression, enhancing late-stage intracellular survival.
Conclusions:
- Brucella melitensis 16M utilizes the VirB IV secretion system to interact with host SUMO-related proteins.
- This interaction interferes with host SUMOylation processes, promoting pathogen survival within host cells.
- SUMO1 and Ubc9 are critical components in the host-pathogen interaction, influencing Brucella's intracellular persistence and host immune responses.
