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Published on: November 16, 2015
MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells
Qingsong Zeng1, Peifang Lai1, Mingqin Huang1
1School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, 510006, China.
Abstract:
V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.
Insights
The outer membrane protein MAM7 from V. parahaemolyticus promotes intestinal inflammation by stimulating macrophage proliferation and cytokine release. This study clarifies MAM7's role as a proinflammatory factor.
Area of Science:
- Microbiology
- Immunology
- Gastroenterology
Background:
- Vibrio parahaemolyticus causes gastroenteritis.
- The outer membrane protein MAM7's role in intestinal inflammation is unclear.
- MAM7 interactions with macrophages require further investigation.
Purpose of the Study:
- To investigate the function of MAM7 in intestinal inflammation.
- To determine MAM7's effect on macrophage immune responses.
- To characterize MAM7 as a potential proinflammatory factor.
Main Methods:
- Recombinant MAM7 expression and purification.
- RAW264.7 cell proliferation assay (CCK-8).
- Nitric oxide (NO) level measurement.
- Quantitative real-time PCR (qRT-PCR) for cytokine mRNA.
- Enzyme-linked immunosorbent assay (ELISA) for cytokine secretion.
Main Results:
- Optimized MAM7 expression yielded a 27-fold increase.
- MAM7 stimulated RAW264.7 cell proliferation and NO production.
- MAM7 significantly upregulated IL-1β, IL-6, and TNF-α mRNA levels.
- MAM7 enhanced IL-6 and TNF-α secretion from macrophages.
Conclusions:
- MAM7 acts as a proinflammatory adhesion factor.
- MAM7 modulates macrophage immune responses.
- MAM7 contributes to intestinal inflammation.
