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Proteomic Profiling of Macrophages by 2D Electrophoresis
Published on: November 4, 2014
Quantitative proteomics revealed modulation of macrophages by MetQ gene of Streptococcus suis serotype 2
Xiaomeng Pei1, Junchi Liu1, Mingxing Liu1
1MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Abstract:
Streptococcus suis serotype 2 (SS2) is a serious zoonotic pathogen; it can lead to symptoms of streptococcal toxic shock syndrome (STSS) in humans and sepsis in pigs, and poses a great threat to public health. The SS2 MetQ gene deletion strain has attenuated antiphagocytosis, although the mechanism of antiphagocytosis and pathogenesis of MetQ in SS2 has remained unclear. In this study, stable isotope labeling by amino acids in cell culture (SILAC) based liquid chromatography-mass spectrometry (LC-MS) and subsequent bioinformatics analysis was used to determine differentially expressed proteins of RAW264.7 cells infected with △MetQ and ZY05719. Proteomic results were verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting for selected proteins. Further research was focused mainly on immune system processes related to downregulated proteins, such as Src and Ccl9, and actin cytoskeleton and endocytosis related upregulated proteins, like Pstpip1 and Ppp1r9b. The proteomic results in this study shed light on the mechanism of antiphagocytosis and innate immunity of macrophages infected with △MetQ and ZY05719, which might provide novel targets to prevent or control the infection of SS2.
Insights
This study investigates how deleting the MetQ gene in Streptococcus suis serotype 2 (SS2) affects macrophage antiphagocytosis. Findings reveal key protein changes impacting innate immunity, offering potential targets for SS2 infection control.
Area of Science:
- Microbiology
- Immunology
- Proteomics
Background:
- Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen causing severe illness in humans and pigs.
- The MetQ gene's role in SS2's antiphagocytic mechanisms and pathogenesis remains poorly understood.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the attenuated antiphagocytosis of the SS2 MetQ deletion strain.
- To identify host cell protein alterations in macrophages infected with SS2, specifically the △MetQ strain.
Main Methods:
- Utilized Stable Isotope Labeling by Amino acids in Cell culture (SILAC) coupled with liquid chromatography-mass spectrometry (LC-MS) for differential protein expression analysis.
- Employed bioinformatics for data analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting for validation.
Main Results:
- Identified differentially expressed proteins in RAW264.7 macrophages upon infection with △MetQ and wild-type SS2 (ZY05719).
- Observed downregulation of immune-related proteins (e.g., Src, Ccl9) and upregulation of actin cytoskeleton and endocytosis-related proteins (e.g., Pstpip1, Ppp1r9b).
Conclusions:
- The proteomic analysis provides insights into the antiphagocytic strategies of SS2 and its impact on macrophage innate immunity.
- Identified proteins like Src, Ccl9, Pstpip1, and Ppp1r9b may serve as novel therapeutic targets for controlling SS2 infections.
