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A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus
Published on: March 28, 2017
Comparative Proteomic Analysis of Differential Proteins in Response to Aqueous Extract of Quercus infectoria Gall in
Radhiah Khairon1, Noraziah Mohamad Zin1, Mariati Abdul Rahman2
1School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
Abstract:
The aim of this study is to analyze the differential proteins in MRSA ATCC 33591 treated with aqueous extract from Q. infectoria gall. Protein extracts were obtained from MRSA cells by sonication and were separated by 2D polyacrylamide gels. Protein spots of interest were extracted from the gels and identified using LC-ESI-QTOF MS. The concentration of Q. infectoria extract used for 2D-gel electrophoresis was subinhibitory concentration. Minimum inhibitory concentration (MIC) value of the extract against MRSA was 19.50 μg/mL with bacteriostatic action at 1x MIC from time-kill assay. However, the extract exhibited dose-dependent manner and was bactericidal at 4x MIC with more than 3 log10 CFU/mL reduction at 4 h. 2D-GE map showed that 18 protein spots were upregulated and another six were downregulated more than twofold (p < 0.05) after treatment with subinhibitory concentration. Out of six proteins being downregulated, four proteins were identified as ferritin and catalase, branched-chain alpha-keto acid dehydrogenase subunit E2, and succinyl-CoA ligase [ADP-forming] subunit beta. Seven upregulated proteins which have been successfully identified were 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain protein, formate C-acetyltransferase, 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ, NAD dependent epimerase/dehydratase family protein, and phosphopantothenoyl cysteine decarboxylase. It is postulated that the main mechanism of aqueous extract from gall of Q. infectoria was most likely involved in energy metabolism and protein stress.
Insights
This study investigated how an aqueous extract from Q. infectoria gall affects proteins in MRSA. The extract altered protein expression, impacting energy metabolism and protein stress responses in the bacteria.
Area of Science:
- Microbiology
- Proteomics
- Natural Product Chemistry
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant public health threat due to its resistance to antibiotics.
- Natural products, such as plant extracts, are being explored as alternative therapeutic agents against resistant bacteria.
- Quercus infectoria gall extract has demonstrated antimicrobial properties, necessitating further investigation into its molecular mechanisms.
Purpose of the Study:
- To identify differentially expressed proteins in MRSA ATCC 33591 upon treatment with a subinhibitory concentration of aqueous Q. infectoria gall extract.
- To elucidate the potential mechanisms of action of Q. infectoria gall extract against MRSA at the proteomic level.
- To understand the impact of the extract on bacterial energy metabolism and stress response pathways.
Main Methods:
- Bacterial culture of MRSA ATCC 33591.
- Preparation of protein extracts using sonication.
- Separation of proteins via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
- Identification of differentially expressed protein spots using Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry (LC-ESI-QTOF MS).
- Determination of Minimum Inhibitory Concentration (MIC) and time-kill kinetics.
Main Results:
- The MIC of Q. infectoria gall extract against MRSA was 19.50 μg/mL, exhibiting bacteriostatic action at 1x MIC and bactericidal effects at 4x MIC.
- 2D-PAGE analysis revealed 18 upregulated and 6 downregulated protein spots (p < 0.05) after treatment with subinhibitory concentrations.
- Identified downregulated proteins included ferritin, catalase, branched-chain alpha-keto acid dehydrogenase subunit E2, and succinyl-CoA ligase [ADP-forming] subunit beta.
- Identified upregulated proteins included enzymes involved in fatty acid biosynthesis (e.g., 3-hydroxyacyl-CoA dehydrogenase, FabZ) and energy metabolism (e.g., formate C-acetyltransferase).
Conclusions:
- The aqueous extract of Q. infectoria gall significantly alters the proteome of MRSA.
- The observed changes suggest that the extract's mechanism of action involves interference with bacterial energy metabolism and protein stress responses.
- These findings support the potential of Q. infectoria gall extract as a source for novel antibacterial agents against MRSA.
