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. 2024 Jun 26:15:1402796.
doi: 10.3389/fmicb.2024.1402796. eCollection 2024.

Proteomic and metabolomic profiling of methicillin resistant versus methicillin sensitive Staphylococcus aureus using a simultaneous extraction protocol

Syrine Boucherabine  1 , Alexander Giddey  2 , Rania Nassar  1   3 , Hamza M Al-Hroub  4 , Lobna Mohamed  1 , Mohammad Harb  4   5 , Nelson Cruz Soares #  4   5   6   7 , Abiola Senok #  1   3
Affiliations

Proteomic and metabolomic profiling of methicillin resistant versus methicillin sensitive Staphylococcus aureus using a simultaneous extraction protocol

Syrine Boucherabine et al. Front Microbiol. .

Abstract

Background: Understanding the biology of methicillin resistant Staphylococcus aureus (MRSA) is crucial to unlocking insights for new targets in our fight against this antimicrobial resistant priority pathogen. Although proteomics and metabolomic profiling offer the potential to elucidating such biological markers, reports of methodological approaches for carrying this out in S. aureus isolates remain limited. We describe the use of a dual-functionality methanol extraction method for the concurrent extraction of protein and metabolites from S. aureus and report on the comparative analysis of the proteomic and metabolomic profiles of MRSA versus methicillin sensitive S. aureus (MSSA).

Methods: Bacterial reference strains MRSA ATCC43300 and MSSA ATCC25923 were used. The conventional urea methodology was used for protein extraction and a methanol based method was used for concurrent proteins and metabolites extraction. Proteomic and metabolomic profiling was carried out using TimsTOF mass spectrometry. Data processing was carried out using the MaxQuant version 2.1.4.0.

Results: This study represents the first report on the utilization of the methanol extraction method for concurrent protein and metabolite extraction in Gram positive bacteria. Our findings demonstrate good performance of the method for the dual extraction of proteins and metabolites from S. aureus with demonstration of reproducibility. Comparison of MRSA and MSSA strains revealed 407 proteins with significantly different expression levels. Enrichment analysis of those proteins revealed distinct pathways involved in fatty acid degradation, metabolism and beta-lactam resistance. Penicillin-binding protein PBP2a, the key determinant of MRSA resistance, exhibited distinct expression patterns in MRSA isolates. Metabolomic analysis identified 146 metabolites with only one exclusive to the MRSA. The enriched pathways identified were related to arginine metabolism and biosynthesis.

Conclusion: Our findings demonstrate the effectiveness of the methanol-based dual-extraction method, providing simultaneous insights into the proteomic and metabolomic landscapes of S. aureus strains. These findings demonstrate the utility of proteomic and metabolomic profiling for elucidating the biological basis of antimicrobial resistance.

Keywords: Staphylococcus aureus; gram positive bacteria; mass spectrometry; metabolomics; proteomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Flowchart of the experiment set up (created using biorender.com).
Figure 2
Figure 2
Profiling of proteins identified. (A) Venn diagram of the overlap as well as unique proteins identified from MRSA ATCC43300 in either of the extraction methods, with 1,190 protein found in both extraction methods and only 4 proteins exclusive to the methanol extraction method compared to 17 proteins exclusive to the conventional urea extraction method. (B) Correlation plot between both extraction methods (Pearson correlation coefficient 0.8; p < 0.05).
Figure 3
Figure 3
Comparison of MRSA protein label-free quantitation (LFQ) intensities obtained using both extraction methods. (A) Boxplots representing the top 12 proteins with differential LFQ levels. (B) Boxplots representing the differences between the properties of the protein with different LFQ levels.
Figure 4
Figure 4
PCA of the proteomes of MSSA vs. MRSA and their respective replicates showing the clustering together of strains.
Figure 5
Figure 5
Comparison of LFQ intensities of methicillin sensitive and resistant Staphylococcus aureus (MSSA and MRSA).
Figure 6
Figure 6
Comparison of LFQ intensities of the top metabolites identified in methicillin sensitive and resistant Staphylococcus aureus (MSSAA and MRSA). (A) Heatmap of the differences in LFQ intensities between top metabolites identified in both strains. (B) Volcano plot representing the LFQ values of shared metabolites between both strains, indicating a log-2 fold change in intensity. Red dots represent higher intensity in MSSA compared to MRSA, while blue dots represent lower intensity in MSSA. (C) Over-representation analysis of enriched pathways done on KEGG based on metabolites identified.
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References

    1. Al-Salhi R., Monfort C., Bonvallot N., David A. (2022). Analytical strategies to profile the internal chemical exposome and the metabolome of human placenta. Anal. Chim. Acta 1219:339983. doi: 10.1016/j.aca.2022.339983, PMID: - DOI - PubMed
    1. Alshehry Z. H., Barlow C. K., Weir J. M., Zhou Y., McConville M. J., Meikle P. J. (2015). An efficient single phase method for the extraction of plasma lipids. Meta 5, 389–403. doi: 10.3390/metabo5020389, PMID: - DOI - PMC - PubMed
    1. Alsoud L. O., Soares N. C., Al-Hroub H. M., Mousa M., Kasabri V., Bulatova N., et al. . (2022). Identification of insulin resistance biomarkers in metabolic syndrome detected by UHPLC-ESI-QTOF-MS. Meta 12:508. doi: 10.3390/metabo12060508, PMID: - DOI - PMC - PubMed
    1. Atshan S. S., Shamsudin M. N., Sekawi Z., Thian Lung L. T., Barantalab F., Liew Y. K., et al. . (2015). Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development. Front. Microbiol. 6:524. doi: 10.3389/fmicb.2015.00524 - DOI - PMC - PubMed
    1. Auer G. K., Weibel D. B. (2017). Bacterial cell mechanics. Biochemistry 56, 3710–3724. doi: 10.1021/acs.biochem.7b00346, PMID: - DOI - PMC - PubMed