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, 6, 19841

Proteomic Response of Methicillin-Resistant S. Aureus to a Synergistic Antibacterial Drug Combination: A Novel Erythromycin Derivative and Oxacillin

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Proteomic Response of Methicillin-Resistant S. Aureus to a Synergistic Antibacterial Drug Combination: A Novel Erythromycin Derivative and Oxacillin

Xiaofen Liu et al. Sci Rep.

Abstract

The use of antibacterial drug combinations with synergistic effects is increasingly seen as a critical strategy to combat multi-drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). In this work, the proteome responses in MRSA under the stress of a sub-inhibitory dose of a synergistic drug combination of a novel erythromycin derivative, SIPI-8294, and oxacillin, were studied by label-free quantitative proteomics. Several control treatment groups were designed to isolate proteome responses potentially related to the synergy: (1) the non-synergistic drug combination of erythromycin and oxacillin, (2) SIPI-8294 only, (3) oxacillin only and (4) erythromycin only. Results showed that 200 proteins were differentially expressed in SIPI-8294/oxacillin-treated cells. Among these proteins, the level of penicillin binding protein 2a, the protein mainly responsible for oxacillin resistance in MRSA, was four times lower in the SIPI-8294/oxacillin group than in the erythromycin/oxacillin group, suggesting that SIPI-8294 may interfere with this known oxacillin resistance mechanism. Moreover, hierarchical clustering analysis of differentially expressed proteins under different treatments revealed that SIPI-8294/oxacillin elicits very different responses than the individual drugs or the non-synergistic erythromycin/oxacillin combination. Bioinformatic analysis indicated that the synergistic effect can be further traced to a disruption in oxidation-reduction homeostasis and cell wall biosynthesis.

Figures

Figure 1
Figure 1. Chemical structures for SIPI-8294 and erythromycin.
The macrolactone ring is in red color and the 5-position disosamine sugar is in blue color. The different functional groups between SIPI-8294 and erythromycin are in pink color for SIPI-8294 and in black color for erythromycin.
Figure 2
Figure 2. Experimental workflow for the sample preparation and data analysis.
Different drug treatment groups were compared with control (no drug treatment) and performed spectral-counting based label-free quantitation.
Figure 3
Figure 3
(a) Volcano plot of differentially expressed proteins in the SIPI-8294/Oxa and Ery/Oxa groups; (b) Numbers of differentially expressed proteins in different treatment groups, SIPI-8294/Oxa, Ery/Oxa, SIPI8294, Ery and Oxa, compared with control (no drug treatment); (c) Venn diagram for differentially expressed proteins in the SIPI-8294/Oxa and the Ery/Oxa treatment groups compared with their individual drug treatment groups; (d) The correlation between the differentially expressed proteins in the SIPI-8294/Oxa and Ery/Oxa groups.
Figure 4
Figure 4. Hierarchical cluster analysis was conducted for all the differentially expressed proteins in the different treatment groups.
SIPI-8294/Oxa, SIPI-8294, Oxa, Ery and Ery/Oxa, as well as the outcome that would be expected if SIPI-8294 and Oxa acted independently (Additive). Each row indicates one treatment group and each column represents one differentially expressed protein (shown in the zoom-in image). The color indicates relative fold changes (up-regulation relative to mean fold change in red, and down-regulation relative to mean fold change in green). The “Additive” group is generated by multiplying the corresponding fold changes of each differentially expressed protein in the SIPI-8294 treatment group and the Oxa treatment group.
Figure 5
Figure 5. Quantification results for the expression of PBP2a (gene: mecA) and β-lactamase (gene: blaZ) at protein level by label-free quantitative proteomics method (a) and at transcription level by Q-RT-PCR method (b).
PBP2a and β-lactamase are the proteins involved in resistance mechanism of bacteria against β-lactam antibiotics. Label-free quantitative proteomics results show that the levels of PBP2a and β-lactamase are lower in the SIPI-8294/Oxa than Ery/Oxa and other treatment groups. The same trend was found at the mRNA level. These results suggest that the synergistic effect mechanism may be related to the interference with the oxacillin resistance mechanism. Asterisks (*) indicate differential expression at p < 0.05.
Figure 6
Figure 6. The differentially expressed proteins in the SIPI-8294/Oxa treatment group involved in nitrogen metabolism pathway classified by KEGG.
narG: nitrate reductase alpha subunit; narH: respiratory nitrate reductase β subunit; nirB: assimilatory nitrite reductase (NAD(P)H) large subunit; NirKS: nitric oxide reductase; glnA: glutamine synthase; gltBD: glutamate synthase. The symbol of (↓) stands for down-regulated protein, and the number ahead is the fold change. nirKS and gltBD were not detected in our experiment.
Figure 7
Figure 7. SEM images for MRSA under different drug treatments.
Ctrl: Normal methicillin-resistance S aureus (MRSA) with no treatment; Oxa: MRSA treated with 1/8 MIC oxacillin (8 μg/ml); SIPI-8294: MRSA treated with 8 μg/ml SIPI-8294; SIPI-8294/Oxa: MRSA treated with the combination of SIPI-8294 and oxacillin (8 μg/ml for SIPI-8294 and 0.03125 μg/ml for oxacillin).

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