Molecular Basis of Rhodomyrtone Resistance in Staphylococcus aureus
Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory gene ( *) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in * alters the activity of the regulator, FarR*, in such a way tha...
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| Published in: | mBio Vol. 13; no. 1; p. e0383321 |
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| Main Authors: | , , , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
American Society for Microbiology
15-02-2022
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory
gene (
*) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in
* alters the activity of the regulator, FarR*, in such a way that not only its own gene,
*, but also the divergently transcribed
gene and genes controlled by the global regulator,
, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as
deletion in either the parent or the RomR strain (RomR Δ
) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the Δ
mutants. Since the PG content in the supernatant (2,244 ng/optical density [OD]) was more than 100-fold higher than that of fatty acids (FA), we assumed that PG interacts with Rom, thereby abrogating its antimicrobial activity. Indeed, by static and dynamic light scattering (SLS and DLS) and isothermal titration calorimetry (ITC) analyses, we could demonstrate that both PG and Rom were vesicular and reacted with each other in milliseconds to form a 1:1.49 [Rom-PG(32:0), where PG(32:0) is PG with C32:0 lipids] complex. The binding is entropically driven and hence hydrophobic and of low specificity in nature. Our results indicate that the cytoplasmic membrane is the actual target of Rom, which is also in agreement with Rom's induced rapid collapse of the membrane potential and decreased membrane integrity.
Antibiotic resistance is a growing public health problem, and alternative antibiotics are urgently needed. Rhodomyrtone (Rom), an antimicrobial compound originally isolated from Rhodomyrtus tomentosa, is active against multidrug-resistant Gram-positive pathogens. However, Rom-resistant (RomR) mutants occur with low frequency. In this study, we unraveled the underlying resistance mechanism, which is based on a point mutation in the
regulator gene, causing overexpression of FarE, which most likely acts as a phospholipid (PG) efflux pump, as large amounts of PG were found in the supernatant and the pellet fraction. We show that PG can bind to Rom, thereby abrogating its antimicrobial activity. The direct interaction of Rom with PG suggests that Rom's actual target is the cytoplasmic membrane. Antibiotics that interact with PG are rare. Since Rom can be chemically synthesized, it serves as a lead compound for synthesis of improved variants. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 The authors declare no conflict of interest. Present address: Carlos Calderón, Escuela de Química, Universidad de Costa Rica, San José, Costa Rica. Present address: Arif Luqman, Biology Department, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia. Li Huang, Miki Matsuo, and Carlos Calderón contributed equally to this work. Author order was determined by the principal investigator. |
| ISSN: | 2150-7511 2150-7511 |
| DOI: | 10.1128/mbio.03833-21 |