Publications
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Singleton, Amanda; Bergum, Olaug Elisabeth Torheim; Scheffold, Jana; Ræder, Synnøve Brandt; Thorfinnsdottir, Lilja Brekke & Røst, Lisa M.
[Show all 9 contributors for this article]
(2025).
A dual-mechanism antimicrobial peptide with antimutagenic activity targets the replisome and induces cell envelope stress.
mSphere.
10(9).
doi:
10.1128/msphere.00068-25.
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To combat the growing threat of multidrug-resistant bacteria, we need to develop novel antibiotics with unique modes of action. This study investigates the antibacterial properties of BTP-001 toward Escherichia coli . BTP-001 targets the β-clamp subunit of the DNA Pol III holoenzyme and is composed of the binding motif APIM linked to a cell-penetrating part composed of 11 arginine residues (R11). Our data indicate that R11 facilitates energy-dependent transport of BTP-001 across the cell membrane, possibly via iron transport systems such as the TonB system. The full-length BTP-001 peptide rapidly disturbs membrane integrity, inducing expression and activation of the Cpx cell envelope stress response. This response likely triggers the production of reactive oxygen species (ROS), contributing to the rapid bactericidal effect, as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, this study confirms that BTP-001 targets DNA replication and reduces resistance development by inhibiting translesion synthesis. In addition, our data suggest that the β-clamp is associated with ribosomal complexes and that BTP-001 disrupts translational processes. In conclusion, BTP-001 exhibits a multifaceted mode of action, which strengthens its potential as a novel therapeutic drug against antibiotic-resistant bacteria.
IMPORTANCE As antimicrobial resistance (AMR) increases, the world needs new antibiotics with new modes of action to avoid cross-resistance. In this study, we investigated how BTP-001, a novel cell-penetrating peptide that contains a protein-binding motif for the essential DNA replication protein β-clamp, kills bacteria. We demonstrate that BTP-001 has a dual mode of action in which it i) targets the β-clamp and inhibits replication and mutagenesis and ii) disrupts the bacterial cell envelope, causing ROS accumulation and rapid cell death. In addition, our data indicate that BTP-001 affects translation, suggesting that the β-clamp may have unknown roles beyond replication. Our data also suggest that the bacterial import of BTP-001, via the cell-penetrating part of the peptide, is dependent on active transport and involves iron uptake mechanisms. BTP-001 has many properties that could be useful for further development as a new antibiotic.
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Bergum, Olaug Elisabeth Torheim; Singleton, Amanda Holstad; Røst, Lisa Marie Ivarsdatter; Bodein, Antoine; Scott-Boyer, Marie-Pier & Rye, Morten Beck
[Show all 9 contributors for this article]
(2024).
SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated.
Frontiers in Microbiology.
15.
doi:
10.3389/fmicb.2024.1373344.
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The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates error-free repair followed by error-prone repair. Here, we have employed a multi-omics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death. Our results show that expression of genes involved in error-free and error-prone repair were both induced shortly after DNA damage, thus, challenging the established perception that the expression of error-prone repair genes is delayed. By combining transcriptomics and a sub-proteomics approach termed signalomics, we found that the temporal segregation of error-free and error-prone repair is primarily regulated after transcription, supporting the current literature. Furthermore, the heterology index (i.e., the binding affinity of LexA to the SOS box) was correlated to the maximum increase in gene expression and not to the time of induction of SOS genes. Finally, quantification of metabolites revealed increasing pyrimidine pools as a late feature of the SOS response. Our results elucidate how the SOS response is coordinated, showing a rapid transcriptional response and temporal regulation of mutagenesis on the protein and metabolite levels.
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Singleton, Amanda Holstad; Bergum, Olaug Elisabeth Torheim; Søgaard, Caroline Krogh; Røst, Lisa M.; Olsen, Cecilie Elisabeth & Blindheim, Fredrik Heen
[Show all 12 contributors for this article]
(2023).
Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates.
Frontiers in Microbiology.
14.
doi:
10.3389/fmicb.2023.1260120.
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The past few decades have been plagued by an increasing number of infections caused by antibiotic resistant bacteria. To mitigate the rise in untreatable infections, we need new antibiotics with novel targets and drug combinations that reduce resistance development. The novel β-clamp targeting antimicrobial peptide BTP-001 was recently shown to have a strong additive effect in combination with the halogenated pyrrolopyrimidine JK-274. In this study, the molecular basis for this effect was examined by a comprehensive proteomic and metabolomic study of the individual and combined effects on Staphylococcus aureus. We found that JK-274 reduced activation of several TCA cycle enzymes, likely via increasing the cellular nitric oxide stress, and BTP-001 induced oxidative stress in addition to inhibiting replication, translation, and DNA repair processes. Analysis indicated that several proteins linked to stress were only activated in the combination and not in the single treatments. These results suggest that the strong additive effect is due to the activation of multiple stress responses that can only be triggered by the combined effect of the individual mechanisms. Importantly, the combination dose required to eradicate S. aureus was well tolerated and did not affect cell viability of immortalized human keratinocyte cells, suggesting a species-specific response. Our findings demonstrate the potential of JK-274 and BTP-001 as antibiotic drug candidates and warrant further studies.
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Olsen, Cecilie Elisabeth; Blindheim, Fredrik Heen; Søgaard, Caroline Krogh; Røst, Lisa M.; Singleton, Amanda & Bergum, Olaug Elisabeth Torheim
[Show all 10 contributors for this article]
(2022).
Halogenated Pyrrolopyrimidines with Low MIC on Staphylococcus aureus and Synergistic Effects with an Antimicrobial Peptide.
Antibiotics.
11(8).
doi:
10.3390/antibiotics11080984.
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Currently, there is a world-wide rise in antibiotic resistance causing burdens to individuals and public healthcare systems. At the same time drug development is lagging behind. Therefore, finding new ways of treating bacterial infections either by identifying new agents or combinations of drugs is of utmost importance. Additionally, if combination therapy is based on agents with different modes of action, resistance is less likely to develop. The synthesis of 21 fused pyrimidines and a structure-activity relationship study identified two 6-aryl-7H-pyrrolo [2,3-d] pyrimidin-4-amines with potent activity towards Staphylococcus aureus. The MIC-value was found to be highly dependent on a bromo or iodo substitution in the 4-benzylamine group and a hydroxyl in the meta or para position of the 6-aryl unit. The most active bromo and iodo derivatives had MIC of 8 mg/L. Interestingly, the most potent compounds experienced a four-fold lower MIC-value when they were combined with the antimicrobial peptide betatide giving MIC of 1–2 mg/L. The front runner bromo derivative also has a low activity towards 50 human kinases, including thymidylate monophosphate kinase, a putative antibacterial target.
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Published
Mar. 16, 2025 12:34 PM
- Last modified
Mar. 19, 2025 11:14 AM
