Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates.

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 ß-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.

Broad-Spectrum Antibacterial Peptide Kills Extracellular and Intracellular Bacteria Without Affecting Epithelialization.

New antibacterial drugs with novel modes of action are urgently needed as antibiotic resistance in bacteria is increasing and spreading throughout the world. In this study, we aimed to explore the possibility of using APIM-peptides targeting the bacterial ß-clamp for treatment of skin infections. We selected a lead peptide, named betatide, from five APIM-peptide candidates based on their antibacterial and antimutagenic activities in both G+ and G- bacteria. Betatide was further tested in minimal inhibitory concentration (MIC) assays in ESKAPE pathogens, in in vitro infection models, and in a resistance development assay. We found that betatide is a broad-range antibacterial which obliterated extracellular bacterial growth of methicillin-resistant Staphylococcus epidermidis (MRSE) in cell co-cultures without affecting the epithelialization of HaCaT keratinocytes. Betatide also reduced the number of intracellular Staphylococcus aureus in infected HaCaT cells. Furthermore, long-time exposure to betatide at sub-MICs induced minimal or no increase in resistance development compared to ciprofloxacin and gentamicin or ampicillin in S. aureus and Escherichia coli. These properties support the potential of betatide for the treatment of topical skin infections.

Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis.

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Novel Peptides Targeting the ß-Clamp Rapidly Kill Planktonic and Biofilm Staphylococcus epidermidis Both in vitro and in vivo.

Antimicrobial resistance is an increasing threat to global health and challenges the way we treat infections. Peptides containing the PCNA interacting motif APIM (APIM-peptides) were recently shown to bind to the bacterial PCNA homolog, the beta (ß)-clamp, and to have both antibacterial and anti-mutagenic activities. In this study we explore the antibacterial effects of these peptides on Staphylococcus epidermidis, a bacterial species commonly found in prosthetic joint infections (PJI). Drug-resistant bacterial isolates from PJIs often lead to difficult-to-treat chronic infections. We show that APIM-peptides have a rapid bactericidal effect which when used at sublethal levels also increase the efficacy of gentamicin. In addition, APIM-peptides reduce development and eliminate already existing S. epidermidis biofilm. To study the potential use of APIM-peptides to prevent PJI, we used an in vivo bone graft model in rats where APIM-peptide, gentamicin, or a combination of the two was added to cement. The bone grafts containing cement with the combination was more effective than cement containing only gentamicin, which is the current standard of care. In summary, these results suggest that APIM-peptides can be a promising new drug candidate for anti-infective implant materials to use in the fight against resistant bacteria and chronic PJI.

APIM-Mediated REV3L⁻PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells.

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.