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 β-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.
Antibiotic resistance is a global problem. Widespread misuse of antibiotics, not only in human medicine but also in animal husbandry, has led to the emergence and spread of bacteria conferring resistance to multiple antibiotics. The World Health Organization (2017, 2018) has published a list of highly virulent bacteria with increasing multidrug resistance (MDR) such as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). New antibiotics are urgently needed to cope with the increasing antimicrobial resistance (AMR) emerging in these pathogens because this is expected to give high annual global mortality and a high economic burden (World Health Organization, 2015).
Bacteria can become antibiotic resistant by harboring mutations in endogenous genes, or by taking up genes. This lack or gain of gene product may give them a functional advantage to resist the antibiotic. Cellular stress, for example, induced by antibiotic treatments, can activate the SOS damage response system (Beaber et al., 2004) and thereby DNA translesion synthesis (TLS) in bacteria (Pham et al., 2001; Goodman, 2002). TLS increases the mutation frequency and is the main cause of increased levels of endogenous mutations (Merrikh and Kohli, 2020). Targeting the SOS response is therefore a potential strategy for inhibiting mutagenesis and development of antibiotic resistance (Yakimov et al., 2021).
Antimicrobial peptides (AMPs) are one of the drug classes emerging as an alternative to conventional antibiotics. They usually act by targeting the bacterial cell wall but can also have intracellular targets. The major hurdle for AMP drug development has been low serum stability and toxicity (Magana et al., 2020). Another concern with AMPs is the development of cross-resistance, as prolonged bacterial exposure to one AMP in sublethal doses is shown to lead to resistance development to a wide variety of other AMPs; however, this is dependent on the nature of the peptides and their target(s) (Andersson et al., 2016).
APIM-peptides are cell-penetrating peptides (CPPs) containing the proliferating cell nuclear antigen (PCNA) interaction motif APIM, which were originally developed as anticancer drugs (Gilljam et al., 2009; Muller et al., 2013; Søgaard et al., 2018). Interestingly, they were found to have antibacterial properties in selected gram-positive (G+) and gram-negative (G–) bacteria (Nedal et al., 2020). This antibacterial property was mainly due to their ability to bind to the bacterial β-clamp via their APIM sequence, thereby inhibiting bacterial DNA replication and TLS. This killed the bacteria or, at sublethal concentrations, reduced their ability to develop resistance against other antibiotics if used in combination treatments (Nedal et al., 2020; Raeder et al., 2021). The APIM-peptide variant ATX-101, which is under development as an anticancer drug, was shown to have a favorable toxicity profile in a recent Phase I study (Lemech et al., 2021). Therefore, the two main concerns with AMPs, i.e., development of resistance and toxicity, may not apply to APIM-peptides.
Skin is the main physical barrier against bacteria. A bruise or an open cut after surgery makes the underlying tissue vulnerable to infection, and accordingly, use of topical antibiotics is shown to prevent infections and accelerate healing. However, the rise and spread of MDR bacteria has led to severe chronic infections in hospitalized patients where current antibiotics are ineffective (Filius and Gyssens, 2002). MDR variants of staphylococci are examples of bacteria that cause recurring infections in hospitalized patients. Staphylococcus epidermidis is a bacterium in the normal skin microbiota (Kloos and Musselwhite, 1975) and S. aureus, which is more virulent (Massey et al., 2006; Otto, 2009), is more common in the microbiota of the upper respiratory tract (Tulloch, 1954). Both species can become opportunistic pathogens post surgery, especially in immunocompromised patients and those with medical implants. S. aureus can in addition thrive intracellularly, making it hard to treat with antibiotics (Tuchscherr et al., 2011).
In wound healing, keratinocytes migrate toward the open gap after 24 h and protect the underlying cells before dermal layers take over and close the gap (Rousselle et al., 2019). In order to develop a drug for topical application, it is important that the reepithelialization capacity of the keratinocytes surrounding the wound area is not severely affected (Pastar et al., 2014). In this study, we selected a lead APIM-peptide, betatide, and examined its antibacterial potential and its effects on epithelialization of keratinocytes in two different cell line-based in vitro infection assays. We also examined the ability of bacteria to develop resistance against betatide and betatide’s activity on resistant and reference ESKAPE pathogens, alone and in combination with selected antibiotics.
All bacterial strains used in this study are listed in Table 1. The reference strains are indicated by their ATCC and CCUG numbers, while the clinical strains, which were obtained from the strain collection at the Department of Medical Microbiology, St. Olav’s (SO) University Hospital, are indicated by their SO codes.
Bacterial strains used in this study.
For the clinical strains, this was essentially done as defined by EUCAST Clinical Breakpoints and guidance (EUCAST, 2021).
APIM-peptides (Innovagen, SE) used in this study have the same N-termini but differ in the composition of linkers and/or CPPs as shown in Table 2. Peptides 1 (RWLVK) and 2 (RWLVK*) are previously used in Nedal et al. (2020). A C-terminus FAM-labeled betatide (Innovagen) was used to study intracellular import. All the concentrations of APIM-peptides given in the different figures are net peptide concentrations, and 4 μg/ml equals approximately 1 μM.
Properties of APIM-peptide variants. MIC, Minimum inhibitory concentration. *All peptides were acetylated on the N-termini and amidated on the C-termini. #Peptides 1 and 2 are named RWLVK and RWLVK*, respectively, in Nedal et al. (2020). "++" to "+++++" denotes degree of reduced viability and mutation frequency relative to untreated control; "–" no effect; "+" tendency, but not a significant reduction; ND, not done. The raw data are shown in Supplementary Figures 1–3.
HaCaT, a human spontaneous immortalized keratinocyte cell line, was cultured in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/L glucose; Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1 mM L-glutamine (Sigma-Aldrich). HEK293, an immortalized embryonic kidney cell line, was grown in DMEM (BioWhittaker, Walkersville, MD, United States) with the same supplements as described above. In addition, Fungizone amphotericin B (2.5 μg/ml; Gibco, Thermo Fisher Scientific, Waltham, MA, United States) and 1 mM antibiotic mixture containing 100 μg/ml penicillin and 100 μg/ml Streptomycin (Gibco) were added to the growth media. The cells were incubated at 37°C in a humidified incubator with 5% CO2.
Minimal inhibitory concentration assay was conducted as recommended by the Clinical and Laboratory Standards Institute (CLSI) (Cockerill et al., 2012), similar to a previous report (Nedal et al., 2020). Briefly, bacterial colonies from blood agar plates were suspended and grown in Cation-Adjusted Mueller-Hinton Broth (CAMHB, 22.5 mg/ml Ca2+, 11 mg/ml Mg2+). The bacterial suspension was adjusted to 0.5 McFarland standard (∼1 × 108 colony-forming units (CFU)/ml) and serial diluted 1:200 in CAMHB (∼5 × 105 CFU/mL). This was subsequently added to polypropylene microtiter plates (Greiner, 100 μl/well, ∼5 × 104 CFU/well) already prepared with betatide and different antibiotics as single agents or in combinations (11 μl/well, twofold serial dilutions). The suspension was plated out on blood agar plates to conf
