Novel Cecropin-4 Derived Peptides against Methicillin-Resistant Staphylococcus aureus

Abstract

Increasing microbial resistance, coupled with a lack of new antimicrobial discovery, has led researchers to refocus on antimicrobial peptides (AMPs) as novel therapeutic candidates. Significantly, the less toxic cecropins have gained widespread attention for potential antibacterial agent development. However, the narrow activity spectrum and long sequence remain the primary limitations of this approach. In this study, we truncated and modified cecropin 4 (41 amino acids) by varying the charge and hydrophobicity balance to obtain smaller AMPs. The derivative peptide C18 (16 amino acids) demonstrated high antibacterial activity against Gram-negative and Gram-positive bacteria, as well as yeasts. Moreover, C18 demonstrated a minimal inhibitory concentration (MIC) of 4 µg/mL against the methicillin-resistant Staphylococcus aureus (MRSA) and showed synergy with daptomycin with a fractional inhibition concentration index (FICI) value of 0.313. Similar to traditional cecropins, C18 altered the membrane potential, increased fluidity, and caused membrane breakage at 32 µg/mL. Importantly, C18 eliminated 99% persisters at 10 × MIC within 20 min and reduced the biofilm adherence by ~40% and 35% at 32 and 16 µg/mL. Besides, C18 possessed a strong binding ability with DNA at 7.8 μM and down-regulated the expression of virulence factor genes like agrA, fnb-A, and clf-1 by more than 5-fold (p < 0.05). Interestingly, in the Galleria mellonella model, C18 rescued more than 80% of larva infected with the MRSA throughout 120-h post-infection at a single dose of 8 mg/kg (p < 0.05). In conclusion, this study provides a reference for the transformation of cecropin to derive small peptides and presents C18 as an attractive therapeutic candidate to be developed to treat severe MRSA infections.

In Vitro and In Vivo Bactericidal and Antibiofilm Efficacy of Alpha Mangostin Against Staphylococcus aureus Persister Cells

“…3903, Corning, NY, USA), we serially diluted 50 ml of alpha mangostin solution (64 -1 mg/ml). To this plate, we added a high density (1.5 OD 600nm ) of overnight culture (S. aureus MW2) and incubated at 37°C for 1 h. After incubation, we discarded the media and washed the wells carefully with PBS to remove any loosely attached or free-floating planktonic cells (Mishra et al, 2016;Peng et al, 2021). We added 20% of XTT to the wells after discarding the planktonic cells and incubated the plate for another 2 h at 37 °C.…”

“…We tested the efficacy of alpha mangostin in inhibiting biofilm formation by S. aureus using the assay described in (Mishra et al, 2016;Peng et al, 2021). We cultivated an overnight culture of S. aureus MW2 in TSB (supplemented with 0.2% glucose and 3% NaCl) media.…”

“…3903, Corning, NY, USA), we added 100 ml of the adjusted bacterial culture and incubated the plates at 37°C for 24 h in static conditions to allow robust biofilm formation. After incubation, we discarded the media and washed the wells carefully with PBS to remove any loosely attached or free-floating planktonic cells (Mishra et al, 2016;Peng et al, 2021). To the established S.aureus MW2 biofilms in 96-well plates, we added serially diluted alpha mangostin (64 -1 mg/ml) in fresh TSB media and incubated the plates for another 24 h. After discarding the media containing planktonic cells and washing the wells with PBS to remove loosely attached or free-floating cells, we added 20% XTT in TSB media to the established S. aureus MW2 biofilms in 96well plates.…”

“…Liver-derived HepG2 and lung-derived A549 cells were used to assess the mammalian cell cytotoxicity of alpha mangostin using previously described methods (Kim et al, 2018b;Peng et al, 2021). The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, MD, USA) and 1% penicillin/ streptomycin (Gibco, MD, USA) and maintained at 37°C in 5% CO 2 .…”

“…The values were represented as the mean of duplicates. The following formula calculated the percentage of hemolysis: (A540 nm in the peptide solution − A540 nm in PBS)/(A540 nm of 1% Triton X-100 treated sample − A540 nm in PBS) × 100 (Peng et al, 2021). Zero and 100% percentage hemolysis were determined in PBS and 0.1% Triton X-100, respectively.…”