Antimicrobial peptide developed with machine learning sequence optimization targets drug resistant Staphylococcus aureus in mice
Biswajit Mishra, … , Paul P. Sotiriadis, Eleftherios Mylonakis
Biswajit Mishra, … , Paul P. Sotiriadis, Eleftherios Mylonakis
Published April 22, 2025
Citation Information: J Clin Invest. 2025;135(12):e185430. https://doi.org/10.1172/JCI185430.
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Research Article Infectious disease Microbiology

Antimicrobial peptide developed with machine learning sequence optimization targets drug resistant Staphylococcus aureus in mice

Abstract

As antimicrobial resistance rises, new antibacterial candidates are urgently needed. Using sequence space information from over 14,743 functional antimicrobial peptides (AMPs), we improved the antimicrobial properties of citropin 1.1, an AMP with weak antimethicillin resistant Staphylococcus aureus (MRSA) activity, producing a short and potent antistaphylococcal peptide, CIT-8 (13 residues). At 40 μg/mL, CIT-8 eradicated 1 × 108 drug-resistant MRSA and vancomycin resistant S. aureus (VRSA) persister cells within 30 minutes of exposure and reduced the number of viable biofilm cells of MRSA and VRSA by 3 log10 and 4 log10 in established biofilms, respectively. CIT-8 (at 32 μg/mL) depolarized and permeated the S. aureus MW2 membrane. In a mouse model of MRSA skin infection, CIT-8 (2% w/w in petroleum jelly) significantly reduced the bacterial burden by 2.3 log10 (P < 0.0001). Our methodology accelerated AMP design by combining traditional peptide design strategies, such as truncation, substitution, and structure-guided alteration, with machine learning–backed sequence optimization.

Authors

Biswajit Mishra, Anindya Basu, Fadi Shehadeh, LewisOscar Felix, Sai Sundeep Kollala, Yashpal Singh Chhonker, Mandar T. Naik, Charilaos Dellis, Liyang Zhang, Narchonai Ganesan, Daryl J. Murry, Jianhua Gu, Michael B. Sherman, Frederick M. Ausubel, Paul P. Sotiriadis, Eleftherios Mylonakis

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Figure 3

Antibiofilm and antipersister activity of CIT-8.

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Antibiofilm and antipersister activity of CIT-8. (A and B) Killing kinet...
(A and B) Killing kinetics of CIT-8 against S. aureus MW2 in (A) exponential phase and (B) gentamicin-induced persister cells at concentrations of 4 and 40 μg/mL, compared with untreated bacterial (BC) control, colony forming units (CFU) counts were monitored for 120 minutes (n = 2, replicated thrice). (C and D) Killing kinetics of CIT-8 against S. aureus strain VRS1 in (C) exponential phase and (D) gentamicin-induced persister cells at 4 and 40 μg/mL, CFU counts were monitored for 120 minutes (n = 2, replicated thrice). We included ciprofloxacin (cipro) (at 10 μg/mL) and linezolid (at 100 μg/mL) as antibiotic controls and bithionol (at 10 μg/mL) as a positive control. (E and F) Disruption of 24 hour established biofilms of (E) MRSA (S. aureus MW2), and (F) VRSA (S. aureus VRS1) by CIT-8, measured as log reductions in bacterial loads on solid membranes treated with 4 and 40 μg/mL of CIT-8 (n = 6, *P < 0.05 by 1-way ANOVA followed by Dunnett’s multiple comparison test). We included 10 μg/mL vancomycin (Vanc) as control. (G and H) Inhibition of S. aureus MW2 biofilm formation by CIT-8 at concentrations ranging from 4–32 μg/mL after 24 hours of treatment, assessed using (G) live-cell viability (XTT assay), and (H) biomass quantification (crystal violet staining) (n = 3, replicated twice). (I and J) Disruption of 24 hours S. aureus MW2 established biofilms by CIT-8 at 4–32 μg/mL, evaluated by (I) reductions in live-cell viability (XTT assay) and (J) biomass loss (crystal violet staining) (n = 3, replicated twice). (K and L) Fluorescence microscopy images (10×) of 24 hour-established S. aureus MW2 biofilms stained with live/dead staining, (K) untreated control, and (L) biofilms treated with 32 μg/ml of CIT-8. Scale bars: 0.05 mm.