Delineating the Mechanism of Action of a Protease Resistant and Salt Tolerant Synthetic Antimicrobial Peptide against Pseudomonas aeruginosa

Rapidly growing antimicrobial resistance (AMR) against antibiotics has propelled the development of synthetic antimicrobial peptides (AMPs) as potential antimicrobial agents. An antimicrobial peptide Nle-Dab-Trp-Nle-Dab-Dab-Nle-CONH2 (P36; Nle = norleucine, Dab = diaminobutyric acid, Trp = tryptophan) potent against Pseudomonas aeruginosa (P. aeruginosa) has been developed in the present study. Rational design strategy adopted in this study led to the improvisation of the therapeutic qualities such as activity, salt tolerance, cytotoxicity, and protease resistance of the template peptide P4, which was earlier reported from our group. P36 exhibited salt tolerant antimicrobial potency against P. aeruginosa, along with very low cytotoxicity against mammalian cell lines. P36 was found to be nonhemolytic and resistant toward protease degradation which qualified it as a potent antimicrobial agent. We have investigated the mechanism of action of this molecule in detail using several experimental techniques (spectroscopic, biophysical, and microscopic) and molecular dynamics simulations. P36 was a membrane active AMP with membrane destabilization and deformation abilities, leading to leakage of the intracellular materials and causing eventual cell death. The interaction between P36 and the microbial membrane/membrane mimics was primarily driven by electrostatics. P36 was unstructured in water and upon binding to the microbial membrane mimic SDS, suggesting no influence of secondary structure on its antimicrobial potency. Positive charge, optimum hydrophobic–hydrophilic balance, and chain length remained the most important concerns to be addressed while designing small cationic antimicrobial peptides.


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All the experiments were performed in triplets. Figure S35: CD spectral study of P31 to P37 in presence of different membrane mimetic environments: SDS mimics the bacterial membrane and DPC mimics the mammalian membrane. 50% TFE promotes the alpha helical propensity.      Figure S1. a), temporally averaged dCOM (averaging over 2 independent MD runs, see Table S1) is plotted. (a) P32-micelle (SDS/DPC) dCOM plot.

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(b) P36-micelle (SDS/DPC) dCOM plot. The black horizontal line is the averaged dCOM obtained from the last 30 ns of the 50ns trajectory that corresponds to stable peptide: SDS complex.

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Supplementary Tables   Table S1. Peptide (P32/P36) in the presence and absence of micelle (SDS/DPC) is considered for MD simulations. Initial structural model (see Figure S27.a), the number of independent replicas (varying the initial velocities), box size, composition and post-equilibrated MD runlength. A total of 810 ns of production MD was performed.   Table S2. Parameters used in MD simulations.

Peptide Purification and Characterization.
Crude peptides were purified by reverse-phase high-performance liquid chromatography (Thermo Scientific Dionex Ultimate 3000) on a semi preparative Biobasic 8 column using binary {CH3CN-H2O (5−100%)} solvent system at a flow rate of 5 mL/min using dual UV detection at 214 and 280 nm. Purity of the peptides were confirmed using a Thermo Scientific Dionex Ultimate 3000 analytical HPLC system and a Thermo Scientific BioBasic 18 analytical column. A flow rate of 1 ml/min and a linear gradient of 10 to100% CH3CN-H2O was used.
The purified peptides were characterized by mass spectrometry on Agilent-Q-TOF 6500 instrument in electrospray ionization positive mode, equipped with Mass Hunter workstation software. The purified peptides were further characterized by 1 H-NMR spectroscopy on a Bruker Ascend Aeon 600 MHz spectrometer at 298 K in D2O solvent. Peptide stock solution was prepared either in water, in sodium phosphate buffer (pH 7.4), or both and filter-sterilized unless stated otherwise.

Microbial Culture.
For antimicrobial activity assay, P. aeruginosa, K. pneumoniae and S. aureus purchased from MTCC, Chandigarh, were grown in nutrient broth and maintained at 310 K under shaking conditions.

Cell Culture
Human normal embryonic kidney (HEK-293) cells were purchased from the National Centre for Cell Science (NCCS, Pune, India). The cell line was maintained in DMEM supplemented with 10% (v/v) fetal bovine serum and 1% penicillin and streptomycin at 310 K in humidified air containing 5% CO2.