KDEON WK-11: A short antipseudomonal peptide with promising potential

Abstract

The plight of antimicrobial resistance continues to limit the availability of antibiotic treatment effective in combating resistant bacterial infections. Despite efforts made to rectify this issue and minimise its effects on both patients and the wider community, progress in this area remains minimal. Here, we de-novo designed a peptide named KDEON WK-11, building on previous work establishing effective residues and structures active in distinguished antimicrobial peptides such as lactoferrin. We assessed its antimicrobial activity against an array of bacterial strains and identified its most potent effect, against Pseudomonas aeruginosa with an MIC value of 3.12 μM, lower than its counterparts developed with similar residues and chain lengths. We then determined its anti-biofilm properties, potential mechanism of action and in vitro cytotoxicity. We identified that KDEON WK-11 had a broad range of antimicrobial activity and specific capabilities to fight Pseudomonas aeruginosa with low in vitro cytotoxicity and promising potential to express anti-lipopolysaccharide qualities, which could be exploited to expand its properties into an anti-sepsis agent.

Keywords: Pseudomonas aeruginosa; antibiofilm activity; antimicrobial peptides; lipopolysaccharides; tryptophan.

FIGURE 1

Bacterial cells viability expressed as CFU/mL after 18 h of peptide treatment. Dotted line represents initial inoculum density. Data represent the mean ± SEM of three independent experiments.

FIGURE 2

Membrane perturbation induced by KDEON as detected by the Sytox Green assay. Time 0 represents peptide addition, after stabilization of the fluorescence signal of the bacterial suspension in 1 μM Sytox Green. Values correspond to one representative experiment of three.

FIGURE 3

Killing kinetic of KDEON peptide against P. aeruginosa ATCC 27853 evaluated by CFU counting after 5, 15, 30 and 60 min of peptide treatment. Dotted line represents initial inoculum density. Data represent the mean ± SEM of three independent experiments.

FIGURE 4

Metabolic activity of P. aeruginosa ATCC 27853 biofilm after treatment with KDEON WK-11 at concentrations of 3.12, 6.25, 12.5, 25, 50 and 100 μM for 2 h in PBS. Data represent the mean ± standard error of the mean (SEM) of three independent experiments.

FIGURE 5

Percentage of biofilm biomass at different sub-MIC concentration of KDEON WK-11 (gold bars) in comparison with the microbial growth (red bars). Biofilm biomass was quantified by CV staining, while the microbial growth was evaluated by reading the absorbance at 590 nm. Data represent the mean ± SEM of three independent experiments.

FIGURE 6

Percentage of pyoverdine production at 1/2, 1/4, and 1/8 x MIC of KDEON WK-11. Data represent the mean ± SEM of three independent experiments.

FIGURE 7

Fold increase in MIC of KDEON WK-11 and colistin against the clinical isolate P. aeruginosa 19595 after 15 cycles of treatment. The experiment was conducted in two independent replicates. The value represent the mean ± standard deviation (SD) of the fold increase in MIC of these two replicates. When SD is 0, the MIC values of both replicates were the same.

FIGURE 8

Electrospray ionisation mass spectra showing Lipid A binding to KDEON WK-11 represented by ion complex formation seen as single peaks expressing the molecular mass of both components. Red circles present at peaks 1103 and 1173 indicate the presence of the peptide-ion complex, proving the binding of KDEON WK-11. Blue circles at peaks 430, 573 and 860 (m/z), indicate the peptide alone (4+, 3+, 2 + charge state respectively of WK11), showing the significant difference between the two, in the presence and absence of ion complex formation. The inset indicates the spectrum of the peptide alone.

FIGURE 9

(A) Predicted LPS-binding of KDEON WK-11; (B) Structural predictions showing the amphipathic character of the helical wheel projection of KDEON WK-11. Blue and yellow colors indicate basic hydrophilic and hydrophobic residuces respectively.

FIGURE 10

Cytotoxicity of KDEON WK-11 as expressed in percentage cell viability assessed by the MTT assay testing varying concentrations of peptide from 1.56 to 50 μM. Data represent the mean ± SEM of three independent experiments.