Photoinactivation of Gram Positive and Gram Negative Bacteria with the Antimicrobial Peptide (KLAKLAK)₂ Conjugated to the Hydrophilic Photosensitizer Eosin Y

Abstract

We test the hypothesis that the antimicrobial peptide (KLAKLAK)(2) enhances the photodynamic activity of the photosensitizer eosin Y upon conjugation. The conjugate eosin-(KLAKLAK)(2) was obtained by solid-phase peptide synthesis. Photoinactivation assays were performed against the Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and multidrug resistant Acinetobacter baumannii AYE, as well as the Gram-positive bacteria Staphylococcus aureus , and Staphylococcus epidermidis . Partitioning assays were performed with E. coli and S. aureus . Photohemolysis and photokilling assays were also performed to assess the photodynamic activity of the conjugate toward mammalian cells. Eosin-(KLAKLAK)(2) photoinactivates 99.999% of 10(8) CFU/mL of most bacteria tested at a concentration of 1 μM or below. In contrast, neither eosin Y nor (KLAKLAK)(2) cause any significant photoinactivation under similar conditions. The increase in photodynamic activity of the photosensitizer conferred by the antimicrobial peptide is in part due to the fact that (KLAKLAK)(2) promotes the association of eosin Y to bacteria. Eosin-(KLAKLAK)(2) does not significantly associate with red blood cells or the cultured mammalian cell lines HaCaT, COS-7, and COLO 316. Consequently, little photodamage or photokilling is observed with these cells under conditions for which bacterial photoinactivation is achieved. The peptide (KLAKLAK)(2) therefore significantly enhances the photodynamic activity of eosin Y toward both Gram-positive and Gram-negative bacteria while interacting minimally with human cells. Overall, our results suggest that antimicrobial peptides such as (KLAKLAK)(2) might serve as attractive agents that can target photosensitizers to bacteria specifically.

Improved Bioactivity of Antimicrobial Peptides by Addition of Amino‐Terminal Copper and Nickel (ATCUN) Binding Motifs

“…Since ROS formation can induce damage to bacterial membranes, [16, 40] we then evaluated whether the diffusible and short-lived ROS generated by the ATCUN–AMPs bound to copper ions (Cu–ATCUN–AMP) could potentially destabilize the bacterial membrane through oxidative stress. The oxidative damage brought about by ROS production was assessed by measuring lipid peroxidation in small unilamellar vesicles (SUVs) that mimic the E. coli outer membrane.…”

“…Conjugation of photosensitizers to AMPs have led to improved antimicrobial action due to the production of ROS upon irradiation with visible light. [16–18] …”

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro

“…Johnson et al reported that the peptide (KLAKLAK) 2 greatly enhances the photodynamic activity of eosin Y, and that eosin-(KLAKLAK) 2 can photoinactivate Gram-positive and Gram-negative strains to a similar extent [41]. Estevão et al have shown that eosin Y and its ester derivatives in micelles are promising photosensitizers for photodynamic applications because increasing hydrophobicity of the photosensitizer promotes a higher interaction with biological membranes [42].…”

Detergent-induced self-assembly and controllable photosensitizer activity of diester phenylene ethynylenes

“…The possibilities of spatially localized generation of reactive oxygen species (ROS) in response to irradiation are only just beginning to be explored, despite the more than 100-y history of phototherapy in modern medicine (1), and are already in the clinic in the form of photodynamic therapy (PDT) for cancers of the skin, esophagus, and organ linings, actinic keratosis, and acne (2,3). Photodynamic destruction of pathogenic bacteria, viruses, and fungi is also under investigation for antibiowarfare applications, passive sanitization of hospital surfaces under room light, and active sanitization of medical devices such as catheters (4)(5)(6)(7)(8). A major drawback of systemically dosed PDT photosensitizers, which are primarily porphyrins or their prodrugs (9), is their accumulation in the skin and eyes leading to long-lasting (weeks to months) posttherapeutic photosensitivity (10).…”