Designed Antimicrobial Peptides Against Trauma-Related Cutaneous Invasive Fungal Wound Infections

Abstract

Cutaneous invasive fungal wound infections after life-threatening dismounted complex blast injury (DCBI) and natural disasters complicate clinical care. These wounds often require aggressive repeated surgical debridement, can result in amputations and hemipelvectomies and have a 38% mortality rate. Given the substantial morbidity associated with cutaneous fungal wound infections, patients at risk need immediate empiric treatment mandating the use of rapidly acting broad-spectrum antimicrobials, acting on both fungi and bacteria, that are also effective against biofilm and can be administered topically. Designed antimicrobial peptides (dAMPs) are engineered analogues of innate antimicrobial peptides which provide the first line of defense against invading pathogens. The antifungal and antibacterial effect and mammalian cytotoxicity of seven innovative dAMPs, created by iterative structural analog revisions and physicochemical and functional testing were investigated. The dAMPs possess broad-spectrum antifungal activity, in addition to being effective against Gram-negative and Gram-positive bacteria, which is crucial as many wounds are polymicrobial and require immediate empiric treatment. Three of the most potent dAMPs-RP504, RP556 and RP557-possess limited mammalian cytotoxicity following 8 h incubation. If these encouraging broad-spectrum antimicrobial and rapid acting results are translated clinically, these novel dAMPs may become a first line empiric topical treatment for traumatic wound injuries.

Keywords: antimicrobial peptides; cutaneous invasive fungal wound infections; host defense peptides; topical treatment; wound infections.

Figure 1

Schematic relative hydrophobicity representation of the designed antimicrobial peptides. Single letter codes of the amino acids are depicted with dark blue representing the most hydrophobic amino acid, phenylalanine (F), through to the hydrophilic amino acids in red, lysine (K). Relative hydrophobicity represented by the energy, kcal/mole, necessary to move an amino acid from an aqueous phase to a lipid bilayer [22]: F, phenylalanine, −3.85; L, leucine, −3.36; I, isoleucine, −3.16; Y, tyrosine, −2.66; M, methionine, −2.34; V, valine, −2.34; W, tryptophan, −1.96; A, alanine, −1.56; C, cysteine, −1.06; G, glycine, −0.14; R, arginine, 2.22; O, ornithine, 3.56; and lysine, K, 3.85.

Figure 2

RP504, RP556 and RP557 induce limited mammalian cell toxicity. Concentration dependent viability analysis of murine L929 fibroblast cells following 8 h incubation. Cells were plated at 1×10⁴ cells/well, allowed to adhere overnight and the specific dAMP added, and cytotoxicity evaluated through 8 h. Cellular toxicity was assayed using a bioluminescent strain of fibroblasts and viability assayed using an IVIS Lumina imaging system (Perkin Elmer). Data shown represent the mean of triplicate replicates; statistically significant (* p < 0.05, ** p < 0.01, **** p < 0.0001), using one-way ANOVA followed by Dunnett’s analysis. For some points, the error bars are shorter than the height of the symbols.

Figure 3

RP553, RP556, RP557 rapidly destroy P. aeruginosa and S. aureus. The bioluminescence of viable P. aeruginosa 19,660 and S. aureus 49,525 cells was quantitated in real time with an IVIS Lumina bioimaging system. Data represent the mean ± SE of triplicate replicates from two independent experiments. For some points, the error bars are shorter than the height of the symbols.