Activity and toxicity of a recombinant LL37 antimicrobial peptide

Abstract

Event Abstract Back to Event Activity and toxicity of a recombinant LL37 antimicrobial peptide Lindsay D. Lozeau1, Denis Kole2, Tanja Dominko3, Marsha Rolle2 and Terri A. Camesano1 1 Worcester Polytechnic Institute, Chemical Engineering, United States 2 Worcester Polytechnic Institute, Biomedical Engineering, United States 3 Worcester Polytechnic Institute, Biology and Biotechnology, United States Introduction: The increasing threat of antibiotic resistance has led to a worldwide call for alternative antimicrobial agents. Chronic wounds cost $24 billion per year [1] and can be populated by over 200 microbes at once [2] and are a special case requiring the development of non-toxic, broadly-active therapeutics at a low cost. Antimicrobial peptides (AMPs) such as human LL37 show promise for fulfilling this need due to their broad activity and wound healing ability. Their unique mechanisms and great diversity make them attractive as an alternative class of antimicrobials; however, clinical applications have been hindered by AMP degradation, high cost and lack of delivery methods. Attaching AMPs to surfaces has been shown to slow their degradation, decrease their toxicity, and allow cost-efficient local delivery [3]. Our goal is to achieve AMP tethering in the wound site to promote healing and prevent infection by creating recombinant LL37 fused to collagen binding domains (CBD). Collagen is prevalent in wounds and scaffold materials used to treat wounds; thus, it provides a suitable substrate for CBD-LL37 tethering. The focus of this study is to evaluate the antimicrobial activity and toxicity of recombinant and synthetic CBD-LL37 compared to unmodified LL37 with no CBD. Methods: Antimicrobial Activity: H1299 cells expressing recombinant CBD-LL37 were grown in RPMI with 10% serum until confluent, followed by addition of serum-free RPMI for 48 or 90hrs and collection of conditioned medium (CM). CM from untransfected cells, synthetic CBD-LL37 and LL37 without the CBD were used as controls. Serial dilutions of each were made and exposed to six microbial strains at 105 cfu/mL. Absorbance (590nm) was measured over time to determine relative bacterial growth rates (A590/Time) or complete inhibition of visible growth, defined by the minimal inhibitory concentration (MIC). Toxicity: CT 1005 fibroblasts were seeded onto 96-well plates, incubated overnight, rinsed and exposed to serial dilutions of synthetic LL37 or CBD-LL37 for 1 hr. An MTT assay was performed to determine cell viability. Results and Discussion: Synthetic LL37 and CBD-LL37 demonstrated MICs of <10uM against E. coli, P. aeruginosa, S. aureus, S. epidermidis, MRSA and B. subtilis, indicating that each AMP has broad activity. The MICs for LL37 and CBD-LL37 against four microbes were not statistically different (ANOVA p<0.05), indicating that the addition of CBD did not largely alter LL37 activity. Recombinant CBD-LL37 in undiluted CM demonstrated slowed growth rates for all microbes compared to untransfected H1299 CM; thus, recombinant production using mammalian cells did not seem to alter activity. Synthetic CBD-LL37 in solution allowed fibroblast proliferation between 0.1-10 uM, had no effect below 0.1 uM and decreased fibroblast proliferation above 10 uM, similar to reported literature values for LL37 [4], indicating that CBD addition alone does not alter LL37 toxicity. Conclusion: Together, these studies show that CBD-LL37 (recombinantly produced and synthetic) has similar activities as LL37, providing a foundation for future collagen-binding assays and the development of AMP-based chronic wound treatments. The authors acknowledge funding from the National Science Foundation IGERT (NSF DGE 1144804); The authors acknowledge funding from the National Science Foundation STTR (NSF IIP 1521294)