Synergistic killing of bacterial pathogens by histones

Project Summary/Abstract The effective treatment of bacterial infections of skin, deep soft tissues and wounds continues to be a major unmet challenge in healthcare settings, especially among patients with chronic diabetes. Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria that are isolated from chronic, non- healing wounds. Antibiotic resistance has arisen in these particular bacteria, causing these infections to become increasingly difficult to treat and giving rise to multi-drug resistant strains, including Methicillin-resistant Staphylococcus aureus (MRSA). The goal of the proposed work is to develop the next generation of antimicrobials for which the design is inspired by a better mechanistic understanding of mammalian antimicrobial defense pathways. We focus our attention on the antimicrobial activities of neutrophil extracellular traps (NETs), which use histones to kill or suppress microbial proliferation. The antimicrobial mechanism of histones has not been understood. The Siryaporn and Gross labs recently reported that the pairing of histones with an additional component found in NETs – the antimicrobial peptide (AMP) LL-37 (cathelicidin) – produces potent antimicrobial synergy. LL-37 forms pores in the bacterial membrane, which enable histones to enter the bacterium and interfere with gene expression. This has an irreversible bactericidal (killing) effect on bacteria. The work proposed here will exploit this discovery by identifying combinations of human histones and membrane-/cell wall-targeting antimicrobials (MTAs) that produce potent antimicrobial activity and synergy. The overall objective of the project is to better understand the mechanism of antimicrobial synergy between histones and MTAs, and to harness it to establish a class of new therapeutics for the treatment of skin infections and wounds. We will accomplish this objective by identifying combinations of human histones with LL-37 and other MTAs that produce the greatest antimicrobial activities and synergies. We will test these against S. aureus, P. aeruginosa, and communities of skin bacteria in vitro (Aim 1). We will attempt to augment the antimicrobial activity by engineering in factors that impact histone function in NETs, specifically chemical modification through citrullination and tethering histones to DNA fibers (Aim 2). To validate our approach, we will test the combinations of histones and MTAs identified in Aims 1 and 2 in a standardized mouse skin infection model (Aim 3). To additionally address the unmet challenge of treating skin infection and wounds in diabetes patients, we will perform the tests in a diabetic mouse model. The results of this work will provide a mechanistic understanding of antimicrobial synergy and develop a strategy to combat the rise of antibiotic resistance. The results of the study could create a new class of antimicrobial therapeutics for the treatment of skin infections and wounds in diabetic and non-diabetic patients. This would represent a game-changer in the approach to antimicrobial treatments.