Antimicrobial Activity Of A Histone Derived Peptide In The Airway Surface Liquid

Abstract

Extracellular histones are viewed as cytotoxic markers in cell death, apoptosis and NETosis. However, extracellular histones have been found to carry out a number of roles outside their function as a damage associated molecular patterns to signal cell death. Extracellular histones have been seen to act as an early release marker of cell stress, influence cell growth, have antimicrobial activity and bioactive peptides. A suggested method for the intentional secretion of histones has been show through amphisomes, with histones being present in the proteome of healthy biological systems. Airway surface liquid (ASL), present in the lumen of the airways plays a critical role in airway defense and innate immunity. We identified a number of histone derived peptides by de novo peptide sequencing of the airway surface liquid. In ASL overlying H441, Calu3 and BMI-1 transduced human bronchial epithelial cells (HBEC) we identified similar histone peptides. This was supported by their presence in sputum samples from people with and without Cystic Fibrosis disease. To test whether the ASL histone peptides had a bioactive role we produced synthetic analogues using SPOT peptide synthesis. The peptides were then tested for effects on human embryonic kidney (HEK 293) cell growth, viability and intracellular Ca2+[Ca2+]i. One candidate, MAT9 when screened suppressed the rise in [Ca2+]i in response to thapsigargin (p=0.001, n=8 with 16 replicates), but did not affect cell viability or cell growth. When MAT9 (10µM) was tested on H441 airway epithelial cells, it increased the thapsigargin-induced rise in [Ca2+]i (P=0.01, n=6, with 12 replicates). This difference in calcium response could be due to a lack of toll-like receptors (known receptors for histone proteins) in HEK cells which are present in H441 cells. Alongside this MAT9 (10µM) inhibited growth of luminescent Pseudomonas aeruginosa and was able to significantly reduce the growth rate over 4 hours at 5µM (P=0.01, n=4, with 16 replicates). Outside of this antibacterial activity, preliminary data showed some activity against Sars-Cov2 replication in Caco-2 cells. This data indicates that MAT9 is a histone derived peptide with bioactivity, and its proline-alanine sequence may represent a chance to enhance this activity by changing its isomerization.