Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacteriumtuberculosis.

Paul Walther,Steffen Stenger,Reiner Noschka,Fanny Wondany,Jens Michaelis,Gilbert Weidinger,Gönül Kizilsavas,Tanja Weil

doi:10.3390/ijms22168392

Abstract

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed “Gran1”). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

Highlights

Granulysin is an antimicrobial peptide (AMP) which is stored in the granules of human T-lymphocytes and natural killer cells [1]
We selected mycobacteria as the target organisms because (i) the parental Granulysin is active against extracellular Mycobacterium tuberculosis (Mtb) [2], (ii) mycobacteria are major human pathogens, (iii) mycobacteria are intrinsically resistant against most classes of antibiotics, (iv) the occurrence of infections with drug resistance strains is increasing, [16] and (v) the available treatment which lasts at least 6 months and consists of a multidrug-regimen with potentially severe side effects
The clinical application of large AMPs such as Granulysin is limited by off-target effects, poor penetration into the cytoplasm of microbial host cells, rapid degradation in human serum, and high manufacturing costs

Summary

Introduction

Granulysin is an antimicrobial peptide (AMP) which is stored in the granules of human T-lymphocytes and natural killer cells [1]. Granulysin mediates tissue damage in graft-versus-host disease and inflammatory skin diseases such as folliculitis, psoriasis, acne, or Steven Johnsons Syndrome [8] Besides these off-target effects, the large size of Granulysin (9 kDa, 74 aa) accounts for rapid degradation in serum, the requirement of high micromolar concentrations for activity, and high costs for manufacturing. It was shown that the peptide “G8” (23–51 aa) lysis the extracellular, gram negative rod Salmonella typhimurium [13] Based on these findings, we hypothesized that “G8” is active against extra- and intracellular Mtb without causing an inflammatory response. We demonstrate that Gran (previously described as “G8”) interacts with the mycobacterial cell wall and exerts antimicrobial activity against extra- and intracellular Mtb without triggering inflammatory immune responses or cell death in human cells or zebrafish embryos

Methods

Results

Conclusion