# Potential Molecular Targets of the Broad-Range Antimicrobial Peptide Tyrothricin in the Apicomplexan Parasite Toxoplasma gondii

## TL;DR

This study explores how the antimicrobial peptide tyrothricin affects the parasite Toxoplasma gondii, identifying potential molecular targets and effects on parasite survival.

## Contribution

The study identifies specific molecular targets of tyrocidine A in T. gondii using DAC-MS-proteomics, revealing multiple pathways affected by the peptide.

## Key findings

- Tyrocidine A inhibited T. gondii proliferation at IC50s < 100 nM, while gramicidin A was inactive.
- Electron microscopy showed cytoplasmic vacuolization and mitochondrial changes in treated parasites.
- DAC-MS-proteomics identified 521 proteins specifically binding to tyrocidine A, including SRS antigens and mitochondrial proteins.

## Abstract

Background: The apicomplexan parasite Toxoplasma gondii causes serious diseases in animals and humans. The in vitro efficacy of the antimicrobial peptide mixture tyrothricin, composed of tyrocidines and gramicidins, against T. gondii tachyzoites was investigated. Methods: Effects against T. gondii were determined by monitoring inhibition of tachyzoite proliferation and electron microscopy, host cell and splenocyte toxicity was measured by Alamar blue assay, and early embryo toxicity was assessed using zebrafish embryos. Differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was employed to identify potential molecular targets in T. gondii cell-free extracts. Results: Tyrothricin inhibited T. gondii proliferation at IC50s < 100 nM, with tyrocidine A being the active and gramicidin A the inactive component. Tyrothricin also impaired fibroblast, T cell and zebrafish embryo viability at 1 µM. Electron microscopy carried out after 6 h of treatment revealed cytoplasmic vacuolization and structural alterations in the parasite mitochondrion, but these changes appeared only transiently, and tachyzoites recovered after 96 h. Tyrothricin also induced a reduction in the mitochondrial membrane potential. DAC-MS-proteomics identified 521 proteins binding only to tyrocidine A. No specific binding to gramicidin A was noted, and four proteins were common to both peptides. Among the proteins binding specifically to tyrocidine A were several SRS surface antigens and secretory proteins, mitochondrial inner and outer membrane proteins associated with the electron transfer chain and porin, and several calcium-binding proteins putatively involved in signaling. Discussion: These results suggest that tyrocidine A potentially affected multiple pathways important for parasite survival and development.