The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

Abstract

The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.

Development and evaluation of cationic amphiphilic antimicrobial 2,5‐diketopiperazines

“…It was shown that LTX109 compared favorably with both terbinafine and amorolfine for the ex vivo treatment of these persistent fungal infections in a very rapid fashion. The fungicidal mode of action of LTX109 on the fungal cells has also been studied and shown to operate via nonspecific destabilization of the fungal plasma membrane . Given these very promising results from the earlier antifungal studies, and a structural resemblance to the highly active antifungal aryl‐alkyl‐lysines recently reported by Haldar and coworkers, it was decided to also screen a selection ( 9 , 12 , 13 , 15 , 16 , and 18 ) of the prepared DKPs against a panel of 5 human pathogenic fungi from the Candida genus (Table ).…”

“…The fungicidal mode of action of LTX109 on the fungal cells has also been studied and shown to operate via nonspecific destabilization of the fungal plasma membrane. 68 Given these very promising results from the earlier antifungal studies, and a structural resemblance to the highly active antifungal aryl-alkyl-lysines recently reported by…”

Antimicrobial peptides under clinical investigation

“…The phase IIa clinical trial was completed by LytixBiopharma for the topical treatment of impetigo and nasal decolonization of MRSA. Additionally, the preclinical study for the treatment of diabetic foot ulcers via topical administration is ongoing [5,66,88].…”

Engineering of membrane complex sphingolipids improves osmotic tolerance of Saccharomyces cerevisiae

“…For example, when sugar and ion transporter, OmpF, was deleted, and the long chain fatty acids transporter, FadL, was overexpressed in E. coli , membrane integrity was enhanced and the fatty acid titer improved (39); (ii) Phospholipids: Membrane integrity can also be altered by modifying the distribution of phospholipid head groups, by adjusting phospholipid saturation, and by altering phospholipid acyl chain length (14, 40). For instance, when pssA, a phosphatidylserine synthase, was overexpressed, PE content increased and membrane integrity was enhanced, as a result of which bio-renewable fuel tolerance and titer was improved (12); (iii) Sterols: Sterol serves as an essential cell membrane component, where deletion of CgMed3AB decreased lanosterol, zymosterol, fecosterol, and ergosterol content, thereby reducing the membrane integrity of Candida glabrata (19); (iv) Sphingolipids: Sphingolipid metabolism plays an important role in membrane integrity (41). When sphingolipid biosynthesis genes were deleted in S. cerevisiae , the resultant strains exhibited resistance to amphipathic peptidomimetic LTX109, which decreased membrane integrity (41).…”