Solubility affects IL-1β-producing activity of the synthetic candidalysin peptide

Abstract

Candidalysin, a peptide toxin produced specifically from hyphae of Candida albicans, plays a crucial role in C. albicans pathogenesis in the oral cavity and vagina. Synthetic peptides have been widely used in previous studies to investigate the bioactivity of candidalysin. Although the solubility of the peptide, which is expected to have a hydrophobic property, has not been well characterized, candidalysin solutions are usually prepared in water. In this study, we prepared the synthetic peptide candidalysin in water (CLw) or in dimethyl sulfoxide (CLd) and compared their cytotoxicity and interleukin (IL)-1β-producing activity to determine whether the activity of the peptide would be affected. In addition, we evaluated whether the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway or other pathways were involved in their activities. Unexpectedly, we found that CLw was not completely solubilized and contained abundant insoluble microparticles. CLw was active at comparably high concentrations (≥ 10 μM). In contrast, CLd is completely solubilized and sufficiently active at low concentrations, that is, 1 μM or less. CLw showed weak cytotoxicity and NLRP3-dependent and cathepsin B-dependent IL-1β-producing activity, whereas CLd showed strong cytotoxicity and cathepsin B-dependent IL-1β-producing activity. Fractionation of CLw revealed that NLRP3-dependent activity was caused by insoluble microparticles. Furthermore, nanoparticle tracking of CLd revealed that the peptide was present as nanoparticles with a size of 96 nm. CLw contained a small amount of such nanoparticles. Thus, the bioactivities of the synthetic peptide candidalysin, especially the IL-1β-producing activity, are affected by the solubility of the peptide depending on the solvent employed. The NLRP3-dependent activity of the synthetic peptide is caused by insoluble microparticles and may not be the intrinsic activity of candidalysin.

Nanobody-mediated neutralization of candidalysin prevents epithelial damage and inflammatory responses that drive vulvovaginal candidiasis pathogenesis

“…Our in vitro data indicated that the nanobody may exhibit an even increased efficacy in vivo , since we observed that nanobodies were more effective at neutralizing damage caused by C. albicans (multiplicity of infection [MOI] 1) compared to the addition of synthetic candidalysin to epithelial cells. This phenomenon might be explained by spontaneous aggregation and clumping of the synthetic toxin in aqueous solution ( 17 ) and slower and more controlled release of lower concentrations of native candidalysin by C. albicans hyphae.…”

“…This structure is likely required to prevent autoaggregation owing to the amphipathic and hydrophobic features of the candidalysin peptide ( 16 ). In fact, synthetic candidalysin spontaneously forms aggregates in aqueous solution ( 17 ).…”

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage byCandida albicans

“…Cell culture. The TR146 human buccal epithelial squamous cell carcinoma cells (MilliporeSigma) 50 were incubated in DMEM/F12 medium (Gibco, #11320033) with 10% fetal bovine serum and 1x penicillin-streptomycin (Gibco, #15140122). A431 vulval epithelial cells (MilliporeSigma) and HEK-293T cells (ATCC) were cultured in DMEM media supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine (Corning, #25005-CL).…”

“…This toxin stimulates an innate epithelial immune response by activating the epidermal growth factor receptor (EGFR) 3,11 , the mitogen-activated protein kinases P38 and extracellular regulated kinases (ERK)1/2 [12][13][14][15] , and the c-Fos transcription factor 12,14 . These signaling pathways trigger the production of downstream inflammatory mediators such as IL-6, GM-CSF, CXCL8 and IL-1β, which recruit phagocytes to foci of infection and enhance their fungicidal activities 2,3,11,16,17 .…”