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Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus., Author summary Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving disease pathology. In this study, we aimed to determine if genetic variation of ECE1 existed across a collection of C. albicans clinical isolates. Using a targeted sequencing approach, we identified a conserved variant candidalysin isoform. We then constructed isogenic chimeric strains harboring swapped Ece1p peptides or HiBiT tags between representative isoforms and showed impaired secretion with the variant that was associated with reduced virulence in vitro and in vivo. We also elucidated a conserved pattern between key sequences near lysine-arginine cleavage sites involved in Ece1p polypeptide processing by the Kex2p enzyme that correlate with predicted and experimental candidalysin secretion. Therefore, we present a novel mechanism for attenuation of C. albicans virulence at the ECE1 locus that may partly underlie differential clinical outcomes and contribute to the balance between commensal and pathogen at the vaginal mucosa.

The acidity-triggered rational membrane (ATRAM) peptide was designed to target acidic diseases such as cancer. An acidic extracellular medium, such as that found in aggressive tumors, drives the protonation of the glutamic acids in ATRAM, leading to the membrane translocation of its C-terminus and the formation of a transmembrane helix. Compared to healthy cells, cancerous cells often increase exposure of the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane. Here we use a reconstituted vesicle system to explore how phosphatidylserine influences the interaction of ATRAM with membranes. To explore this, we used two new variants of ATRAM, termed K2-ATRAM and Y-ATRAM, with small modifications at the non-inserting N-terminus. We observed that the effect of PS on the membrane insertion pK and lipid partitioning hinged on the sequence of the non-inserting end. Our data additionally indicate that the effect of PS on the insertion pK does not merely depend on electrostatics, but it is multifactorial. Here we show how small sequence changes can impact the interaction of a peptide with membranes of mixed lipid composition. These data illustrate how model studies using neutral bilayers, which do not mimic the negative charge found in the plasma membrane of cancer cells, may fail to capture important aspects of the interaction of anticancer peptides with tumor cells. This information can guide the design of therapeutic peptides that target the acidic environments of different diseased tissues.Statement of SignificanceCurrent targeted therapies for cancer have limited success due to drug resistance. Resistance often arises after mutation of the receptor being targeted. A more general target is needed to prevent drug resistance. Most aggressive solid tumors have an extracellular medium. We propose that extracellular acidity is promising for improved targeted therapies. The acidity-triggered rational membrane (ATRAM) inserts in membranes only under acidic conditions. However, it is now known how the lipid changes that occur in the plasma membrane of cancer cells impact the membrane insertion of ATRAM. Here we perform biophysical experiments that show that PS, a lipid exposed in the cancer cell, can impact the membrane insertion of ATRAM. We also uncovered a region of the peptide important for insertion.

The acidity-triggered rational membrane (ATRAM) peptide was designed to target acidic diseases such as cancer. An acidic extracellular medium, such as that found in aggressive tumors, drives the protonation of the glutamic acids in ATRAM, leading to the membrane translocation of its C-terminus and the formation of a transmembrane helix. Compared to healthy cells, cancerous cells often increase exposure of the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane. Here we use a reconstituted vesicle system to explore how PS influences the interaction of ATRAM with membranes. To explore this, we used two new variants of ATRAM, termed K2-ATRAM and Y-ATRAM, with small modifications at the noninserting N-terminus. We observed that the effect of PS on the membrane insertion pK and lipid partitioning hinged on the sequence of the noninserting end. Our data additionally indicate that the effect of PS on the insertion pK does not merely depend on electrostatics, but it is multifactorial. Here we show how small sequence changes can impact the interaction of a peptide with membranes of mixed lipid composition. These data illustrate how model studies using neutral bilayers, which do not mimic the negative charge found in the plasma membrane of cancer cells, may fail to capture important aspects of the interaction of anticancer peptides with tumor cells. This information can guide the design of therapeutic peptides that target the acidic environments of different diseased tissues.