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Candida albicans is a fungal pathobiont colonizing mucosal surfaces of the human body, including the oral cavity. Under certain predisposing conditions, C. albicans invades mucosal tissues activating EGFR-MAPK signalling pathways in epithelial cells via the action of its peptide toxin candidalysin. However, our knowledge of the epithelial mechanisms involved during C. albicans colonization is rudimentary. Here, we describe the role of the transcription factor early growth response protein 1 (EGR1) in human oral epithelial cells (OECs) in response to C. albicans . EGR1 expression increases in OECs when exposed to C. albicans independently of fungal viability, morphology, or candidalysin release, suggesting EGR1 is involved in the fundamental recognition of C. albicans , rather than in response to invasion or "pathogenesis." Upregulation of EGR1 is mediated by EGFR via Raf1, ERK1/2, and NF-κB signalling but not PI3K/mTOR signalling. Notably, EGR1 mRNA silencing impacts on anti- C. albicans immunity, reducing GM-CSF, IL-1α and IL-1β release, and increasing IL-6 and IL-8 production. These findings identify an important role for EGR1 in priming epithelial cells to respond to subsequent invasive infection by C. albicans and elucidate the regulation circuit of this transcription factor after contact.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans , and the related species C. dubliniensis , and C tropicalis , suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C . albicans isolates, 10 C . dubliniensis isolates, and 78 C . tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species., Importance: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis , thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans , C. dubliniensis , and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity., Competing Interests: The authors declare no conflict of interest.

Candida albicans is a fungal pathobiont colonising mucosal surfaces of the human body, including the oral cavity. Under certain predisposing conditions, C. albicans invades mucosal tissues activating EGFR-MAPK signalling pathways in epithelial cells via the action of its peptide toxin candidalysin. However, our knowledge of the epithelial mechanisms involved during C. albicans colonisation is rudimentary. Here, we describe the role of the transcription factor early growth response protein 1 (EGR1) in human oral epithelial cells (OECs) in response to C. albicans . EGR1 expression increases in OECs when exposed to C. albicans independently of fungal viability, morphology, or candidalysin release, suggesting EGR1 is involved in the fundamental recognition of C. albicans, rather than in response to invasion or 'pathogenesis'. Upregulation of EGR1 is mediated by EGFR via Raf1, ERK1/2 and NF-κB signalling but not PI3K/mTOR signalling. Notably, EGR1 mRNA silencing impacts on anti- C. albicans immunity, reducing GM-CSF, IL-1α and IL-1β release, and increasing IL-6 and IL-8 production. These findings identify an important role for EGR1 in priming epithelial cells to respond to subsequent invasive infection by C. albicans and elucidate the regulation circuit of this transcription factor after contact.

In 2016, the first peptide toxin in any human fungal pathogen was identified. It was discovered in Candida albicans and was named candidalysin. Candidalysin is an amphipathic cationic peptide that damages cell membranes. Like most lytic peptides, candidalysin shows alpha-helical secondary structure. As the helicity and the membrane lytic activity of candidalysin are key factors for pathogenicity, here we describe in vitro approaches to monitor both its membrane-lytic function and the secondary structure. First, membrane permeabilization activity of candidalysin is measured in real time by direct electrical recording. Second, the secondary structure and helicity of candidalysin are determined by circular dichroism spectroscopy. These biophysical methods provide a means to characterize the activity and physical properties of candidalysin in vitro and will be useful in determining the structural and functional features of candidalysin and other similar cationic membrane-active peptides., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)