Candidalysin: From Mechanism of Action to Biomarker Development and Therapeutic Response

Candida albicans, an opportunistic human fungal pathogen, is one of the predominant fungal species colonizing the human mucosa, skin, mouth, gastrointestinal tract, vagina, and so on.^[1,2] It can harmlessly exist in the human mucous membrane as a commensal, on the one hand, and transition into a pathogen that causes severe candidiasis disease when the immune system and microbiome are compromised on the other hand.^[3] Candidiasis disease can vary from a superficial skin infection to a life-threatening whole-body infection. For example, C. albicans colonized in the oral cavity of the human body can lead to severe oral candidiasis in immunodeficient patients.^[4,5] And vulvovaginal candidiasis shows the characteristics of frequently occurring disease, easy recurrence, and difficult radical cure.^[6] About 75% of women in the world will be confronted with at least one vulvovaginal candidiasis in their lifetime, affecting their physical and mental health.^[7]

C. albicans is a dimorphic commensal fungus and can transition from yeast to invasive filamentous hyphae, which is a critical determinant of its pathogenesis and virulence. The transition into hyphae promotes its penetration into human tissues and organs, which results in epithelial damage and immune activation.^[8] However, the mechanism of induction of epithelial inflammatory responses and cell damage by the hyphae remained unclear until 2016. Moyes et al^[9] revealed a mechanism through which hyphae secreted a toxin that was called candidalysin. Candidalysin is a 31 amino acid peptide toxin derived from the gene ECE1 that was identified as a hyphal-specific protein. This is the first cytolytic peptide toxin found in the human fungal pathogen.^[9,10] This review will focus on the identification of candidalysin and the roles of candidalysin during C. albicans infection, as well as on how the accrued knowledge can be used for the potential development of biomarkers and therapeutic targets.

During mucosal and systemic infections, only the invasive hyphal form of C. albicans induces Mitogen-activated protein kinase phosphatase-1 (MKP1) phosphorylation (p-MKP1), c-Fos, cytokines (interleukins [IL]-1α, IL-6, granulocyte-colony stimulating factor [G-CSF]), and damage in epithelial cells.^[11]ECE1, a hyphal-related gene, encodes membrane proteins. The growth of C. albicans and the conformation of hyphae do not completely depend on the expression of ECE1.^[12] However, the expression of ECE1 gene is associated with cell elongation. As cells continue to elongate, ECE1 expression increases either.^[13]ECE1 is highly expressed by C. albicans as it encodes a secreted protein, Ece1p, which is necessary for epithelial activation, damage, and mucosal pathogenesis. Ece1p can produce eight secreted peptides by cleavage at seven xxx (KR)-processing sites. However, only Ece1-III (62–93), a secreted cytolytic peptide toxin, induces p-MKP1, c-Fos, cytokines, and damage response. This peptide toxin was thus named candidalysin. The immature candidalysin is generated by Ece1p, which is first processed via Kex2p, and then the terminal Arg93 of the immature candidalysin is removed by Kex1p and turned into mature candidalysin. The mature candidalysin is then secreted from hyphae.^[10] Candidalysin is critical and sufficient for activation of the epithelial immune response, damage, and mucosal infection.^[11] Candidalysin is required for epithelial damage and is the critical factor in fungal invasion. Therefore, candidalysin mediates the transcellular location of C. albicans through the intestinal mucosa layer.^[14] Candidalysin acts like a switch for the epithelial immune response and is the active form of Ece1p.^[15]

C. albicans hyphae can induce endocytosis and active penetration through adhesion and engulfment by the host. The endocytosis is mediated via interaction between various epithelial receptors and fungal adhesin Als3 and its partner Ssa1. The epithelial cells are then activated by the cytolytic peptide toxin candidalysin, which damages epithelial membranes and triggers damage response pathways.^[12] Candidalysin invades the epithelial cell membrane via pore-like structure formation, which leads to lactate dehydrogenase (LDH) release and calcium influx.^[10] At the same time, the host immune responses are activated and cytokines and chemokines are secreted to activate epidermal growth factor receptor (EGFR). Then the downstream mitogen-activated protein kinases (MAPK) and c-Fos signaling pathways are activated. Once the epithelial immune response is activated through MKP1 activation, the chemokine and cytokine are released after c-Fos activation,^[10] which will recruit innate immune cells like neutrophils, macrophages, and Th17 cells.^[16] A series of innate immune responses is activated to prevent the infection and clear the fungal pathogen. The Th17 cell is a new lineage of CD4+ effector T helper cells that selectively produce IL-17 and IL-22.^[10] The macrophage cells will phagocytose and clear C. albicans through oxidative and nitrosative mechanisms.^[12]

Candidalysin is crucial for mucosal inflammation, systemic fungal infection, and immune activation. In epithelial signaling activated by candidalysin through c-Fos activation, chemokines and cytokines recruit innate immune cells, including neutrophils and Th17 cells.^[11]

The activation of Th17 immunity is a hallmark of the oral immune response to C. albicans, with secretion of IL-17A, IL-17F, and IL-22.^[17] In oral epithelial cells, C. albicans can activate the proliferation of innate IL-17 TCRαβ+ cells, named nTh17 cells. Candidalysin can drive the proliferation of nTh17 cells, which are not antigen-specific. And the classic fungal pattern recognition receptors are not involved in the downstream proliferation of nTh17 cells and induction of IL-17. In addition, candidalysin functions synergistically with IL-17 to promote the expression of proinflammatory mediators including multiple IL-1 cytokines.^[18] They can further promote neutrophil recruitment to the oral site.^[11] In contrast, in vaginal candidiasis, neutrophil-driven immunopathology mediated by candidalysin aggravates the disease.^[19,20]

Candidalysin also drives C. albicans systemic infections.^[11] Vellanki et al^[21] demonstrated that candidalysin can induce FGF-2 secretion from human endothelial cells. Drummond et al^[22] found that candidalysin elicited IL-1b and CXCL1 secretion from CARD9+ microglial cells, resulting in the subsequent recruitment of CXCR2-expressing neutrophils to the brain to diminish the infection. Kasper et al^[23] showed that candidalysin provided the second signal to activate the NLRP3 inflammasome, and then mediated caspase-1–dependent maturation and secretion of IL-1b. Notably, candidalysin can independently induce cytolysis.

Currently there is an urgent need to search for novel diagnostic biomarkers for early detection of invasive C. albicans infections and for potential antifungal therapeutic targets as C. albicans develops drug resistance to almost all commercially available antifungals. In clinical practice, diagnoses of invasive candidiasis are mainly based on cultures of blood samples collected under sterile conditions. Mannan and (1,3)-b-D-glucan (BDG) have proven to be valuable diagnostic serum biomarkers, but their sensitivity or validity is still controversial.^[24]

The discovery of candidalysin and functional characterization of this cytolytic peptide toxin greatly improved fungal infection diagnosis and its future clinical application. As a secreted peptide, candidalysin can be developed as a potential diagnostic biomarker with convenient sampling from patients. We may detect C. albicans infections via measurement of candidalysin-Ag and anti-candidalysin antibodies by enzyme-linked immunosorbent assay (ELISA) with highly diagnostic sensitivity.

As a therapeutic target, target of rapamycin complex 1 (TORC1) is an essential regulator of cell growth in C. albicans. It is required for C. albicans morphogenesis, biofilm formation, and virulence.^[25] Dai et al^[26] found that Hsp90 is involved in apoptosis of C. albicans by regulating the calcineurin-caspase apoptotic pathway. Thus activating the Hsp90-mediated apoptosis pathway may be an effective way to combat invasive candidiasis. However, therapies targeting TOR or Hsp90 may also elicit cell damage to humans because of the conserved homologous partner of these proteins in humans. Unlike these targets, candidalysin is a unique peptide that is expressed only by C. albicans. Humans can be protected from the drugs targeting candidalysin with regard to its target specificity. In addition, candidalysin is a virulence factor in invasive candidiasis that can both damage host cells directly and activate immunity. The intriguing properties of candidalysin provide potential therapeutic targets for an antifungal strategy. Targeting the expression, induction, and pathogenicity of candidalysin might be a promising and effective therapeutic path. As candidalysin originated from the protein Ece1p, encoded by the ECE1 gene, the ECE1 gene appears to be a drug target for the prevention and control of C. albicans infection. Haque et al^[27] found that sophorolipid (SL) downregulated the expression of hyphae-specific genes including ECE1, and this possibly explains the inhibitory effect of SL on hyphae and biofilm formation. Kurakado et al^[28] concluded that minocycline decreased the expression of hyphae-specific genes HWP1 and ECE1, inhibiting the C. albicans yeast-to-hyphae transition.^[28] Furthermore, during the immunoactivation triggered by candidalysin, morphology-dependent host FGF-2 signaling^[21] and EGFR^[29] can also be potential therapeutic targets. Said et al^[30] demonstrated that garlic can alter the expression of putative virulence factor genes SIR2 and ECE1 in vulvovaginal C. albicans isolates, suggesting a possible mechanism of phytotherapy with garlic. And Chu et al^[31] found in their research that candidalysin was cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.^[31]

Every year, millions of infections, from superficial mucosal infections to deadly systemic infections, have been emerging across the world.^[32–37] The first human fungal toxin, candidalysin, can damage mucosal epithelial cells and activate a danger response signaling pathway via enzymatic immunoassay (ERK)/MKP1 and p38/cFos. This will further trigger downstream proinflammatory cytokine responses and activate epithelial immunity. The high mortality rates associated with C. albicans infection call for novel diagnostic biomarkers as an early detection method. Therefore, it is crucial to develop inhibitors of candidalysin production and activity for effective antifungal therapy. Conventional serum biomarkers such as mannan and BDG lack diagnostic specificity or validity. And previous therapeutic targets like TOR (mTOR in humans) and Hsp90 only exist in the host as a result of the general expression of these genes in eukaryotic cells. Advances in understanding the mechanism of fungal pathogenesis facilitate the development of novel diagnostic approaches and therapy against C. albicans infections. However, there is no report on the expression of candidalysin in patients infected with C. albicans. Therefore it continues to require further investigation. More importantly, C. albicans infections should be taken seriously during the coronavirus disease 2019 (COVID-19) pandemic as a result of the high rate of concurrent bacterial or fungal infections.^[37]