Candidalysin: discovery and function in Candida albicans infections

Candidalysin is a cytolytic peptide toxin secreted by the invasive form of the human pathogenic fungus, Candida albicans. Candidalysin is critical for mucosal and systemic infections and is a key driver of host cell activation, neutrophil recruitment and Type 17 immunity. Candidalysin is regarded as the first true classical virulence factor of C. albicans but also triggers protective immune responses. This review will discuss how candidalysin was discovered, the mechanisms by which this peptide toxin contributes to C. albicans infections, and how its discovery has advanced our understanding of fungal pathogenesis and disease.


Introduction
Deaths per annum from fungal infections are greater than the global mortality due to malaria or breast cancer and are similar to deaths due to tuberculosis or HIV [1,2]. As such, the major challenges facing medical mycology highlight the need to better understand the biological processes that promote fungal pathogenesis and host immunity, and to translate this knowledge into the development of novel immunotherapies, vaccines and diagnostics [1][2][3][4]. One of the most important human fungal pathogens is Candida albicans, which causes millions of skin, mucosal (mouth, vagina, gut) and life-threatening systemic infections each year [1,2]. Recently, it was discovered that the invasive (hyphal) form of C. albicans secretes a cytolytic peptide toxin, named candidalysin [5••]. Before this, human fungal pathogens were not known to possess such toxins. This review will focus on how candidalysin was commensal and pathogenic states of opportunistic pathogens. However, the precise mechanism by which epithelial cells sense the 'danger' remained unclear.

Discovery of candidalysin
The abovementioned studies made it abundantly clear that activation of MAPK (c-Fos/ MKP1) and subsequent production of proinflammatory cytokines in OECs was hypha dependent. To identify the hyphal factor responsible for these events, a library of C. albicans mutants were screened to identify strains that could form hyphae normally but were unable to induce damage, c-Fos/MKP1 or cytokines [5••]. Remarkably, the screen identified only a single mutant with these highly specific characteristics, namely a C. albicans strain deficient in ECE1 (extent of cell elongation 1). ECE1 had long been known to be a highly expressed, hypha-associated gene encoding a unique protein (Ece1p; 271 amino acids, 28.9 kDa) [31], and was identified as a core filamentation gene expressed under most hypha-inducing conditions [32]. The Moyes et al. study also found that an ECE1-deficient strain induced significantly reduced damage and immune activation in a zebrafish swimbladder model of mucosal infection and was avirulent in an immunocompromised murine model of oropharyngeal candidiasis (OPC) [5••].
Ece1p has intriguing structural characteristics, most notably seven lysine-arginine (KR) motifs regularly dispersed throughout the full-length protein ( Figure 1). These KR motifs are known processing sites for the kexin, Kex2p, suggesting that Ece1p was cleaved by Kex2p into at least eight smaller peptides and secreted [33]. Application of the eight Ece1p peptides onto OECs identified a single 32 amino acid (aa) peptide that accounted for damage induction, c-Fos/MKP1 activation and cytokine production to a similar extent as wild-type C. albicans hyphae [5••]. Further investigations demonstrated that the terminal arginine of the peptide was removed by a carboxypeptidase, Kex1p, to produce a mature 31 aa peptide, the secretion of which from C. albicans hyphae was confirmed by liquid chromatographymass spectrometry. Sitedirected mutagenesis experiments demonstrated that Kex2pmediated proteolysis of Ece1p after Arg61 and Arg93, but not after other KR processing sites within Ece1p, was critical for peptide generation and infection in vitro and in vivo [34•]. Finally, the functional importance of the peptide was confirmed using a C. albicans strain that was deficient only in the peptide-encoding region of ECE1, which was unable to induce damage, c-Fos/MKP1 activation or cytokine production [5••].
The peptide had striking features in that it was amphipathic, α-helical and possessed two amyloidogenic regions (Figure 1). The peptide lysed red blood cells, formed lesions in synthetic membranes and induced calcium influx in OECs, confirming that it was cytolytic [5••]. Hence, the peptide was named candidalysin and is the first cytolytic peptide toxin identified in any human fungal pathogen. Importantly, the epithelial response to candidalysin is dose-dependent, supporting the concept that the host response to C. albicans during infection requires sufficient numbers of candidalysin-producing hyphae. As such, this was the first study to define the molecular link between hypha formation and pathogenicity, and correlated pathogenicity with the ability of C. albicans to damage and induce immune responses predominantly through candidalysin activity [5••] (Figure 1).

Candidalysin activates epithelial signalling via EGFR
Toxins activate epithelial cells via numerous mechanisms ranging from damage-mediated and/or receptor-mediated mechanisms [35,36]. To identify potential surface receptors activated by candidalysin, a microarray screen was undertaken, which identified the epidermal growth factor receptor (EGFR) family as being significantly affected by C. albicans infection [22]. EGFR (ErbB1/Her1) is a membrane-bound tyrosine kinase, which together with ErbB2 (Her2), ErbB3 (Her3) and ErbB4 (Her4), constitute the EGFR/ErbB family [37,38]. EGFR is distributed diversely in the body and can trigger signalling via several major pathways associated with growth, cell proliferation, survival, angiogenesis, differentiation and motility [37,39] and EphA2 (ephrin type-A receptor 2) [50], these EGFR functions may be pivotal for the balance between commensalism, disease and restoration of health in the context of mucosal fungal infections ( Figure 2).

Candidalysin is critical for mucosal immune activation in vivo
A defining feature of the oral immune response to C. albicans is the activation of The neutrophil response is essential for immunity to mucosal candidiasis [62,63]. IL-17 is a potent activator of neutrophils, acting indirectly through induction of CXC chemokines and G-CSF on non-hematopoietic cells [64].  65,66], these studies collectively reveal that candidalysin plays a critical role in driving protective innate immunity via Th17 cells and neutrophils during OPC (Table 1).
Unlike oral candidiasis, vulvovaginal candidiasis is a disease of otherwise healthy individuals and does not seem to strongly involve an IL-17 response [68]. However, robust recruitment of neutrophils is a hallmark of vaginal candidiasis and appears to exacerbate disease rather than clear the infection [69,70]. Notably, neutrophil-driven immunopathology was recently shown to be mediated by candidalysin, as mice intravaginally challenged with ECE1-deficient and candidalysin-deficient C. albicans strains showed significant decreases in neutrophil recruitment, damage, and pro-inflammatory cytokine expression [70]. The study was the first to link vaginitis immunopathogenesis with the capacity of candidalysin to damage the vaginal mucosa.
The role of candidalysin during C. albicans gut infections is less clear. Several studies ascribe the major source of systemic candidiasis to the commensal C. albicans gut population [71][72][73]. In vitro data indicate that C. albicans translocation is a dynamic fungaldriven process initiated by invasion (active penetration) and followed by cellular damage and loss of epithelial integrity [74]. C. albicans translocation via the transcellular route required candidalysin-induced epithelial damage, but low-level fungal translocation occurred via a paracellular route in a candidalysin-independent manner. While the requirement of candidalysin for C. albicans gut translocation needs to be confirmed in vivo, this study showed that a peptide toxin can drive translocation of a human pathogenic fungus across the intestinal barrier.

Candidalysin is critical for systemic infection and immune activation in vivo
Phagocytes such as macrophages and dendritic cells are critically important for efficient clearance of C. albicans infections and initiation of inflammatory responses [75]. Once phagocytosed, C. albicans forms hyphae, resulting in inflammasome activation, cell lysis and escape. Inflammasome activation is a two-step process, requiring an initial priming step and a second, inflammasomeactivating step [76][77][78]. Using human and mouse primary monocyte-derived macrophages and dendric cells, candidalysin was shown to provide the second signal to activate the NLRP3 inflammasome, resulting in caspase1-dependent maturation and secretion of IL-1β [79••]. However, candidalysin-induced cytolysis occurred independently of inflammasome activation and pyroptosis. Thus, the study identified candidalysin-induced cell damage as an additional mechanism of C. albicans-mediated cell death in addition to pyroptosis [80][81][82] and the growth of glucose-consuming hyphae [83]. NLRP3 inflammasome activation by candidalysin was recently confirmed using primary macrophages [84•]. NLRP3 inflammasome activation also promotes the immunopathogenesis of vulvovaginal candidiasis [85] but a role for candidalysin has not yet been formally demonstrated.
Candidalysin also drives C. albicans systemic infections. The C-type lectin receptor/Syk adaptor CARD9 is known to facilitate protective antifungal immunity within the central nervous system (CNS) through neutrophil recruitment [86]. Recently, candidalysin was shown to induce IL-1β and CXCL1 secretion from CARD9 + microglial cells in a p38/c-Fosdependent manner, and that they function to recruit CXCR2-expressing neutrophils to the brain to control the infection [87••]. The work revealed an intricate network of hostpathogen interactions that promotes CNS antifungal immunity via candidalysin activity and provided novel mechanistic insights into how human CARD9-deficiency is associated with CNS fungal disease.
Finally, candidalysin also induces FGF-2 secretion from human endothelial cells and drives angiogenesis during murine systemic infections [88]. As to why candidalysin promotes angiogenesis is intriguing but it is notable that candidalysin also activates EGFR signalling [40••], which is associated with angiogenesis [37,39].

Conceptual aspects of candidalysin production
Why does C. albicans produce candidalysin? Evidence so far points towards a dual role for candidalysin in C. albicans pathogenesis. One on hand, candidalysin suits the description of a classical virulence factor in that it directly damages host cells [89]. On the other hand, candidalysin is an immunomodulatory molecule that is sensed by the host to initiate a protective response (via neutrophils and Type 17 immunity); such molecules have been  [90,91]. The balance of this virulence/avirulence encounter, namely damage induction versus immune protection, dictates the outcome of infection. This is elegantly addressed in the damage-response framework [92], which was recently utilised to conclude that C. albicans infections fit all six classifications of the framework [93]. Given that candidalysin is critical for driving damage and immunity/immunopathology in all infection models tested, candidalysin is probably a pivotal factor in the outcome of this virulence/avirulence encounter.
Another conceptual aspect is whether candidalysin also acts as a commensal factor. C. albicans is adapted to life in the host, which is typified by asymptomatic commensal carriage. Indeed, gene expression analysis directly from patient samples indicated that both yeast and hyphal morphologies are present during asymptomatic colonisation of human mucosal surfaces [94][95][96]. Intriguingly, in a murine gastrointestinal colonisation model, competitive infection experiments revealed that commensal fitness may inversely correlate with the gene network associated with morphogenesis [97••]. This apparent antagonism between commensalism and hyphal growth is supported by the observation that serial passage of C. albicans through the murine gastrointestinal tract resulted in the loss of hyphaforming ability in the absence of a competitive microbiota [98••]. Furthermore, gut-evolved C. albicans strains that lost the ability to form hyphae exhibited reduced virulence in vitro and in vivo. Given this, it may be unsurprising that commensal fitness inversely correlates with morphogenesis, since hypha formation will lead to candidalysin secretion, damage and immune activation, which will ultimately lead to fungal clearance or immunopathology. Therefore, it may not be in the fungus' interests to secrete high levels of candidalysin when colonising host surfaces. This is supported by data showing that a threshold level of candidalysin activity is required to damage epithelial cells and drive immune responses [5••, 17•,19,70]. Hence, the commensal lifestyle of C. albicans may be promoted by reduced hypha formation accompanied by low levels of candidalysin secretion, which may function to acquire nutrients from intracellular sources (through non-damaging pore formation) or by promoting colonisation through direct antimicrobial activity on the local microbiota. On the other hand, a pathogenic lifestyle may be promoted when C. albicans hyphal burdens increase accompanied by high levels of candidalysin secretion, or in immunocompromised individuals that exhibit defective anti-Candida immunity. These conceptual aspects for a role of candidalysin in C. albicans infections will no doubt be addressed more fully in the coming years.
• of special interest  Europe PMC Funders Author Manuscripts candidalysin interacts with the cell membrane to form pore-like structures that results in membrane damage (LDH release) and calcium influx. (b) These events lead to the activation of matrix metalloproteinases and the release of epidermal growth factor receptor (EGFR) ligands, which ultimately leads to EGFR activation. (c) EGFR activation leads to induction of MAPK signalling (via p38, ERK1/2) and the activation of c-Fos. MKP1 activation (via ERK1/2) contributes to the regulation of the epithelial immune response (as it dephosphorylates p38). (d) c-Fos activation leads to chemokine and cytokine release and the subsequent recruitment of innate immune cells, including neutrophils and innate Type 17 cells (e.g. natural Th17 cells). Neutrophils phagocytose and kill the fungus and innate type 17 cells release IL-17 and IL-22. Together, these innate cells promote fungal clearance, activate epithelial tissues and improve barrier function, resulting in reduction in fungal burdens and/or clearance of the infection (commensalism).