Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence

ABSTRACT Candida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38–mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]–MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity.


FIG 1
Alanine substitutions at positions 61 and 93 of Ece1p render C. albicans incapable of damaging or activating TR146 oral epithelial cells in vitro. (A) Epithelial cell damage induced by C. albicans Ece1p alanine substitution mutants. Epithelial cells were exposed to Ece1p alanine substitution mutants for 24 h, and levels of cell damage were assessed by LDH assay. Statistics are applied relative to the ece1Δ/ΔϩECE1 parental control (n ϭ 5 biological repeats). (B) Western blot analysis of epithelial cells infected with C. albicans alanine substitution mutants. Epithelial cell lysates (20 g total protein) were probed with anti-c-Fos and anti-p-MKP1 antibodies. One representative blot presented (from n ϭ 3 biological repeats). (C) Analysis of c-Fos DNA binding activity from epithelial cells infected with C. albicans alanine substitution mutants. Statistics are applied relative to the vehicle control (n ϭ 3 biological repeats). (D to H) Quantification of cytokines (IL-1␣, IL-1␤, IL-6, G-CSF, and GM-CSF) secreted from epithelial cells in response to alanine substitution mutants of C. albicans Ece1p. Statistics are applied relative to ece1Δ/ΔϩECE1 parental control (n ϭ 3 biological repeats). (A and C to H) Data are presented as fold change relative to vehicle control (dashed line) ϩ standard deviation (SD). Statistical significance was calculated using one-way ANOVA with a post hoc Dunnett comparison test. ****, P Յ 0.0001; ***, P Յ 0.001; **, P Յ 0.01; *, P Յ 0.05. macrophage colony-stimulating factor (GM-CSF) (3). Therefore, we quantified the secretion of these cytokines following infection with the panel of alanine substitution mutants ( Fig. 1D to H). Epithelial cells responded to the WT, ece1Δ/ΔϩECE1 parental control, and mutant strains that do not affect the release of candidalysin (R31A, R126A, R160A, R194A, and R228A), by secreting significant levels of IL-1␣, IL-1␤, IL-6, G-CSF, and GM-CSF. In contrast, epithelial cytokine secretion was abolished in response to infection with the mutant strains predicted to affect the release of candidalysin (R61A, R93A, R61A ϩ R93A, and ALL KA). Taken together, these data indicate that Arg61 and Arg93 within C. albicans Ece1p are essential for the release of candidalysin and the induction of epithelial damage and immune activation in vitro.
R61A and R93A mutations in C. albicans Ece1p result in attenuated secretion of candidalysin. To confirm whether mutation of Kex2p recognition sites within Ece1p impaired candidalysin secretion, hyphal growth was induced in mutant and control strains, and hypha-secreted peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The number of peptide spectrum matches (PSMs) corresponding to candidalysin was determined (Table 1; amino acid sequences of detected peptides and nomenclature conventions are presented in Table S1 in the supplemental material).
The C. albicans reference strain SC5314 (16) and the isogenic WT strain BWP17ϩCIp30 secreted candidalysin in the absence of epithelial cells under hyphainducing conditions, and BWP17ϩCIp30 secreted candidalysin when cultured in the presence of epithelial cells for 3 h and 18 h. No candidalysin PSMs were detected from an ece1Δ/Δ null mutant or a mutant lacking the candidalysin-encoding region of ECE1 (ece1Δ/ΔϩECE1 Δ184 -279 ). Substantial candidalysin PSMs were obtained from the ece1Δ/ ΔϩECE1 parental control, and candidalysin was the most abundant peptide detected in the strains harboring alanine substitutions that did not flank candidalysin (R31A, R126A, R194A, and R228A; the exception was the R160A strain, where candidalysin was the second most abundant peptide). In contrast, secretion of candidalysin from strains that harbored alanine substitutions which flanked candidalysin (R61A, R93A, R61A ϩ R93A, and ALL KA) was absent or severely attenuated (Table 1). These data demonstrate that Kex2p recognition of arginine residues at positions 61 and 93 within Ece1p is required for efficient release of candidalysin.
LC-MS/MS analysis of peptides secreted from R61A and R93A mutants demonstrated that while candidalysin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK) displayed minimal PSM values relative to total PSMs (Table 1), larger "fusion" peptides containing the mutated Kex2p recognition site (KA) were detected in the exhausted culture medium (Data Set S1), suggesting that Kex2p was unable to process Ece1p at these mutated locations. Fusion peptides were secreted from all alanine substitution mutants except the R126A and R228A mutants. Collectively, these data indicate that the KR motifs of Ece1p are required for enzymatic processing of the full-length protein into peptide fragments and that Arg61 and Arg93 are critical for the secretion of candidalysin.
C. albicans Kex1p is required for candidalysin maturation, epithelial damage, and immune activation. The candidalysin toxin was initially predicted to terminate in a dibasic lysine-arginine (KR) motif based upon Kex2p substrate specificity, and a peptide corresponding to this sequence (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) was capable of damaging epithelial cells and activating the c-Fos/MKP1 signaling circuits in vitro (3). However, subsequent analysis of the hypha-secreted peptides revealed that secreted candidalysin lacks the C-terminal arginine residue (3). This observation implied the potential involvement of a carboxypeptidase enzyme and a second processing step. In addition to the Kex2p endoproteinase, the carboxypeptidase Kex1p is also located in the Golgi complex of yeast (13), and an ortholog of Kex1p exists in C. albicans. Given these observations, Kex1p was therefore a compelling candidate for the removal of the C-terminal arginine residue and production of mature candidalysin and was confirmed to do so in a previous study (3).
Further analysis of the hypha-secreted peptides of a kex1Δ/Δ null mutant (Table 1) revealed an approximately 7-fold reduction in candidalysin PSM values relative to total PSM values compared with a kex1Δ/ΔϩKEX1 reintegrant control (3.2% versus 23.5%, respectively). Importantly, analysis of WT and the kex1Δ/ΔϩKEX1 strain revealed that mature candidalysin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK) was the predominant peptide secreted from both strains. However, upon disruption of the KEX1 gene (kex1Δ/Δ), immature candidalysin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) was the dominant peptide produced (Table S2). Collectively, these data confirm that C. albicans Kex1p carboxypeptidase activity is required for the production of mature candidalysin.
To determine whether C. albicans Kex1p activity was required for cellular damage, signal pathway activation, and the induction of proinflammatory cytokines, we infected TR146 epithelial monolayers with a kex1Δ/Δ null mutant. In contrast to the WT and  Table S1. PSM, peptide spectrum match; NA, not applicable. Full details of LC-MS/MS data sets and sequence alignments are provided in Data Set S1. e Samples were prepared as previously described (3) unless otherwise specified. kex1Δ/ΔϩKEX1 reintegrant control strains, the kex1Δ/Δ null mutant was unable to cause damage or induce c-Fos production/DNA binding, MKP1 phosphorylation, or cytokine secretion from epithelial cells (Fig. S4). These data demonstrate that Kex1p processing is also required for epithelial damage and host recognition of candidalysin.
Saps are not required for Ece1p processing or candidalysin production. Like Kex2p, Kex1p is likely to have multiple targets, including other proteases. The observations made with the kex1Δ/Δ null mutant could therefore be an indirect effect of the absence of Kex1p, which could be required for full activity of other proteases possibly involved in the maturation of candidalysin. The secreted aspartyl proteinases (Saps) are a family of enzymes that exhibit broad substrate specificity (17). Pepstatin A is a potent inhibitor of most Sap activity (although Sap7p is not inhibited by pepstatin A, and Sap9p and Sap10p exhibit reduced sensitivity to such inhibition under certain physiological conditions [18,19]), and epithelial cells treated with pepstatin A are partially protected from fungal damage in vitro (20). In light of these observations, we investigated whether Saps may play a role in Ece1p processing and candidalysin production. Therefore, we cultured WT C. albicans under hypha-inducing conditions in the presence of pepstatin A and analyzed the secreted peptides using LC-MS/MS (Table 1). Candidalysin was secreted from WT hyphae in the presence of pepstatin A, and no reduction in candidalysin PSMs was observed following Sap inhibition. These observations suggest that Saps and/or other extracellular aspartyl proteinases (e.g., Bar1p [21]) targeted by pepstatin A are not required for Ece1p processing or candidalysin production, at least when inhibited extracellularly.
Arg93 is required for efficient processing of C. albicans Ece1p. Candidalysin possesses a hydrophobic N-terminal region (Ece1p 62-85 ; SIIGIIMGILGNIPQVIQIIMSIV) and a hydrophilic C terminus (Ece1p 86Ϫ92 ; KAFKGNK) ( Fig. S1B). A previous in vitro analysis of mutated candidalysin peptides indicated that the positively charged C terminus of candidalysin is required for epithelial damage (3). Having demonstrated that Arg61 and Arg93 are required for efficient enzymatic processing of Ece1p, we questioned whether replacement of arginine with alanine at position 93 could nevertheless result in successful (albeit inefficient) proteolytic cleavage of mutated Ece1p, generating a secreted candidalysin toxin with a modified C terminus (amino acid sequence SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKA; Ece1p62-93 KA ). LC-MS/MS analysis of hypha-secreted peptides from the R93A mutant revealed the presence of this modified candidalysin toxin terminating in lysine-alanine (Data Set S1). However, the PSM value for this modified toxin was very low (PSM ϭ 14; complete LC-MS/MS data set is provided in Data Set S1), and infection of epithelial cells with the R93A mutant did not induce cellular damage, MKP1 phosphorylation, c-Fos DNA binding, or cytokine secretion ( Fig. 1).
To determine whether direct application of Ece1p62-93 KA was capable of causing epithelial damage and immune activation, we treated TR146 cells with different concentrations (70, 15, and 1.5 M; ranging from lytic to sublytic concentrations of mature candidalysin [3]) of Ece1p62-93 KA peptide and quantified LDH activity, c-Fos/p-MKP1 responses, and cytokine secretion ( Fig. 2). At lytic concentrations, Ece1p62-93 KA peptide caused dose-dependent epithelial damage ( Fig. 2A) and induced c-Fos and p-MKP1 responses (Fig. 2B) and secretion of cytokines ( Fig. 2C to G). These data affirm the ability of Ece1p62-93 KA to cause epithelial damage and immune activation in vitro and suggest that the lack of epithelial damage and signaling observed in response to the R93A mutant arose from inefficient processing of Ece1p resulting in a severe reduction in Ece1p62-93 KA secretion.
Ece1p processing is required for the induction of mucosal immune responses in vivo. Mucosal responses to infiltrating pathogenic C. albicans hyphae culminate in the secretion of immunomodulatory cytokines and chemokines that collectively drive innate immune responses leading to fungal clearance (reviewed in reference 22). To determine whether enzymatic processing of C. albicans Ece1p is a driver of early-phase host immune responses in vivo, we used a nonimmunosuppressed murine model of oropharyngeal candidiasis (OPC). WT mice were infected with positive-control strains (WT and ece1Δ/ΔϩECE1), negative-control strains (ece1Δ/Δ and ece1Δ/ΔϩECE1 Δ184 -279 ), a candidalysin-secreting alanine substitution mutant (R31A), and alanine substitution mutants that showed a marked reduction in candidalysin secretion (R61A, R93A, R61A ϩ R93A, and ALL KA) for 24 h. Gene expression of Ccl20, Il1b, Il6, and Csf3 from infected tongue tissue was assessed by quantitative PCR (qPCR) (Fig. 3).
Mice infected with the WT or ece1Δ/ΔϩECE1 strain or a candidalysin-secreting alanine substitution mutant (R31A) responded by expressing increased levels of Ccl20, Il1b, Il6, and Csf3 compared to the vehicle (negative) control ( Fig. 3A to D). In contrast, mice infected with the R61A, R93A, R61A ϩ R93A, and ALL KA mutants were significantly attenuated in their ability to induce the expression of at least one proinflammatory gene compared with the ece1Δ/ΔϩECE1 parental control. Indeed, the impaired response was similar to that of negative-control strains that did not express ECE1 (ece1Δ/Δ) or that lacked the candidalysin-encoding region of the ECE1 gene (ece1Δ/ ΔϩECE1 Δ184 -279 ). These data confirm the importance of Ece1p processing and the secretion of candidalysin for the early-phase activation of mucosal immune responses to C. albicans hyphae in vivo.
Arg61 and Arg93 of C. albicans Ece1p are required for mucosal infection in vivo. A C. albicans ece1Δ/Δ mutant and a mutant lacking the candidalysin-encoding region of ECE1 (ece1Δ/ΔϩECE1 Δ184 -279 ) are severely diminished in their ability to cause murine OPC and disease in a zebrafish swim bladder model of mucosal infection (3). To determine whether processing of Ece1p at Arg61 and Arg93 was required for successful fungal infection in vivo, we challenged immunosuppressed WT mice with positivecontrol strains (WT and ece1Δ/ΔϩECE1), negative-control strains (ece1Δ/Δ), and selected alanine substitution mutants (R61A ϩ R93A and ALL KA) and quantified fungal burdens in tongue tissue after 24 or 48 h. Mice infected with the WT or ece1Δ/ΔϩECE1 parental control strain exhibited high levels of fungal burdens compared to sham-infected (negative) controls after 24 h (Fig. 4A). Mice infected with the ALL KA mutant produced a statistically significant reduction in fungal burdens consistent with the ece1Δ/Δ mutant. The R61A ϩ R93A mutant also exhibited a (nonsignificant) reduction in fungal burdens compared to the ece1Δ/ΔϩECE1 parental control. Repeat experiments at 48 h showed that the ALL KA mutant (the R61A ϩ R93A mutant was not tested) maintained a significant reduction in fungal burdens compared with the ece1Δ/ΔϩECE1 strain (Fig. 4B). Taken together, these data demonstrate the importance of Ece1p processing in C. albicans pathogenicity in oral infections.
Candidalysin Maturation from C. albicans Ece1p ® Kex2p in vitro (6), this study investigated the importance of Ece1p processing for the production of candidalysin and C. albicans pathogenicity.
The endoproteinase Kex2p cleaves protein substrates and model peptides after dibasic arginine-arginine (RR) and lysine-arginine (KR) motifs (8). Analysis of the C. albicans genome has identified 147 potential Kex2p substrates, including Saps, the hypha-wall protein Hwp1p (7), and Ece1p (6). However, a kex2Δ/Δ null mutant has severely attenuated fitness (11) and is unable to damage and activate epithelial cells due to its inability to form hyphae (3). Since hypha formation is strongly associated with ECE1 gene expression (3,14), the lack of epithelial cell activation by the kex2Δ/Δ null mutant cannot be attributed to defective Ece1p processing alone. Therefore, to address the importance of Kex2p for Ece1p processing, we created a panel of alanine substitution mutants in Ece1p in which each Kex2p recognition site (KR) was mutated, individually or in combination. With this approach, we were able to circumvent any off-target effects imposed by KEX2 disruption and address three outstanding questions regarding Ece1p processing and candidalysin production: (i) which KR motifs are important for candidalysin production and maturation, (ii) whether sequential Kex2p processing from the N or C terminus of Ece1p is required for candidalysin release, and (iii) whether Ece1p processing by Kex2p is critical for C. albicans pathogenicity and mucosal infection.
C. albicans mutants (R61A, R93A, R61A ϩ R93A, and ALL KA) harboring replacements of Arg61 and Arg93 with alanine within Ece1p, which directly flank candidalysin (see Fig. S2 in the supplemental material), were unable to induce epithelial damage, c-Fos production, DNA binding, MKP1 phosphorylation, and cytokine secretion in vitro (Fig. 1), which are key readouts of candidalysin activity (3). The same inability to induce these phenotypes was also observed in C. albicans mutants lacking the ECE1 gene (ece1Δ/Δ) (Fig. 1) or the candidalysin-encoding region of ECE1 (ece1Δ/ΔϩECE1 Δ184 -279 ) (3). Notably, mutation of arginine residues that did not directly flank candidalysin (R31A, R126A, R160A, R194A, and R228A) failed to abrogate these responses. LC-MS/MS analysis of hypha-secreted peptides revealed that mutations at Arg61 and Arg93 markedly reduced candidalysin secretion, whereas mutations at all other arginine residues did not prevent secretion (Table 1). These data demonstrate that only the Arg residues that directly flank the candidalysin region (Arg61 and Arg93) are required for the release of candidalysin from Ece1p and that these processing events are essential for the ability of C. albicans hyphae to cause epithelial damage and immune activation. Importantly, the data also demonstrate that sequential Kex2p processing from the N or C terminus of Ece1p is not required for the release of candidalysin from the Ece1p preproprotein.
Analysis of hypha-secreted peptides demonstrated that, in all cases, mutants with an altered KA motif still secreted the modified fusion peptide into the extracellular milieu (Table 1), strongly suggesting that secretion pathways are intact in all of these strains. Likewise, the fact that candidalysin was secreted from R31A, R126A, R160A, R194A, and R228A mutants (Table 1) indicates that candidalysin release from Ece1p is dependent upon processing at Arg61 and Arg93 alone and is not influenced by mutations of Kex2p recognition sites that do not directly border the toxin.
Mature candidalysin (terminating in K) is the predominant toxin secreted from WT C. albicans. Given that the endoproteinase Kex2p cleaves protein substrates after lysine-arginine (KR) motifs (8), it became apparent that a second cleavage event was occurring that resulted in the removal of the C-terminal arginine residue from immature candidalysin. The removal of C-terminal arginine residues from proteins and peptides is the function of the carboxypeptidase Kex1p (23)(24)(25). To confirm that removal of the C-terminal arginine residue from immature candidalysin was due to the function of Kex1p (3), we analyzed the hypha-secreted peptides of a kex1Δ/Δ null mutant compared to its matched revertant strain. The dominant peptide secreted from the kex1Δ/Δ null mutant was immature candidalysin (terminating in KR), whereas the kex1Δ/ΔϩKEX1 revertant showed WT-like secretion patterns (Table S2). This demonstrates that Kex1p activity is an important requirement for candidalysin maturation. However, the biological reason for removal of the C-terminal arginine residue by Kex1p is unclear, as both immature (Ece1-III 62-93KR ) and mature (Ece1-III 62-92K ) candidalysin are able to damage and activate epithelial cells, with Ece1-III 62-93KR being even more cytolytic than mature candidalysin at lower concentrations (3). We thus postulate that the removal of the C-terminal arginine must confer an evolutionary advantage to C. albicans, either as a commensal or as a pathogen. Furthermore, it is likely that Kex1p (like Kex2p) targets multiple proteins and peptides in addition to immature candidalysin, which are also required for fungal fitness and virulence. These observations suggest that the immature candidalysin secreted from C. albicans kex1Δ/Δ null mutant hyphae is not present in sufficient concentrations to cause damage to host epithelial cells if other fungal attributes are dysfunctional. This could account for the attenuated damage potential of the kex1Δ/Δ null mutant.
Exhausted culture medium from epithelial cells infected with WT C. albicans failed to induce detectable damage on freshly cultured epithelial cells (not shown). The most likely explanation for this observation is that the concentration of candidalysin secreted into the extracellular environment is insufficient to cause plasma membrane destabilization. Indeed, we propose that in addition to correct processing of C. albicans Ece1p, an epithelial invasion pocket produced by an invading hypha is also required in order for secreted candidalysin to reach the concentrations necessary to cause epithelial damage (3).
We also questioned whether extracellular aspartyl proteases, including Saps (17) and Bar1p (21), could be involved in Ece1p processing and/or candidalysin maturation. Culture of WT C. albicans under hypha-inducing conditions in the presence of the aspartyl protease inhibitor pepstatin A followed by LC-MS/MS analysis of the hyphasecreted peptides revealed that candidalysin secretion was unaffected (Table 1). Furthermore, C. albicans mutant strains unable to express SAP2, SAP7, and SAP9/10 were observed to cause epithelial damage and activation (3), suggesting that processing of Ece1p and secretion of candidalysin were unaffected by these enzymes.
To determine whether defective Ece1p processing impacted C. albicans pathogenicity in vivo, we utilized a murine model of OPC. Since mice are immunologically naive to C. albicans, we first investigated the ability of selected substitution mutants to induce proinflammatory gene expression in the tongue tissue of immunocompetent mice. Only those C. albicans mutants harboring substitutions at Arg61 and Arg93 (R61A, R93A, R61A ϩ R93A, and ALL KA), which flank candidalysin, were severely diminished in their ability to induce early-phase (24 h) gene expression, similar to the ece1Δ/Δ mutant (Fig. 3). This demonstrates that Ece1p processing at Arg61 and Arg93 and subsequent candidalysin secretion are crucial for the induction of immune responses against C. albicans in vivo. Given that C. albicans is not a natural colonizer of mice, we next investigated the ability of the R61A ϩ R93A and ALL KA substitution mutants to colonize tongue tissue in an immunosuppressed OPC model. Both mutants showed a reduced capacity to infect tongue tissue at 24 h, with significant reductions in fungal burdens observed with the ALL KA substitution mutant (Fig. 4A). Additional experiments with the ALL KA substitution mutant indicated that fungal burdens remained low after 48 h (Fig. 4B). We noted that while the R61A ϩ R93A and ALL KA mutants induced almost identical epithelial phenotypes in vitro (Fig. 1), they induced subtly different phenotypes in the context of OPC in vivo after 24 h (Fig. 4A), with the ALL KA mutant exhibiting a greater reduction in fungal burdens than the R61A ϩ R93A mutant. This raises the possibility that candidalysin may have some residual activity when fused to an adjacent peptide but not when contained within full-length Ece1p. Alternatively, Ece1p processing at sites other than Arg61 and Arg93, and hence other Ece1p-derived peptides, may have a role in fungal pathogenesis in vivo. Indeed, the role (if any) of the other noncandidalysin peptides derived from Kex2p-Kex1p processing of Ece1p remains to be determined. Investigations are under way to address this question.
In summary, this study demonstrates that the sequential two-step posttranslational processing of C. albicans Ece1p by kexin-like proteinases is a critical event required for candidalysin production, immune activation, and C. albicans pathogenicity. Site-specific proteolytic degradation of Ece1p by the endoproteinase Kex2p releases immature candidalysin, SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (Ece1p 62Ϫ93 ), terminating with a C-terminal arginine (Arg93). Following release from Ece1p, the immature toxin is further processed by the carboxypeptidase Kex1p, which removes a C-terminal arginine to yield mature candidalysin, SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK (Ece1p 62Ϫ92 ) (Fig. S1C). The release of candidalysin from Ece1p can now be added to a growing list of protein zymogens and peptide precursors that are targeted for proteolysis to produce smaller biologically active molecules, including fungal killer toxins (23), hydrophobic plant repellent peptides (26), mating pheromones (27), and mammalian prohormones (28). Given the fundamental role of candidalysin in C. albicans virulence and the presence of kexin-like proteinases in other human-pathogenic fungi, kexin-mediated processing events and/or candidalysin itself may provide novel targets for the development of new therapeutic drugs to treat fungal infections.

MATERIALS AND METHODS
Fungal strains. All fungal strains used in this study are presented in Table S3 in the supplemental material (3,16,29,30).
Oligonucleotides. Oligonucleotide primers were purchased from Integrated DNA Technologies (Belgium). The sequence of primers used in this study is provided in Table S4.
Mammalian cell culture. Experiments were performed using the TR146 human oral epithelial cell line (31)  Infection of epithelial cells with C. albicans. Prior to infection, confluent TR146 epithelial cells were serum starved overnight, and all experiments were carried out in serum-free DMEM-F-12 medium. For Western blotting, epithelial cells were infected with C. albicans strains at a multiplicity of infection (MOI) of 10 for 2 h. For cytokine and damage assays, cells were infected at an MOI of 0.01 for 24 h. For c-Fos DNA binding assays, cells were infected at an MOI of 10 for 3 h. Following infection, cells were cultured at 37°C and 5% CO 2 .
Construction of alanine substitution mutants in C. albicans Ece1p. The plasmid CIp10-ECE1 (3,32), containing the ECE1 gene and its upstream and downstream intergenic regions, was used as a parental template for site-directed mutagenesis. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis system (Agilent). Alanine substitutions in the ECE1 gene were screened by restriction endonuclease digestion, and mutations were confirmed by DNA sequencing. Mutagenized constructs were linearized by digestion with StuI and concentrated by ethanol precipitation prior to transformation.
Transformation of C. albicans. A uridine auxotrophic ece1 null mutant [ece1Δ/Δ (ura Ϫ )] was transformed with 15 g of linearized construct using a lithium acetate method modified from reference 33. Transformants were selected on SD agar medium and were restreaked onto fresh SD agar three times to ensure stability, and genomic DNA was extracted using phenol-chloroform-isoamyl alcohol and glass bead lysis. Successful integration of each construct into the C. albicans genome (at the RPS1 locus) was confirmed by PCR amplification across the 5= and 3= integration sites.
Epithelial cell damage assay. Damage to epithelial monolayers was determined by quantification of lactate dehydrogenase activity using a CytoTox 96 nonradioactive cytotoxicity assay (Promega) according to the manufacturer's instructions. Porcine lactate dehydrogenase (Sigma) was used to create the standard curve.
Transcription factor (c-Fos) DNA binding assay. Epithelial cells were differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Active Motif) according to the manufacturer's instructions, and 5 g of nuclear extract was quantified for c-Fos DNA binding activity using a TransAM DNA binding assay (Active Motif) according to the manufacturer's instructions.
Growth curve analysis. C. albicans mutant and control strains were cultured in YPD liquid medium overnight at 30°C in a shaking incubator (180 rpm). Cultured cells were washed twice in sterile phosphate-buffered saline (PBS), and absorbance (600 nm) was adjusted to 0.1 in YPD liquid medium, using a Biochrom WPA CO8000 cell density meter. For each analysis, 200 l of adjusted culture was added to three individual wells of a 96-well plate. The plate was sealed and maintained at 30°C, and the absorbance (600 nm) was determined every 30 min using a Tecan Infinite 200 Pro plate reader (Tecan Instruments).
Hyphal length analysis. C. albicans mutant and control strains were cultured in YPD liquid medium overnight at 30°C in a shaking incubator (180 rpm). Cultured cells were washed twice in sterile PBS and adjusted to a concentration of 5 ϫ 10 4 ml Ϫ1 in RPMI 1640 medium. For each analysis, 1 ml of adjusted culture was added to 2 individual wells of a 24-well plate, each containing a glass coverslip. The plate was incubated at 37°C, 5% CO 2 , for 3 h, and after incubation, the wells were washed once with PBS and fungal cells were fixed in 4% paraformaldehyde, washed again, and stained with calcofluor white. Coverslips were mounted on microscopy slides, and images were taken of at least 50 fungal cells using fluorescence microscopy. Hyphal length was measured using the software ImageJ (34). Hypha branches were included in the measurement, and nonfilamentous cells were assigned a hyphal length equal to 0.
Inhibition of secreted aspartic proteinase activity. C. albicans BWP17ϩCIp30 was cultured under hypha-inducing conditions in the presence of 50 M pepstatin A (Sigma) to inhibit the activity of Saps. A control culture treated with an equivalent volume of ethanol (vehicle) was prepared in parallel.
Candidalysin Maturation from C. albicans Ece1p . Mass spectra were searched for both unspecific cleavages (no enzyme) and tryptic peptides up to 4 missed cleavages. The precursor mass tolerance was 10 ppm, and the fragment mass tolerance was 0.02 Da. At least two unique peptides per protein, a false discovery rate of Ͻ1%, and cross-correlation (Xcorr) validation (from 2.0 at z ϭ 2 up to 3.0 at z ϭ 6) were required for positive protein hits.
RNA extraction from fungi. C. albicans mutant and control strains were cultured in YPD liquid medium overnight at 30°C in a shaking incubator (180 rpm). Cells were washed twice in PBS, and the concentration was adjusted to 1 ϫ 10 7 cells ml Ϫ1 in 25 ml RPMI 1640 (hypha inducing) or 5 ml YPD (yeast). For hyphal samples, fungal suspensions were distributed in 150-cm 2 petri dishes and incubated at 37°C and 5% CO 2 for 3 h. After incubation, medium and nonadherent Candida cells were discarded. Adherent Candida cells were rinsed once with ice-cold PBS, loosened with a cell scraper, and collected. For yeast samples, fungal suspensions were cultured at 30°C for 3 h in a shaking incubator (180 rpm). After incubation, cells were collected by centrifugation (3,000 ϫ g for 2 min at 4°C) and resuspended in 10 ml ice-cold PBS. Hyphae and yeast samples were washed again with 1 ml ice-cold PBS and centrifuged (3,000 ϫ g, 2 min, 4°C), the supernatant was removed, and cell pellets were snap-frozen in liquid nitrogen. Frozen Candida pellets were thawed in 600 l RLT buffer (Qiagen) containing 1% ␤-mercaptoethanol, mixed with 300 l acid-washed glass beads (diameter, 0.5 mm), and bead beaten twice at 5,500 rpm for 15 s. Lysates were centrifuged for 2 min at 20,000 ϫ g, 4°C, the supernatant was mixed with an equal volume of 70% ethanol (prepared in diethyl pyrocarbonate [DEPC]-water), and total RNA was isolated using the RNeasy minikit (Qiagen) according to the manufacturer's instructions. RNA integrity and concentration were confirmed using a Bioanalyzer (Agilent).
Quantification of C. albicans ECE1 gene expression. RNA (500 ng) was treated with DNase (Epicentre), and cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). cDNA samples were used for qPCR with EvaGreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for ACT1 and ECE1-F2 and ECE1-R for ECE1 [Table S4]) were used at a final concentration of 500 nM. qPCR amplifications were performed using a CFX96 thermocycler (Bio-Rad). ECE1 expression was calculated using the threshold cycle (ΔΔC T ) method, with ACT1 as the reference gene and C. albicans reference strain SC5314 (yeast morphology) as the control sample.
Nonimmunosuppressed model of OPC infection. BALB/c mice were purchased from Harlan and housed at King's College London. A murine model of oropharyngeal candidiasis (OPC) (35) was modified to investigate early-phase gene expression responses to selected C. albicans alanine substitution mutants and controls. Briefly, nonimmunosuppressed female BALB/c mice (6 to 8 weeks old, 22 to 25 g) were sedated for 75 min with an intraperitoneal injection of 110 mg/kg of body weight ketamine and 8 mg/kg xylazine, and a swab soaked in sterile saline (vehicle) or 1 ϫ 10 7 CFU ml Ϫ1 of C. albicans yeast culture (WT, ece1Δ/Δ, ece1Δ/ΔϩECE1, ece1Δ/ΔϩECE1 Δ184 -279 , R31A, R61A, R93A, R61 ϩ R93A, and ALL KA) in sterile saline was placed sublingually for 75 min. After 24 h, mice were sacrificed, the tongue was excised, and RNA was extracted as described below.
RNA extraction from murine tissue. Murine tongue tissue was homogenized in RLT lysis buffer (Qiagen) containing 1% ␤-mercaptoethanol using a gentleMACs dissociator (Miltenyi Biotec), and RNA was extracted using an RNeasy Plus minikit (Qiagen) according to the manufacturer's instructions.
Quantification of gene expression from murine tissue. RNA (600 ng) was treated with Turbo DNase (Invitrogen), and cDNA was synthesized using Superscript IV reverse transcriptase (Invitrogen). cDNA samples were used for qPCR with FIREpol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne). Primers (complementary to murine beta-actin, Ccl20, Il1b, Il6, and Csf3 [Table S4]) were used at a final concentration of 400 nM. qPCR amplifications were performed using a RotorGene qPCR system (Corbett). Gene expression was calculated using the two-standard-curve method with murine beta-actin as the reference gene.
Murine model of oropharyngeal candidiasis. BALB/c mice were purchased from The Jackson Laboratory and housed at the University of Pittsburgh. Mice were injected subcutaneously with 225 mg/kg cortisone acetate (prepared in 0.05% Tween 20-PBS solution) 1 day before infection. The OPC experiment was performed the following day. Mice were sedated with an intraperitoneal injection of ketamine-xylazine solution (15 mg ml Ϫ1 ketamine, 1.5 mg ml Ϫ1 xylazine prepared in sterile saline). A 2.5 mg cotton ball was soaked in C. albicans solution (1 ϫ 10 7 CFU ml Ϫ1 in sterile PBS) and placed sublingually for 75 min as described in reference 36. Oral swabs were obtained before every experiment to verify the absence of commensal fungi. Mice were sacrificed, the tongue was excised, tissue homogenates were prepared on a gentleMACS dissociator (Miltenyi Biotec), and CFU were determined by plating serial dilutions on YPD agar supplemented with 50 g ml Ϫ1 ampicillin.
Statistical analysis. All data were analyzed by one-way analysis of variance (ANOVA) with a post hoc Dunnett comparison test. In all cases, P Յ 0.05 was taken to be significant. Where data are expressed as "fold change versus vehicle control," a log transformation was performed prior to performing statistical analysis, to ensure a normal distribution of data.
Ethics statement. Murine infections were performed under UK Home Office project license PPL 70/7598 in dedicated animal facilities at King's College London and under U.S. license 14125154 (with modification number IM-14125154-21). All protocols were approved by the King's College London ethical review board and the University of Pittsburgh IACUC. Power analysis was used to predetermine sample size. No method of randomization was used to allocate animals to experimental groups. Mice in the same cage were part of the same treatment. The investigators were not blind during outcome assessment.