A C. albicans TRAPP Complex-Associated Gene Contributes to Cell Wall Integrity, Hyphal and Biofilm Formation, and Tissue Invasion

ABSTRACT While endocytic and secretory pathways are well-studied cellular processes in the model yeast Saccharomyces cerevisiae, they remain understudied in the opportunistic fungal pathogen Candida albicans. We previously found that null mutants of C. albicans homologs of the S. cerevisiae early endocytosis genes ENT2 and END3 not only exhibited delayed endocytosis but also had defects in cell wall integrity, filamentation, biofilm formation, extracellular protease activity, and tissue invasion in an in vitro model. In this study, we focused on a potential C. albicans homolog to S. cerevisiae TCA17, which was discovered in our whole-genome bioinformatics approach aimed at identifying genes involved in endocytosis. In S. cerevisiae, TCA17 encodes a transport protein particle (TRAPP) complex-associated protein. Using a reverse genetics approach with CRISPR-Cas9-mediated gene deletion, we analyzed the function of the TCA17 homolog in C. albicans. Although the C. albicans tca17Δ/Δ null mutant did not have defects in endocytosis, it displayed an enlarged cell and vacuole morphology, impaired filamentation, and reduced biofilm formation. Moreover, the mutant exhibited altered sensitivity to cell wall stressors and antifungal agents. When assayed using an in vitro keratinocyte infection model, virulence properties were also diminished. Our findings indicate that C. albicans TCA17 may be involved in secretion-related vesicle transport and plays a role in cell wall and vacuolar integrity, hyphal and biofilm formation, and virulence. IMPORTANCE The fungal pathogen Candida albicans causes serious opportunistic infections in immunocompromised patients and has become a major cause of hospital-acquired bloodstream infections, catheter-associated infections, and invasive disease. However, due to a limited understanding of Candida molecular pathogenesis, clinical approaches for the prevention, diagnosis, and treatment of invasive candidiasis need significant improvement. In this study, we focus on identifying and characterizing a gene potentially involved in the C. albicans secretory pathway, as intracellular transport is critical for C. albicans virulence. We specifically investigated the role of this gene in filamentation, biofilm formation, and tissue invasion. Ultimately, these findings advance our current understanding of C. albicans biology and may have implications for the diagnosis and treatment of candidiasis.

related processes. Our findings provide insight into the role of the TRAPPII tethering complex for C. albicans filamentation, vacuolar functions, cell wall integrity, and virulencerelated properties.

RESULTS
Identification and comparison of S. cerevisiae TCA17 and C. albicans C4_06460C. A search of the Candida Genome Database listed the C. albicans gene C4_06460C as an ortholog to S. cerevisiae TCA17 (35). C. albicans C4_06460C is an uncharacterized gene located on chromosome 4 that has a 675-bp open reading frame (ORF), encoding a 224-amino-acid product. Comparison of protein sequences from S. cerevisiae TCA17 and C. albicans C4_06460C on SnapGene exhibited modest overall alignment, with 28.75% identity and 40.63% similarity.
To further elucidate the evolutionary relationships, the protein sequences of potential homologs in seven other Candida species were aligned with C. albicans C4_06460C and S. cerevisiae TCA17 using Muscle5 on SnapGene (Fig. 1A). This comparison revealed several regions of cross-species conservation, particularly between residues 26 and 42 and residues 74 and 90 of the C. albicans product. Furthermore, proline (P) 36 and lysines (K) 75 and 173 are absolutely conserved across all nine species. P27 and K138 also demonstrated conservation in eight of the nine species examined. All these species also had an asparagine-proline (NP) motif. Interestingly, one of the prolines (P36), the three conserved lysines, and the NP motif are also conserved in the human homolog TRAPP2CL (Fig. 1C), suggesting evolutionary selection for these residues over time.
The protein modeling program AlphaFold (https://alphafold.ebi.ac.uk/) calculated a striking similarity in the three-dimensional structures of C. albicans C4_06460C and S. cerevisiae TCA17 (Fig. 1B). Both proteins are predicted with high confidence to have three alpha-helical regions and five antiparallel beta-strands. This same secondary and tertiary structure is found in the human homolog TRAPP2CL. In addition to the conserved structures, the Candida protein has several loops of less structured protein sequence that are not found in either the Saccharomyces or human homologs.
Given the partial homology to S. cerevisiae TCA17, the presence of conserved residues across eukaryotic species, and the conservation of the predicted secondary and tertiary structures, C. albicans C4_06460C was considered and referred to as TCA17.
Construction of the C. albicans tca17D/D mutant and reintegrant strains. Using a reverse genetics approach, we investigated the function of C. albicans TCA17. We generated the C. albicans tca17D/D null mutant (KO) and the corresponding reintegrant (KI) strain through a PCR-based, C. albicans-adapted CRISPR-Cas9 strategy. PCR of genomic DNA indicated several candidate KI and KO transformants but confirmed the correct KO strain composition only in transformant 41 (Fig. S1 in the supplemental material). These results were supported by Southern blotting (data not shown). Therefore, null mutant strain 41 was selected for further experimentation as the KO strain, and KI1 was arbitrarily selected as the KI strain.
Contribution of C. albicans TCA17 to growth, viability, and cell morphology. We next analyzed the growth of the C. albicans tca17D/D mutant compared to that of the wild-type (WT) and complemented KI strains. When grown on yeast extract peptone dextrose (YPD) agar plates at 30°C, 37°C, and 42°C, the KO strain displayed a very slight reduction in growth compared to the control and complemented strains and also demonstrated an absence of temperature sensitivity ( Fig. 2A). Small fluctuations can be attributed to subtle variability in cell dilution and pipetting. However, when grown in liquid YPD medium at 30°C, a slight growth delay was observed for the KO strain, particularly between 8 h and 12 h of growth (Fig. 2B). The doubling times for the WT, KI, and KO strains were calculated as 3.17 6 0.09 h, 3.00 6 0.05 h, and 3.53 6 0.13 h, respectively.
Cell morphology was studied by first staining cells with Calcofluor white, a dye that binds chitin and cellulose in yeast cell walls, followed by visualization using differential interference contrast (DIC) and fluorescence microscopy. Yeast cells of the KO strain appeared to be enlarged relative to cells of the WT and KI strains (Fig. 2C). Quantitative analysis of cell length determined the average cell length for the KO strain to be 7.661 6 0.323 mm, which was significantly longer than that observed for the WT (5.396 6 0.182 mm, P , 0.001) and KI (5.590 6 0.184 mm, P , 0.001) strains (Fig. 2D). Similarly, the average cross-sectional cell area for the KO strain (38.483 6 3.063 mm 2 ) was significantly larger than those of the WT (20.131 6 0.928 mm 2 , P , 0.001) and KI (20.166 6 0.781 mm 2 , P , 0.001) strains (Fig. 2E). Our findings indicate that C. albicans TCA17 may play a role in cell growth and morphology.
Contribution of C. albicans TCA17 to stress tolerance. To determine if the growth and morphological defects are related to cell wall integrity, we characterized the growth of the tca17D/D mutant in response to cell wall stressors and antifungal agents. While no growth defect was observed on plates containing sodium dodecyl sulfate (SDS), Calcofluor white, or lithium chloride, the null mutant saw a dramatic lack of growth compared to the WT and KI strains on medium containing Congo red, which disrupts fungal cell walls (Fig. 3). By contrast, the mild reduction of growth seen in the KO strain on the SDS, Calcofluor white, and salt osmotic stressor plates is attributable to the general growth defect on the standard rich medium (YPD) and yeast nitrogen base (YNB) minimal medium control plates (Fig. 3).
In response to three common antifungal drugs (amphotericin B, fluconazole, and caspofungin), the tca17D/D null mutant displayed no altered sensitivity to amphotericin B and fluconazole (Fig. 4). The slightly reduced growth in the KO strain is consistent with the previously described minor growth defect on YPD. However, a pronounced growth defect in the KO strain was observed on YPD agar plates with 0.05 mg/mL caspofungin. While all three (WT, KI, and KO) strains saw growth at a concentration of 0.025 mg/mL and minimal growth at a concentration of 0.1 mg/mL, only the WT and KI strains grew in the presence of 0.05 mg/mL caspofungin. Unlike fluconazole and amphotericin B, which both disrupt fungal membranes, caspofungin interferes with cell wall integrity via inhibition of glucan synthase (36). The increased sensitivity of the KO strain to cell wall-active agents Congo red and caspofungin reveals that C. albicans TCA17 contributes to maintenance of cell wall integrity.
Membrane-related endocytosis is intact in the C. albicans tca17D/D mutant. We investigated whether defects in endocytosis and intracellular membrane trafficking also accompanied the observed cell wall impairments in the tca17D/D mutant. The lipophilic fluorescent dye N-(3-triethylammoniumpropyl)-4-(6-[4-(diethylamino)phenyl] hexatrienyl) pyridiniumdibromide (FM4-64) inserts into the outer membrane and is internalized through clathrin-based endocytosis, allowing visualization of the process of transport toward the vacuole via endocytic intermediates.
We found that the tca17D/D mutant did not exhibit delayed active endocytosis. After 30 min of endocytosis, the stain accumulated in the vacuolar membrane of all three strains, indicating that FM4-64 was successfully endocytosed to the vacuole by the KO strain with similar efficiency and timing as the WT and KI strains (Fig. 5). These results are consistent with previous findings seen in Fig. 4 that the KO strain did not display altered susceptibility when grown in the presence of fluconazole, whose function depends on cellular internalization (37). The normal growth of the KO strain on medium containing fluconazole coupled with the lack of observed endocytic defects suggest that C. albicans TCA17 does not act directly within endocytic membrane trafficking pathways. However, FM4-64 staining confirmed the enlarged vacuolar phenotype of the KO strain, as previously demonstrated. Several key conserved residues are illustrated within boxes, including prolines (P27 and P36; orange), lysines (K75, K138, and K173; blue), and an asparagine-proline motif (NP; purple). (B) AlphaFold models of the putative protein structure of C. albicans C4_06460C and S. cerevisiae YEL048C. Notably, the program identified in both proteins three alpha-helical regions and five beta-sheet stretches with "high confidence" (dark blue color), which are found in a similar tertiary arrangement. In addition, C. albicans has loops that less rigorously conform to helical algorithms (light blue) as well as regions that are unstructured (orange and yellow). (C) The protein alignment extends the homology to human TRAPP2CL. The conserved residues are boxed as above. The regions of highest amino acid homology coincide with the secondary structures identified by AlphaFold in all three homologs. The boxes below the alignments delimit those structures as found in C. albicans. C. albicans TCA17 plays a role in filamentation and biofilm formation. C. albicans hyphal and biofilm formation are dependent on proper cell wall composition. To determine whether these cell wall defects also impact filamentation, we tested the ability of the tca17D/D mutant to form hyphae on solid filamentation medium, including fetal calf serum (FCS), RPMI 1640, medium 199 (M199), and Spider medium (Fig. 6A). We found that the null mutant demonstrated a marked reduction in filamentous growth, whereas the WT and KI strains saw robust filamentous growth on and around the spotted colony. When grown in liquid RPMI 1640 medium overnight at 37°C, the null mu-  tant predominantly formed pseudohyphae compared to true hyphal growth observed in the control and complemented strains (Fig. 6B).
To assess the effects of defective filamentation on biofilm formation, we next assessed biofilm metabolic activity. Relative to the WT and KI strains, biofilm metabolic activity decreased by 67.4 6 0.35% in the KO strain (Fig. 6C). In addition, when visualized by light microscopy, the null mutant formed minimal biofilm that was sparse, patchy, and poorly adherent compared to the dense sheet-like film seen in the WT and KI strains (Fig. 6D).  Taken together, these results indicate that C. albicans TCA17 is necessary for proper hyphal and biofilm formation.
Dissolution of cell-cell adhesions is impaired in the C. albicans tca17D/D mutant. Hyphal and biofilm formation, along with secretion of extracellular proteases, are key attributes required for C. albicans virulence and pathogenesis. Given the role of S. cerevisiae TCA17 in secretion (31) and the defects in filamentation and biofilm formation in the C. albicans tca17D/D mutant, we next investigated the role of C. albicans TCA17 in virulence using a human vaginal keratinocyte (VK-2) model of infection. C. albicans virulence is characterized by its ability to target tight junctions between host cells and destroy cell-cell adhesions. This activity is partially facilitated by secreted aspartic proteases Sap4 to Sap6 that digest E-cadherin, a mammalian adhesion protein present at tight junctions (38,39). Therefore, we infected VK-2 cells with C. albicans WT, KI, or KO strains for 6 h and 24 h and visualized the labeled E-cadherin using fluorescence microscopy. While E-cadherin-labeled tight junctions remained intact 6 h after infection with all three C. albicans strains, no E-cadherin staining was observed in VK-2 cells infected with WT and KI strains after 24 h (Fig. 7A). By contrast, E-cadherin-labeled puncta were still present in VK-2 cells incubated with the KO strain after 24 h, albeit to a slightly diminished degree compared to at 6 h (Fig. 7A).
Impaired dissolution of cell-cell adhesions for the null mutant was also studied via Western blotting to determine the presence and absence of E-cadherin in VK-2 cells at 0, 6, and 24 h postinfection (Fig. 7B). E-cadherin levels were similar in VK-2 cells infected with WT, KI, and KO strains at 6 h. However, while E-cadherin was virtually undetected in WTand KI-infected cells after 24 h, E-cadherin levels remained stable in KO-infected cells, consistent with the microscopy results. These findings suggest that deletion of C. albicans TCA17 is associated with reduced virulence, as measured through the diminished ability to disrupt cell-cell adhesions in host VK-2 cells.
C. albicans TCA17 influences the ability to kill host cells. In addition to disruption of tight junctions, C. albicans has the ability to kill host cells. We sought to further define the role of TCA17 in C. albicans pathogenesis. Using a microplate live/dead assay (Invitrogen, Waltham, MA), we tested the ability for the tca17D/D mutant to kill host cells by measuring VK-2 cell growth after infection with WT, KI, or KO strains. At 6 h postinfection, almost 100% of VK-2 cells were still alive after exposure to any of the three strains (Fig. 8A). At 24 h, the WT and KI strains killed about 50% and 70% of VK-2 cells, respectively, but nearly all of the VK-2 cells remained alive after KO infection (Fig. 8B). Thus, the KO strain was unable to kill VK-2 cells in vitro, indicating that TCA17 contributes substantially to the virulence-related capacity of C. albicans.

DISCUSSION
Although S. cerevisiae TCA17 (YEL048C) has been identified as an ortholog of C. albicans C4_06460C, analyses to determine functional homology have yet to be performed (35). Sequence alignment between C. albicans C4_06460C and S. cerevisiae TCA17 demonstrated a modest overall alignment score on UniProt. However, analysis of potential homologs between C. albicans, S. cerevisiae, seven other Candida species, and humans revealed several regions of conservation. Interestingly, none of the conserved regions corresponded to any identified protein domains, and no domains have been annotated on the translated product from C. albicans C4_06460C. Proline residues P23 and P36 of the C. albicans product are conserved across at least eight of the nine aligned species. As the conformation of proline side chains often introduces a kink in the peptide backbone, these prolines may play an important role in protein structure and function. Additionally, three conserved lysine residues are found across Candida species, which are also conserved in the human homolog, TRAPP2CL. In proteins, lysine residues can undergo a number of posttranslational modifications, including acetylation, methylation, and ubiquitination, to regulate enzyme activity, protein-protein interactions, stability, and localization (40). Therefore, these residues may serve as key sites of modification for proper protein function in eukaryotes. Furthermore, a conserved NP motif was observed at N181 and P182 of the C. albicans product. The tripeptide motif NPF has been shown to interact with endocytic machinery and is present in the C. parapsilosis, C. orthopsilosis, C. auris, C. glabrata, and human homologs as well as the C. albicans early endocytosis gene ENT2 (8,41,42). Mutation of this motif to NPV has been reported to abolish binding between endocytic adaptor proteins (42). Notably, both C. albicans C4_06460C and S. cerevisiae TCA17 have leucine as the third amino acid (NPL), suggesting that the protein may not function directly in endocytosis. While bioinformatic analysis of C. albicans C4_06460C provides limited indication of its cellular function, the presence of conserved residues and secondary and tertiary structures across Candida species and S. cerevisiae suggests that this gene is the TCA17 homolog in C. albicans.
C. albicans growth is dependent on its ability to adapt its physiology to external nutrient conditions (43). Our results revealed that deletion of TCA17 in C. albicans is not lethal, but the tca17D/D mutant (KO) exhibited slightly reduced and delayed growth on both solid and liquid media. In addition, altered cellular morphology was observed, with the KO strain demonstrating a significantly larger cell size than the wild-type (WT) and complemented (KI) strains. The increased cell size could be due to a number of factors. This "goliath cell" phenotype has been previously described in C. albicans under zinc-restricted conditions, where growth was also reduced in a zinc-dependent manner, suggesting that growth defects and enlarged cells may arise from defective or absent nutrient trafficking pathways (44). Curiously, the zinc-starved cells were hyperadherent to polystyrene surfaces, which contrasts with the weak biofilm observed in our KO strain (44). Furthermore, diminished growth may be associated with structural defects in the cytoskeleton, which could be related to intracellular nutrient trafficking. Septin proteins are a family of cytoskeletal filament-forming proteins that regulate hyphal morphogenesis and cytokinesis in C. albicans (45,46). Deletion of the septin gene CDC11 leads to partial defects in cytokinesis and enlarged cells (46). Moreover, cytoplasmic division occurs in the late stage of mitosis to physically separate a parent cell into two daughter cells, doubling the cell count. Therefore, the increased doubling time in the KO strain may be due to impaired cytokinesis. Thus, the growth defect displayed by the KO strain may be directly associated with the goliath phenotype, as failures in cytokinesis would result in larger cells and fewer cell divisions.
The tca17D/D mutant also exhibits an aberrantly large vacuole relative to the control and complemented strains. Several vacuolar mutants exhibit enlarged vacuoles in C. albicans, including mutants lacking VAM2, which encodes a subunit of the HOPS tethering complex, or VPS34, a phosphatidylinositol-3-kinase gene (47,48). Null mutants of either gene demonstrate defects in virulence, which was also observed in our tca17D/D mutant. Loss of Rab GTPases Vps21 and Ypt52 led to intact but expanded vacuoles in C. albicans (49). Therefore, membrane fusion and tethering play a role in proper vacuolar morphology, supporting the hypothesis that C. albicans TCA17 may function in the TRAPPII tethering complex. Taken together, C. albicans TCA17 is required for proper cell growth. Growth and morphological defects in the KO strain may be attributed to issues in nutrient transport, cytokinesis, vacuolar integrity, or exocytosis. Additional investigations are needed to elucidate the mechanisms that lead to these abnormalities.
In addition, C. albicans TCA17 is involved in stress tolerance. Impaired growth of the KO strain in response to cell wall stressors (caspofungin and Congo red), but not plasma membrane stressors (fluconazole and amphotericin B), suggests that TCA17 contributes to cell wall integrity. Specifically, fluconazole interferes with ergosterol biosynthesis to impair plasma membrane integrity, and amphotericin B binds ergosterol in the fungal cell membrane leading to pore formation, but the antifungal drug caspofungin inhibits glucan synthase to interfere with fungal cell wall integrity (50)(51)(52). Additionally, Congo red interacts with b-linked glucans and chitin to impede cell wall assembly (53). However, as Calcofluor white also inhibits chitin synthesis, it is unclear why the KO strain only showed increased sensitivity to Congo red, although the two dyes have slightly different chemical structures (54). It is also possible that C. albicans TCA17 is involved in glucan but not chitin biosynthesis.
In comparison to our previous studies on C. albicans ENT2 and END3, TCA17 appears to play a lesser role in cell wall maintenance, as ent2D/D and end3D/D null mutants exhibited sensitivity to a wider range of stressors, including amphotericin B, Calcofluor white, SDS, and osmotic stressors (7,8). The tca17D/D mutant did not show increased sensitivity under salt stress conditions (55). Interestingly, our results directly contrast with preliminary observations from our lab on the C. albicans early endocytosis gene PAL1, which demonstrated increased resistance to caspofungin and Congo red (unpublished data). C. albicans Ent2, End3, and PalI are all proposed as coat proteins in clathrin-mediated endocytosis. The differences in response to cell wall stressors between the endocytosis genes and TCA17 indicates that this gene likely functions in pathways distinct from endocytosis, despite still being necessary for WT cell wall integrity. In fact, defects in endosome-to-Golgi retrograde trafficking have been shown to alter the cell wall proteome and reduce virulence in C. albicans, which may be directly related to TCA17 and the TRAPPII complex (56).
Most interestingly, the C. albicans tca17D/D mutant exhibited a significant decrease in true hyphae and biofilm formation, producing filaments that appear more pseudohyphal in morphology. Therefore, C. albicans TCA17 plays a critical role in mediating hyphae formation and, consequently, biofilm formation. Although these findings can be partially attributed to the mild growth defect seen in yeast form, the impaired growth is unlikely to account for the magnitude of reduced filamentation and biofilm formation observed. The C. albicans tca17D/D null mutant was only able to transition between yeast and pseudohyphal forms, even under strong inducing conditions. Pseudohyphae in the tca17D/D null mutant appeared more swollen with larger vacuoles, consistent with the enlarged cells in the yeast form. Defects in cytoskeleton transport and cytokinesis can result in irregular hyphal morphology, as the septin ring at the cell junctions of hyphae distinguishes yeast and pseudohyphal growth from hyphal growth in C. albicans (57). The germination and extension of the C. albicans germ tube, which develops into hyphal filaments, also depend on the formation of enlarged vacuolar compartments (58,59). Repression of vacuolar transport genes VAC1, VAM2, and VAM3 results in sparsely branched hyphae but larger hyphal compartments, emphasizing the function of the vacuole in the filamentation process (47). Hence, the enlarged vacuole but lack of true hyphae in the KO strain may also be due to impaired cell division, potentially the same mechanisms causing defects in yeast growth and vacuolar morphology.
Given the involvement of C. albicans TCA17 in pathogenic mechanisms like filamentation and biofilm formation, its impact on C. albicans virulence was expected when assayed indirectly in our in vitro models. Indeed, the tca17D/D mutant showed impaired dissolution of cell-cell adhesion and ability to kill host cells. These findings are likely a product of reduced active penetration, which partially depends on physical force exerted by elongating hyphae. In fact, C. albicans mutants unable to form hyphae have been reported to exhibit significantly reduced epithelial cell damage compared with mutant strains still capable of forming hyphae in vivo, supporting our results (60). Therefore, the lack of true hyphae formed by the KO strain may be directly associated with its attenuated virulence.
Interestingly, the reduced virulence in the null mutant is not due to defects in the secretion of secreted aspartyl proteases (Saps). C. albicans Saps have previously been shown to destroy epithelial cell-associated E-cadherin (61,62). However, the WT, KI, and KO strains had similar-sized zones of bovine serum albumin (BSA) degradation (Fig. 7C), suggesting similar levels of Sap activity. Taken together, C. albicans TCA17 is critically involved in virulence-related mechanisms, likely due to its effects on filamentation, although its role in the secretion of different virulence factors has yet to be explored.

MATERIALS AND METHODS
Identification of the C. albicans homolog of S. cerevisiae TCA17. Using the Candida Genome Database (http://www.candidagenome.org), we retrieved the predicted protein and DNA sequences for the uncharacterized ORF C4_06460C in C. albicans and identified its single potential ortholog in S. cerevisiae as TCA17. The protein and DNA sequences for S. cerevisiae TCA17 were taken from the Saccharomyces Genome Database (http://www.yeastgenome.org). The protein sequences were aligned according to the Smith-Waterman method using the protein alignment function on SnapGene (http:// www.snapgene.com) (63). Secondary and tertiary protein structure modeling was done by AlphaFold (https://alphafold.ebi.ac.uk/) (64, 65).
Deletion of C. albicans TCA17 and Southern blotting. Table 1 lists the oligonucleotides and primers used for gene mutations and Southern blotting in this study. We generated a C. albicans tca17D/D null mutant (KO) and the knock-in reintegrant strain (KI) using the AHY940 wild-type strain and the CRISPR-Cas9 protocol developed by the Hernday laboratory (66). Successful transformations and correct strain construction were verified through PCR and Southern blotting of genomic DNA. For Southern blotting, 5 mg of genomic DNA was digested with BamHI and ScaI overnight and loaded onto a 1% agarose gel. Blotting was performed with a Hybond nylon membrane (Amersham) using standard protocols. The DNA probe was constructed by PCR using digoxigenin (DIG)-labeled dNTPs (Roche, Basel, Switzerland) and visualized with a DIG luminescent detection kit (Roche, Basel, Switzerland).
Strains, media, and cell culture. The C. albicans strains used in this study are listed in Table 2. Liquid cell cultures of C. albicans strains were established in YPD (1% yeast extract, 2% peptone, and 2% glucose) or in YNB minimal medium (0.67% yeast nitrogen base, amino acids, and 2% glucose) and incubated at 30°C with shaking at 250 rpm, unless otherwise specified. For agar plate assays, solid media were prepared by adding 2% agar, and the inoculated plates were incubated at 30°C or 37°C.
Human vaginal keratinocyte cells VK-2/E6E7 (ATCC CRL-2616, Manassas, VA) were grown in keratinocyte serum-free medium (SFM; Invitrogen, Waltham, MA) in a cell culture incubator at 37°C with 5% CO 2 . Cells were split once a week and fed every 2 days as per the manufacturer's instructions.
Preparation of genomic DNA and plasmid isolation. Genomic DNA was extracted from yeast cells using the MasterPure yeast DNA purification kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's instructions. Plasmids were transformed and maintained in competent Escherichia coli DH5a cells (Invitrogen, Waltham, MA). Plasmid extraction was conducted following the provided protocol for the Qiagen plasmid miniprep system (Qiagen, Germantown, MD) from overnight cultures of transformed E. coli cells grown in LB medium (1% tryptone, 0.5% glucose, and 1% NaCl) with 100 mg/mL ampicillin at 37°C. The plasmids generated and used in this study are listed in Table 3.
Cell growth assay and plate assays for response to environmental stress and filamentation. To investigate the effect of TCA17 on cell growth, C. albicans tca17D/D or control and complemented strains were first grown in YPD overnight at 30°C. Cell cultures were subsequently counted and diluted in YNB to a concentration of 1 Â 10 6 cells/mL; 100 mL of the corresponding cell dilutions were loaded in 96-well plates in triplicate, with three replicates per strain. The growth curve was determined at 30°C using a BioTek Synergy H1 microplate reader that recorded the extinction at an optical density at 600 nm (OD 600 ) at 30-min intervals over 16 h. The data were analyzed, and the average OD 600 values were graphed over time using Excel (Microsoft).
Growth rates were also assessed qualitatively using agar plate assays as previously described (13,67). Overnight cell cultures grown in YPD at 30°C were counted and diluted to a concentration of 1 Â 10 8 cells/mL in YPD. Five 5-fold serial dilutions were then performed, and 5 mL of cells from each concentration were spotted onto agar plates. Growth on YPD plates at 30°C, 37°C, and 42°C was observed after 48 h. Response to cell wall stressors was assayed after 48 h at 30°C on YNB plates containing 140 mg/mL Congo red or 100 mg/mL Calcofluor white and on YPD plates containing 0.02% SDS, 100 mM lithium chloride, or 500 mM sodium chloride (all from Sigma-Aldrich, St. Louis, MO). In addition, sensitivity to antifungal drugs was assessed on YPD agar medium containing amphotericin B (0.11, 0.33, and 1 mg/mL), fluconazole   Immunofluorescence microscopy of C. albicans strains. We documented cell morphology of the yeast and hyphal forms as described in Rollenhagen et al. (8) and Pringle et al. (69). Yeast growth was supported by standard overnight culturing of cells in liquid YPD at 30°C, while filamentous growth was promoted by incubating cells overnight in RPMI 1640 medium at 37°C with shaking. Cells from the overnight cultures were mixed 2:1 with Calcofluor white (Sigma-Aldrich, St. Louis, MO), placed on a glass slide, and visualized with a 63Â lens objective through both the differential interference contrast (DIC) channel and the 49,6-diamidino-2-phenylindole (DAPI) filter of a Zeiss Axio Imager M1 fluorescence microscope.
Cell size was analyzed by evaluating the length and area of cells imaged via DIC light microscopy. The cell length was approximated as the major axis of each cell and measured with the ruler tool in Adobe Photoshop (Adobe, Inc., San Jose, CA). The cross-sectional cell area was measured by using the quick selection tool in Adobe Photoshop to encircle each cell and recording the area enclosed. Measurements were documented for 100 cells, and the average cell length and area were graphed in Excel (Microsoft Corporation, Redmond, WA).
Analysis of endocytosis with FM4-64. As an actively endocytosed lipophilic membrane dye, N-(3triethylammoniumpropyl)-4-(6-[4-(diethylamino)phenyl]hexatrienyl) pyridiniumdibromide (FM4-64; EMD Millipore, Temecula, CA) was used to assay membrane-related endocytosis and vacuole morphology as previously described (8). Briefly, FM4-64 at a final concentration of 2 mM was added to ice-cold cell cultures grown to the exponential phase. The cells were incubated on ice for 20 min for dye uptake and washed in ice-cold YPD before being incubated at room temperature for 5, 15, and 30 min. An aliquot of cells was taken at each time point and maintained in ice-cold 12 mM sodium azide (Sigma-Aldrich, St. Louis, MO) to stop membrane transport and observe the process over time. Staining of the vacuoles was visualized using a Zeiss Axio Imager M1 fluorescence microscope with standard Texas Red filters for FM4-64. Images were analyzed for the presence and rate of vacuolar membrane staining.
Analysis of adherence and biofilm formation in C. albicans endocytosis mutants. The reduction of 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT), a tetrazolium salt, was used to assay for biofilm metabolic activity according to published methods (70,71). Briefly, overnight cell cultures of C. albicans tca17D/D or control and complemented strains in YPD were diluted to a concentration of 1 Â 10 6 cells/mL in RPMI 1640 to promote biofilm growth. One hundred microliters of the cell dilutions was loaded into a CellBIND 96-well microplate (Corning, Inc., Corning, NY) in triplicate with 3 separate biological replicates and incubated for 48 h at 37°C. Films were washed with phosphate-buffered saline (PBS) and incubated with XTT-menadione substrate (10 mM menadione stock diluted 1:10,000 in XTT) at 37°C for 2 h. The supernatant was subsequently transferred to a new 96-well plate, where the absorbance at 490 nm was read using a BioTek Synergy H1 microplate reader (Winooski, VT). The relative biofilm activities for the wild-type, knock-in, and knockout strains were graphed, with the absorbance of wild-type biofilms arbitrarily set as 100%. Statistical significance was assessed using the Student's t test in Microsoft Excel. Biofilms were visualized via DIC light microscopy on a Nikon Eclipse Ti inverted microscope.
Immunofluorescence microscopy of cocultured VK-2 cells with C. albicans strains. VK-2 cells in keratinocyte serum-free medium (SFM) were grown on glass coverslips at 37°C and 5% CO 2 and infected with C. albicans tca17D/D or control and complemented strains at a multiplicity of infection (MOI) of 0.01 for 6 and 24 h, as previously described (8,72). A 1:100 dilution of an E-cadherin primary antibody (R&D, Minneapolis, MN) in 0.2% gelatin-PBS was then used to stain the VK-2 cells for 1 h. VK-2 cells were subsequently washed and incubated with an anti-rat Alexa Fluor 488 secondary antibody (Invitrogen, Waltham, MA) for 1 h. Coverslips were mounted on slides with an antifade mounting solution containing DAPI for nuclear visualization. Images were taken using a Zeiss Axio Imager M1 microscope with standard enhanced green fluorescent protein (eGFP) and DAPI filters and assessed for the presence of E-cadherin.
Western blotting and detection of E-cadherin. Western blotting was performed with a standard protocol as described in Kurien and Scofield (73) using protein lysates from VK-2 cells infected with C. albicans tca17D/D mutant or control and complemented strains for 6 and 24 h in keratinocyte SFM at 37°C and 5% CO 2 . The blot was then hybridized overnight in 1Â Tris-buffered saline casein blocking buffer (Bio-Rad, Hercules, CA) with a 1:500 dilution of an E-cadherin primary antibody (R&D, Minneapolis, MN) or 1:1,000 dilution of a tubulin antibody (Invitrogen, Waltham, MA) as a loading control. After washing, the blot was incubated with an anti-mouse horseradish peroxidase (HRP) secondary antibody (Invitrogen, Waltham, MA) for 1 h. Protein detection was performed using Clarity Max Western ECL substrates (Bio-Rad, Hercules, CA) and imaged with a ChemiDoc imaging system (Bio-Rad, Hercules, CA).
Live/Dead viability assay. VK-2 cells were grown to 30% confluency in keratinocyte SFM in 96-well plates. Overnight cultures of C. albicans strains in YPD were cocultured with VK-2 cells at a concentration of 5 Â 10 5 cells/mL for 6 and 24 h. The Invitrogen Live/Dead viability assay (Waltham, MA) was performed according to the manufacturer's protocol, and the data were analyzed using Excel (Microsoft Corporation, Redmond, WA) as per the manufacturer's instructions.