Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.


Introduction
C. albicans is a commensal fungus of the human orogastrointestinal and vaginal tracts [1,2]. When the immune system is compromised, or when the normal microbiota are disrupted, debilitating and often recurring mucosal diseases can result. C. albicans, the most common clinical Candida isolate, uses adhesins, hypha formation, phenotypic switching and production of extracellular hydrolytic enzymes to interact with its human host [3][4][5][6]. Among C. albicans adhesins is the Als (agglutinin-like sequence) family, encoded by eight distinct genetic loci (ALS1 to ALS7, ALS9; [7,8]). Als proteins have a similar structure, including an N-terminal secretory signal sequence, followed by an NT domain of approximately 320 amino acids, a T domain of approximately 104 amino acids, a TR domain of head-to-tail copies of a Ser/Thr-rich repeated sequence, and a Ser/Thr-rich C domain of variable size and sequence. Mature Als molecules are large glycoproteins that are linked to b-1,6 glucan in the C. albicans cell wall [9]. For example, the estimated sizes for mature Als1 and Als3 are 600 and 440 kDa, respectively.
Because of its proposed similarity to functional domains of other adhesion proteins, the Als NT domain is often studied in the absence of the remainder of the mature molecule. X-ray crystallography and NMR were used to solve the structure of the Als9-2 (amino acids  and Als1 (amino acids 18-329) respectively [10]. This work showed highly similar structures for the two proteins that feature two tandem Dev-IgG type immunoglobulin domains and a cavity, with an invariant Lys residue, which binds to the C-terminal carboxyl group of peptides with broad binding specificity. It is very likely that these structural features will be found in the corresponding fragments from the remaining proteins in the Als family, although the other structures must still be solved. Als proteins, particularly Als3, contribute to biofilm formation, mediate epithelial invasion and induce epithelial cell damage [11]. Als3 has been the focus of considerable investigation since it is produced so abundantly on the surface of germ tubes and hyphae [12], providing a potential intersection between adhesive function and hypha formation. Hypha formation is also very important in mucosal pathogenicity [13][14][15].
As part of our studies of interactions between C. albicans and oral epithelial cells, we discovered a mechanism that enables oral epithelial cells to discriminate between C. albicans yeast and hyphae via a mitogen-activated protein kinase (MAPK) signaling pathway [16][17][18][19]. This discriminatory mechanism targets C. albicans hyphae and constitutes activation of the MAPK phosphatase MKP1 and c-Fos transcription factor, which are involved in the induction and regulation of a proinflammatory cytokine response. Since the ALS gene family is expanded in C. albicans and has adhesion/invasion functions, we sought to determine the role of this family in epithelial adhesion and induction of cell damage. Furthermore, given that Als3 is abundant on hyphae, we wanted to determine whether Als3 is the moiety that mediates activation of the MAPKbased MKP1/c-Fos signaling pathway leading to cytokine induction.
Adherence assay and morphological analysis TR146 oral epithelial cell monolayers were grown to confluence in six-well tissue culture plates and serum-starved overnight. 100, 200 or 300, depending on the assay, C. albicans yeast cells were added to replicate wells containing 1 ml serum-free DMEM for 90 min at 37uC and 5% CO 2 . C. albicans contact with epithelial cells is a strong inducer of hypha formation, and by 90 min significant germ tube formation has already occurred. Following incubation, non-adherent C. albicans cells were removed by aspiration. Each well was washed twice with 1 ml PBS, and overlaid with molten Sabouraud's dextrose agar (SDA) at 45uC. Plates were then incubated at 30uC for 24 h, and the resulting colonies counted. Replicate control plates of the initial inoculum were also incubated and the resulting colonies counted. Percent adherence was calculated as (mean adherent CFU/mean total CFU)6100. The experiments were repeated on two separate occasions. For morphological analysis, CAI4 and Dals3 strains were inoculated onto TR146 monolayer cultures at a MOI of 10. After 90 min monolayers were fixed in 10% buffered formalin and examined by differential interference contrast (DIC) microscopy at 6400.

Cell damage assay
TR146 confluent monolayers were grown in 24-well tissue culture plates. C. albicans yeast cells were added to TR146 monolayers and incubated at 37uC and 5% CO 2 for 24 h. A fungal:epithelial cell multiplicity of infection (MOI) between 0.01 and 10 was used. Control wells contained PBS alone. Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to manufacturer's instructions. A recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve and sample values extrapolated from the curve. The assay was conducted using a single well on three separate occasions. Replicate LDH measurements were made on the single well. Data were analyzed using a two-tailed ttest with p,0.05 considered significant.

Measurement of cytokine levels
C. albicans cells were added to TR146 monolayers, grown in 24well tissue culture plates, and incubated as described above. At 24 h, culture supernatants were used to measure cytokine levels (IL-1a, IL-6, G-CSF, GM-CSF) using the Fluorokine MAP cytokine multiplex microbead assay kits (R&D Systems), coupled with the Bioplex 200 TM machine according to the manufacturer's protocol. The assay was conducted using a single well on three separate occasions. Replicate cytokine measurements were made on the single well. Data were analyzed using a two-tailed t-test with p,0.05 considered significant.

Western blotting
Western blotting was used to assess phosphorylation of MKP1 and induction of c-Fos in TR146 monolayers that were incubated with C. albicans. C. albicans yeast cells were added to the TR146 monolayers, grown in 12-well tissue culture plates, and incubated for 2 h at 37uC and 5% CO 2 . Epithelial cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing inhibitors of protease (Perbio, UK) and phosphatase (Sigma-Aldrich, UK). The lysate was incubated on ice for 30 min and centrifuged for 10 min in a refrigerated microfuge. Supernatants were assayed for total protein using the BCA Protein Quantitation kit (Perbio, UK). Protein (15 mg) was separated on a 12% NuPAGE Bis:Tris minigel (Invitrogen, UK) before transfer to a PVDF membrane (GE Healthcare). a-actin was used as a loading control. Membranes were incubated with primary (1:1000 dilution) and secondary (1:10,000 dilution) antibodies and developed using Immobilon chemiluminescent substrate (Millipore, UK). Rabbit monoclonal antibodies to human phospho-MKP1 and c-Fos were purchased from Cell Signaling Technologies (New England Biolabs, UK). Mouse monoclonal antibody to human a-actin was purchased from Millipore and goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunologicals Ltd (Stratech Scientific, UK). The assay was conducted on four independent occasions, inoculating a single TR146 well with each C. albicans strain.

Stimulation of epithelial monolayers with recombinant Als3
Soluble, purified NT-Als3 was prepared as described previously [12]. Briefly, a vector encoding the first 329 amino acids of Als3 and a hexa-His tail was transformed into Pichia pastoris. Soluble Als3 was secreted from the recombinant strain, producing a protein (amino acids 18 to 329) from which the secretory signal sequence was cleaved. The protein was purified by His-Trap column chromatography (GE Healthcare), dialyzed against phosphate buffered saline and its concentration determined using the Bradford method (Bio-Rad). The NT-Als3 protein was added to TR146 monolayers in the context of the cell damage, cytokine induction, MKP1 phosphorylation and c-Fos assays described above. Concentrations added ranged from 0.3-10 mg/ml. Assays were conducted on two separate occasions. Controls included PBS alone. Data analysis followed the methods described above.

Dals3 has reduced adhesion and damage to oral epithelial cells
The TR146 oral epithelial carcinoma cell line is one of the most common cell lines currently used to investigate C. albicans-oral epithelial cell interactions [13,14,[16][17][18]29,30]. We tested each of the Dals mutants for its ability to adhere to TR146 cells after 90 min of co-incubation. Only the Dals3 mutant was defective in adhesion in this assay, with approximately 30-40% reduced adhesion compared to control strains (Fig. 1a). Notably, the percentage of Dals3 cells forming germ tubes and the morphology of the germ tubes could not be distinguished from the wild-type strain (Fig. 1b). The Dals3 strain was also the only one of the set of Dals mutants to show significantly decreased damage to TR146 cells following 24 h of incubation (Fig. 2a). Although damage to TR146 cells was reduced by nearly 75% for the Dals3 strain, a small amount of epithelial cell damage was still observed. Because adhesion is a prerequisite for invasion and subsequent cell damage, this result is consistent with the adhesion assay data. Notably, at 24 h, the Dals3 strain showed decreased adhesion to the TR146 monolayer compared with the control (data not shown).
Because the Dals3 mutant showed significant reductions in the adhesion and cell damage assays, the assays were repeated to include the control strain 2322, into which an ALS3 allele was reintegrated. Restoration of wild-type adhesion (Fig. 1b) and cell damage (Fig. 2b) for this strain suggested that the results observed for the Dals3 mutant were attributable directly to loss of Als3. Variation between biological replicates was evident when comparing the various assays. For example, cell damage data for the Dals3 mutant varied between experiments (compare Fig. 2a and b); however, in both instances the trends were the same and damage caused by the Dals3 strain was reduced significantly compared to the control. It was also unclear why the ALS3 reintegrant strain 2322 showed higher LDH levels than strains SC5314 and CAI4, however, the strain clearly demonstrated rescue of the cell damage phenotype.
Reduced capacity of Dals3 to induce epithelial cytokines One marker of epithelial activation in response to an invading pathogen is the induction of a pro-inflammatory cytokine response. The ability of Dals mutants to induce cytokines from epithelial cells has not been investigated previously. Because of their role in adhesion to epithelial cells, one would expect that Alsepithelial receptor interactions at the cell surface might trigger cytokine production. We assessed whether Dals mutants were able to induce cytokine production in TR146 cells 24 h following inoculation with C. albicans at a MOI of 0.01. In this assay, only the Dals3 mutant was deficient in inducing G-CSF, GM-CSF, IL-6 and IL-1a (Fig. 3a), which we previously demonstrated as being the main read-out cytokines induced by C. albicans for TR146 cells [16]. Repetition of the cytokine assays using the ALS3 reintegrant control strain showed that the results observed for the Dals3 mutant were due to the loss of Als3 (Fig. 3b).

Dals3 induces MKP1 phosphorylation and c-Fos production
Epithelial cells discriminate between the yeast and hyphal form of C. albicans via a MAPK-based mechanism that targets the hyphal form, which involves MKP1 and c-Fos activation [16]. Given that the Dals3 mutant forms normal germ tubes/hyphae in the epithelial cell culture conditions used in these experiments, but was deficient in adhesion ( Fig. 1), damage induction ( Fig. 2) and cytokine production (Fig. 3), we hypothesized that Als3 may be the target on C. albicans hyphae that mediates discrimination between the yeast and hyphal form. Therefore, we screened the ability of the Dals mutants to induce MKP1 phosphorylation or c-Fos production. Because 2 h was the time point previously shown to be optimal for characterization of the discriminatory response [16], it was used for this assay. MKP1 phosphorylation and c-Fos production were induced by the Dals strains to a similar degree as by wild-type/parent cells (Fig. 4). This result indicated that deletion of individual ALS genes did not affect activation of epithelial cells via MPK1 or c-Fos and that Als3 was unlikely to be the hyphal moiety targeted by epithelial cells to discriminate between the yeast and hyphal form of C. albicans. Given that the Dals3 mutant was markedly reduced in its ability to cause epithelial damage (Fig. 2), the data also suggested that MKP1 or c-Fos activation was, in part, not directly correlated to damage as indicated previously [16].

Increasing the fungal burden of Dals3 rescues damage and cytokine phenotypes
Our previous work demonstrated that epithelial activation and cytokine production in TR146 cells is dependent on fungal burdens [16]. Because the Dals3 mutant exhibited poor epithelial adherence, we hypothesized that this phenotype may account for the poor ability of Dals3 to induce damage and a cytokine response, since damage and cytokine induction require sustained hypha formation and epithelial activation [13,17,18]. Thus, it was possible that the deficiencies in the epithelial damage and cytokine responses for the Dals3 strain may be because the threshold level of activation was not reached due to insufficient numbers of Dals3 cells adhering to and invading the epithelial cells.
First, we tested whether the percentage of adherence was increased if higher initial doses of Dals3 yeast cells were used. Although we did not observe an increase in the percentage of adherent cells by increasing the MOI, a greater number of Dals3 cells adhere to the epithelial cells as a result (Fig. 5a). The Dals3 shows a 50% reduction in adherence compared with the wild type strain CAI4. By increasing the number of Dals3 cells by 50% (from ,200 (low dose) to ,300 (high dose)) we observed a similar amount of adhered cells as with the parent strain when 50% fewer cells are used. This demonstrates that while the percentage of adherent cells does not change, more cells adhere if the number of cells is increased.
Next, we attempted to rescue the damage and cytokine phenotypes by increasing the Dals3 dose to the threshold activation level. We first inoculated TR146 monolayers for 24 h with SC5314, CAI4 and Dals3 at a MOI ranging from 0.01 to 10. Increasing the Dals3 burden 10-fold (from MOI of 0.01 to 0.1) was sufficient to initiate cell damage, albeit at a lower level than the wild-type/control strains (Fig. 5b). However, at a MOI of 10, Dals3 induced damage at similar levels as the wild-type/control strains, potentially indicating that a similar number of Dals3 cells were adhering and invading epithelial cells as the wild-type/ control strains at an MOI of 0.1. The importance of fungal burdens was further supported by the observation that the LDH levels caused by SC5314 and CAI4 peaked and reached a plateau at a MOI of 1. Presumably, most or all TR146 cells were degraded at 24 h using this MOI, so increasing the MOI to 10 could not further increase epithelial damage. The Dals3 strain continued to increase LDH levels to a MOI of 10, consistent with the observation that the strain adhered poorly and required a greater fungal cell number to cause epithelial damage.
Next, we investigated whether the increased fungal burden could induce epithelial cytokine production by infecting TR146 cells for 24 h with C. albicans SC5314, CAI4 or Dals3 using MOIs of 0.01 and 0.1. Similar to the cell damage results, increasing fungal burden 10-fold was sufficient to induce cytokine production (Fig. 5c). For IL-1a, the increased MOI of the Dals3 strain produced cytokine levels similar to the control strains. Since IL-1a is associated with cell damage [31,32], this result was consistent with the data that demonstrated increases in LDH when the Dals3 strain was used at a MOI of 0.1 (Fig. 5b). Together, the data suggested that the lack of damage and cytokine production observed for Dals3 at 24 h at lower MOIs was most likely due to its poor ability to adhere to and invade oral epithelial cells. Increasing the MOI restores all phenotypes by ensuring sufficient numbers of Dals3 cells adhere thus reaching the threshold level for activation. This also further demonstrates that a threshold level is required for epithelial cell activation (supporting our previous work [16], which is dependent on how many cells initially adhere. Furthermore, because cell damage and cytokine production were restored by increasing Dals3 burden, it was unlikely that Als3 was a hyphal factor that directly induced epithelial damage and cytokine production. Although Als3 was unlikely to be the hyphal factor that directly activates epithelial cells, we hypothesized that C. albicans hyphal adhesion/anchoring to epithelial cells was a prerequisite for epithelial activation. In other words, sufficient numbers of hyphae first need to adhere to epithelial cells to enable the hyphal factor that activates MKP1/c-Fos to be present in sufficient quantity. Because we previously determined the threshold level of MKP1-C-Fos activation by wild-type C. albicans (SC5314) to be around a MOI of 1 [16] and that Dals3 adheres poorly to oral epithelial cells (Fig. 1), we reduced the Dals3 MOI from 10 to 1 with the prediction that MKP1/c-Fos activation would be reduced. As predicted, C. albicans Dals3 at a MOI of 1 only minimally induced MKP1 phosphorylation and c-Fos (Fig. 5d), further supporting the importance of fungal burdens and threshold levels of activation in C. albicans infection.

Recombinant NT-Als3 does not induce epithelial damage, cytokines, MKP1 phosphorylation or c-Fos production
The phenotypes observed for the Dals3 mutant suggested that Als3 might be required for initial epithelial adhesion, but not for direct induction of cell damage, cytokines or activation of the epithelial hypha discriminatory response. To test these ideas further, we stimulated TR146 epithelial monolayers with a purified recombinant portion of the Als3 protein [12]. This protein, called NT-Als3, includes amino acids 18 to 329 of the Als3 molecule and has an adhesive function [10]. We predicted that NT-Als3 would not induce cell damage, cytokine production, MKP1 phosphorylation or c-Fos because (i) Dals3 activated both MKP1 phosphorylation and c-Fos similar to the wild-type control at standard MOIs (Fig. 4) and (ii) damage and cytokine production were restored by increasing the Dals3 fungal burden (Fig. 5). NT-Als3 did not induce cytokine production (Fig. 6a), cell damage ( Fig. 6b), MKP1 phosphorylation or c-Fos activation (Fig. 6c) despite testing multiple concentrations of protein. These data supported the conclusion that the N-terminal adhesive domain of Als3 was not responsible for directly activating immune responses or hyphal discrimination in oral epithelial cells.

Discussion
The goal of this work was to investigate the role of Als proteins in epithelial adhesion and damage, and cytokine production. We also evaluated whether Als3 is the moiety that mediates activation of the MAPK-based MKP1/c-Fos signaling pathway. We found that Als3 makes an important contribution to C. albicans adhesion to TR146 oral epithelial cells, and subsequent epithelial damage, and that loss of Als3 results in reduced capacity of C. albicans to induce epithelial cytokines. The reduction in epithelial damage and cytokine production observed for the Dals3 strain was rescued by increasing the fungal burden in association with the epithelial cells. We found that Dals3 cells are still able to induce MKP1 phosphorylation and c-Fos production, indicating Als3 is not the target on C. albicans hyphae that mediates discrimination between the yeast and hyphal form. We also found that the soluble NT domain of Als3 was not sufficient to induce epithelial damage, cytokine production, MKP1 phosphorylation or c-Fos production.
The ability of Dals strains to adhere to TR146 cells was studied previously in a different context. TR146 cells are the same as those used to produce recombinant human oral epithelium (RHE), marketed by SkinEthic (www.skinethic.com). The interaction between individual Dals strains and oral RHE was described in previous publications [21][22][23][24]. The Dals3 strain was noted to have markedly reduced adhesion relative to the control strain. This assessment was made microscopically by counting C. albicans cells adhered to thick sections of the RHE. Using this analysis, the Dals2/P Mal -ALS2 strain also showed decreased adhesion to TR146, compared with the control. Microscopic inspection of thick sections also showed decreased RHE destruction by the Dals3 strain, and also by the Dals1 strain at 8 h post-inoculation.
Other assays have drawn conclusions about adhesion of Dals mutants to epithelial cells. Using freshly collected buccal epithelial cells (BECs), the Dals3 strain showed significantly reduced adhesion relative to the control. Unexpectedly, the Dals5, Dals6, and Dals7 strains showed nearly a two-fold increase in adhesion to BECs. Phan et al. [33] used a differential fluorescence assay to identify association between C. albicans and epithelial cells from the FaDu pharyngeal carcinoma cell line. They concluded that Als3 is not involved in adhesion to epithelial cells, but is involved in invasion. In the adhesion assays described here, only the Dals3 strain showed a significant decrease in adhesion, compared with control strains. Overall, a variety of approaches has been used to address the role of Als proteins in interactions between C. albicans and epithelial cells, with some variability in the conclusions. Undoubtedly, the differing assay configurations, such as whether yeast cells or germ tubes are inoculated into the assay, assay timing, and the fungal burden used for each study, contribute to these differences. Regardless of the differences, the conclusion that Als3 plays a greater role in epithelial cell interactions than the remainder of the Als proteins, is consistent across the body of evidence, and our results further support this conclusion.
These data raise the question of why Als3 has a greater role in epithelial interactions between C. albicans germ tubes and epithelial cells than the other Als proteins. Among the possible explanations are relative Als protein abundance and spatial distribution on the germ tube, and functional interchangeability between the various Als proteins. Studies of ALS gene expression provided the first clues to the possible relative abundance of Als proteins (reviewed in [7]). Some ALS genes are regulated by large increases and decreases in transcriptional level, while others are transcribed consistently at lower levels. ALS3 has a large increase in transcription when C. albicans yeast cells are placed into growth conditions that promote hypha formation [34]. Analysis using Als3-specific monoclonal antibodies visualized the great abundance and broad distribution of Als3 over the entire surface of C. albicans hyphae [12]. This abundance and distribution promote the greatest opportunity for contact with epithelial cells, potentially resulting in the greatest phenotypic effect on adhesion.
Previously, a potential role for Als1 and Als5 in epithelial adhesion was suggested [35], which seems inconsistent with our results that demonstrate lack of an adhesive change in C. albicans strains from which ALS1 or ALS5 were deleted. However, results in the earlier study were observed for S. cerevisiae strains in which each ALS gene was overexpressed. The overexpression strains have a uniform, thick coat of Als protein, unlike the wild-type abundance and distribution of the Als1 and Als5 on wild-type C. albicans [36,37]. Understanding the differences in protein presence in each experimental approach provides consistent conclusions from seemingly disparate results: Als1 or Als5 can mediate epithelial cell adhesive interactions, given a sufficient abundance on the cell surface. These observations further solidify our conclusion that the greatest phenotypic effect is observed for Als3, because of its abundance and broad coverage of C. albicans hyphae. Experiments to address the functional interchangeability of Als proteins have not been fully explored.
Since adhesion is a prerequisite for invasion and Als3 is also an invasin, the reduced ability of Dals3 to adhere is sufficient to account for its significantly reduced ability to cause subsequent cell damage. Because the Dals3 mutant is able to form normal germ tubes and hyphae, its inability to adhere and invade epithelial cells most likely explains why the Dals3 mutant was also unable to induce cytokines, since cytokine induction requires sustained adhesion and invasion by hyphae [13,17,18]. This conclusion is supported by data showing that induction of epithelial damage and cytokine production could be restored by simply increasing the fungal burden of Dals3 10-fold (from MOI of 0.01 to 0.1). Therefore, our data support the notion that a threshold level first needs to be reached to fully activate epithelial cells and that this depends on how many cells have initially adhered. Notably, the lack of invasion by Dals3 at lower MOIs (0.01) also correlates with the lack of IL-1a production (a cytokine associated with cell damage), which again could be restored by increasing the fungal burden of Dals3 10-fold (to MOI of 0.1).
Recently, we found that epithelial cells are strongly activated by C. albicans hyphae via a MAPK-based mechanism constituting MKP1 phosphorylation and c-Fos activation, contributing to cytokine production and associated with cell damage [16] thus initiating a 'danger response'. It is known that C. albicans entry into epithelial cells via induced endocytosis is mediated via E-cadherin through a mechanism that is actin-dependent and requiring clathrin [38]. Therefore, although the predominant function of Als3 is to mediate epithelial adherence and entry, we hypothesized that once inside the epithelial cells Als3 may trigger the epithelial cell danger response mechanism (specifically MKP1 phosphory-lation and c-Fos production), thus enabling epithelial cells to discriminate between the yeast and hyphal form. However, the Dals3 mutant was able to induce MKP1 phosphorylation and c-Fos equally as well as wild-type/parent cells whilst NT-Als3 was Figure 5. Adhesion, cell damage, cytokine production and activation of MKP1 and c-Fos by C. albicans Dals3 at different MOIs. C. albicans CAI4 yeast cells (200 cfu) and C. albicans Dals3mutant yeast cells (200 cfu -low dose or 300 cfu -high dose) were added to TR146 monolayers and incubated for 90 min to determine the number of colonies and the percentage of adherence (A). C. albicans Dals3 mutant, the parent strain CAI4 and wild-type strain SC5314 were added to TR146 oral epithelial cells under standard culture conditions for 2 h or 24 h at a fungal:epithelial cell MOI ranging between 0.01 and 10. Induction of cell damage assessed by LDH release (B) and the production of cytokines by multiplex microbead assay (luminex) (C) were assayed in the cell culture medium supernatants at 24 h. (D) Induction of MKP1 phosphorylation and c-Fos at 2 h (MOI of 1). Data represent mean values 6 SEM and are representative of duplicate experiments. * p,0.05 compared to CAI4. doi:10.1371/journal.pone.0033362.g005 unable to directly induce MKP1 phosphorylation or c-Fos. These data indicate that Als3 does not activate the yeast-hyphal discriminatory signaling response in oral epithelial cells and that its role is most likely confined to promoting C. albicans adhesion and to mediating initial invasion of C. albicans via induced endocytosis. However, we note that these conclusions are based in part on NT-Als3 (albeit supported by the Dals3 data), so we cannot exclude that other regions of the Als3 protein may be involved in epithelial cell activation through other indirect mechanisms. Additional investigations are underway to identify the C. albicans hypha factor that mediates activation of the MAPK-based MKP1/ c-Fos signaling pathway.