Distinct stages of <i>C</i>. <i>albicans</i>-epithelial infection.

<p>(<b>a</b>) In experimental epithelial infections, <i>C</i>. <i>albicans</i> yeasts form hyphae upon contact with epithelia and adhere tightly to the host cells. This is mediated by a number of adhesins, including members of the Als family and Hwp1. (<b>b</b>) This is followed by initial epithelial invasion via two routes—(i) fungal-driven active penetration and (ii) host-mediated induced endocytosis. (<b>c</b>) Elongating and branching hyphae result in extensive interepithelial invasion. Surprisingly, this invasion itself does not cause damage to the epithelium. (<b>d</b>) Simultaneous secretion of the fungal peptide toxin, Candidalysin (red pentagons), lyses the host epithelia and causes tissue destruction.</p

<i>C. albicans</i>-induced epithelial cell signalling.

<p>Oral epithelial cell discrimination of <i>C. albicans</i> yeast from hyphae is enabled via differential MAPK signalling. Recognition of yeast triggers activation of PI3K (green) and NF-κB (blue) as well as weak, transient activation of all three MAPK pathways (red). This MAPK activation leads to a transient activation of the c-Jun transcription factor via JNK/ERK1/2 signalling, with as-yet-unknown transcriptional effects. Activation of the PI3K pathway leads to activation of the epithelial damage protection and/or prevention response. Exposure of epithelial cells to <i>C. albicans</i> hyphae leads to the strong activation of MAPK signalling, resulting in the activation of the c-Fos transcription factor via the p38 pathway. At this point, regulation of MAPK signalling is initiated by the induction and stabilisation of the MAPK phosphatase, MKP1 (via the ERK1/2 pathway), which acts to regulate p38 and JNK signalling. Activation of c-Fos in the presence of NF-κB and PI3K signalling leads to the production of cytokines and inflammatory mediators, thereby activating immune responses to <i>C. albicans</i>.</p

<i>C. albicans</i> recognition and protection at mucosal surfaces.

<p>In health (left panel) <i>C. albicans</i> resides in the commensal state, which is characterised by low fungal burdens. <i>C. albicans</i> is recognised but an activation threshold is not reached; thus, the fungus is tolerated without activating epithelial immune responses. During infection (right panel), <i>C. albicans</i> burdens increase and an activation threshold is reached when a sufficient hyphal biomass is present. Immune recognition of <i>C. albicans</i> hyphae occurs via unknown PRR mechanisms but results in the activation of NF-κB, MAPK, and PI3K signalling pathways. Signalling via p38/c-Fos enables discrimination between yeast and hyphae whilst all three pathways (NF-κB, MAPK, and PI3K) promote immune activation, particularly via p38/c-Fos. Finally, PI3K signalling activates epithelial damage protection/prevention mechanisms. Cytokines and chemokines secreted by epithelial cells in response to <i>C. albicans</i> hypha invasion and damage recruit and activate immune cells. IL-8 recruits neutrophils that are in turn activated by GM-CSF, G-CSF, and IL-1 family members. Neutrophils protect directly through phagocytosis and NET (neutrophil extracellular trap) formation and indirectly via immunological cross-talk with epithelial TLR4. CCL20 and β-defensin 2 secretion recruits mucosal-homing CCR6-expressing dendritic cells, which will process fungal antigens and activate Th immunity, including Th17 cells. TGFβ may also act with IL-1α and IL-6 to induce Th17 differentiation. IL-17 production by Th17 cells increases neutrophil activity and IL-22 production promotes epithelial barrier function. Together, these innate and adaptive immune response mechanisms ultimately clear the fungus or reduce fungal burdens below the activation threshold, thereby re-establishing the commensal phenotype.</p

Induction of c-Jun phosphorylation, c-Fos and cytokine production in A431 vaginal epithelial cells is dependent on <i>C. albicans</i> hypha formation.

<p>(A) <i>C. albicans</i> SC5314 (wild type), CAI4 (parent control), Δ<i>efg1/cph1</i> (non-filamentous) and Δ<i>nrg1</i> (hyperfilamentous) were added to A431 vaginal ECs under standard culture conditions for 30 min and 2 h. Total protein was isolated and induction of c-Jun phosphorylation and c-Fos assessed by Western blotting. Bands are shown alongside an α-actin loading control. (B) Cytokine protein production by A431 ECs 24 h post-infection with all four strains as measured by multiplex microbead assay (luminex). A <i>C. albicans</i>:EC MOI of 10∶1 (A) and 0.01 (B) was used. Data are representative (A) or mean ± SEM (B) of three independent experiments. Statistical analysis (B) of SC5314, CAI-4, Δ<i>efg1/cph1</i> and Δ<i>nrg1</i> infected versus PBS-treated controls was performed using the ANOVA test with Bonferroni post-hoc analysis. *** = p<0.001.</p

Effect of fungal burdens on MKP1, c-Fos and c-Jun activation.

<p>A431 vaginal ECs were infected for 2 h with <i>C. albicans</i> SC5314 at MOI's ranging between 0.01 and 10. Total protein was isolated and phosphorylation of MKP1 and c-Jun and c-Fos induction was assessed by Western blotting. Bands are shown alongside an α-actin loading control. Data are representative of four independent experiments.</p

<i>C. albicans</i> infection of A431 epithelial cells activates NF-κB and MAPK signaling.

<p><i>C. albicans</i> SC5314 was added to A431 vaginal ECs under standard culture conditions for 5, 15, 30, 60 min and 2 h. Total protein was isolated and phosphorylation of (A) IκBα or (B) p38, JNK, ERK1/2 and MKP1 were assessed by Western blotting. Bands are shown with an α-actin loading control. A <i>C. albicans</i>:EC MOI of 10∶1 was used. Data are representative of three independent experiments.</p

Expression of MKP1 and c-Fos in vaginal RHE.

<p>Increase in MKP1 and c-Fos expression in vaginal epithelium is associated with <i>C. albicans</i> SC5314 hypha formation, with minimal activation at the surface by 3 h and gradually increased activation co-localising in the epithelial layers where hyphae penetrate and invade at 6, 12 and 24 h (dark brown staining). Resting levels of MKP1 and c-Fos expression can be seen in areas without <i>C. albicans</i> at each time point (sectional control).</p

Activation of MAPK transcription factors by <i>C. albicans</i> in A431 vaginal epithelial cells.

<p>(A) Resting levels of DNA binding activity (absorbance values) of AP-1 transcription factor members in nuclear extracts from A431 ECs, measured by TransAm ELISA. (B) Changes in DNA binding activity of c-Jun, Elk1, c-Fos, and MEF2 in nuclear extracts of A431 ECs 30 and 180 min post-infection with <i>C. albicans</i> SC5314 by TransAm ELISA. These MAPK-activated transcription factors have previously been identified as showing altered DNA binding activity in oral ECs. Data are represented as fold change relative to resting levels at 0 h. A <i>C. albicans</i>:EC MOI of 10∶1 was used. Data represent mean values ± SEM and are representative of a minimum of three independent experiments. Statistical analysis (B) of raw data for infected versus uninfected cells was performed using the unpaired, two-tailed t-test * p<0.05.</p

Cytokine activation and cell damage induced by <i>C. albicans</i> in A431 vaginal epithelial cells and TR146 oral epithelial cells.

<p>(A) <i>C. albicans</i> SC5314 was added to monolayers of A431 ECs for 24 h and the cell culture medium collected and assessed for cytokine proteins by multiplex microbead assay (luminex) or ELISA (CCL20). (B) LDH (lactate dehydrogenase) release (measure of cell damage) from A431 ECs 24 h post-infection with <i>C. albicans</i> SC5314. (C) Comparison of cytokine protein release by A431 ECs and TR146 ECs 24 h post-infection with <i>C. albicans</i> SC5314. A <i>C. albicans</i>:EC MOI of 0.01 was used for all the experiments. Data are mean values ± SEM of three independent experiments. Statistical analysis of infected versus uninfected epithelial cells (A & B) or oral versus vaginal epithelial cells (C) was performed using the unpaired, two-tailed t-test. * p<0.05, ** p<0.01, *** p<0.001.</p

Fkh2 is differentially phosphorylated between yeast and hyphal growth.

<p>A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Figs. 1B–D</a> αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Fig. 1E</a> is shown with an independent replicate in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.s002" target="_blank">S2 Fig.</a></p