Dissecting the Invasion Routes of C. Albicans Hyphae into Oral Epithelial Cells
In order to assess the relative contributions of the two known invasion mechanisms of C. albicans
, we selectively blocked either induced endocytosis with the microfilament inhibitor cytochalasin D (cytD) or active penetration by killing the fungal cells with thimerosal (Thim). The remaining invasion potential in the presence of cytD is host actin-independent and predominantly driven by fungal activity (active penetration only). When thimerosal was used to kill C. albicans
hyphae, the remaining invasion is solely driven by epithelial cell activity (induced endocytosis only). The effectiveness of this approach was investigated by analyzing the invasion potential of thimerosal-treated fungal cells into cytochalasin D-treated epithelial cells. With this approach, no epithelial invasion occurred within the 3 h time of the experiment, indicating that cytochalasin D fully blocked induced endocytosis within the course of the experiment and that at least one route must be available for fungal invasion into epithelial cells. Note that for determining induced endocytosis, killed C. albicans
hyphae, rather than yeast cells, were used because yeast cells are not endocytosed by epithelial cells 
Electron Microscopy of Induced Endocytosis and Active Penetration
Previous scanning electron microscopy (SEM) studies of viable hyphae invading oral epithelial cells have demonstrated both depressions of epithelial cell surfaces (indicative of active penetration) and membrane ruffling and epithelial cell protrusions (indicative of induced endocytosis) 
. To further dissect the cellular events associated with these two invasion mechanisms, we employed transmission electron microscopy (TEM) of (1) oral epithelial cells co-incubated with thimerosal killed C. albicans
hyphae (induced endocytosis only), (2) cytD treated epithelial cells co-incubated with viable C. albicans
(active penetration only) and (3) untreated epithelial cells co-incubated with viable C. albicans
(both invasion mechanisms). Thimerosal killed hyphae (induced endocytosis only) were engulfed by oral epithelial cells and tightly surrounded by the host membrane (, upper panel). Membrane ruffling was visible around engulfed hyphae (, lower panel). In contrast, oral epithelial cells treated with cytD (active penetration only) did not exhibit filopod formation or membrane ruffling (). Invasion of hyphae occurred not only vertically (tip-first), but also laterally, with few direct physical contacts between hyphae and epithelial surface structures ( upper picture). Furthermore, invading hyphae were not tightly surrounded by host membranes in the presence of cytD. Rather we observed invaginations and broader spaces between penetrating hyphae and host membranes (, lower pictures). Untreated oral epithelial cells invaded by viable hyphae (both invasion routes possible) generally reflected that of active penetration (invaginations and broader spaces between hyphae and host membranes). However, we also observed intermediate examples, with filopod-like structures and membrane ruffling on epithelial surfaces, but also the presence of broader spaces between hyphae and host membrane () similar to pictures shown previously 
. We also tested the effect of complete epithelial inactivation on invasion by killing the epithelial cells with paraformaldehyde. In contrast to invasion of living cells by viable C. albicans
, where we observed defined spaces between the fungal cell wall and the host membrane, invasion of killed epithelial cells was characterized by disrupted cellular structures around the invading hypha, with no evidence of an intact host membrane ().
Transmission electron microscopy of C. albicans epithelial invasion.
Relative Contributions of Active Penetration, Induced Endocytosis and C. Albicans Invasins to Epithelial Invasion
We next sought to quantifiably evaluate the relative contributions of induced endocytosis and active penetration to C. albicans
invasion. We therefore employed our selective inhibition approach and analyzed the invasion potential of C. albicans
wild type and mutants lacking the two known fungal invasins, Als3 and Ssa1 
In agreement with previous studies 
, killed hyphae (induced endocytosis only) were internalized by oral epithelial cells in a time dependent manner (Wt, Thimerosal, ). Occasionally, we observed both viable and killed hyphae which had been entirely internalized (), demonstrating that induced endocytosis can result in complete uptake of C. albicans
. Indeed, a noteworthy proportion of killed hyphae were internalized (2.5 and 12.2% at 2 and 3 h, respectively – Wt, Thimerosal); however, the comparative invasion potential of untreated living fungi was much higher (32.7 and 69.4% at 2 and 3 h, respectively – control, ). As described previously, we did not observe internalization of killed yeast cells; these cells remained on the epithelial surface (
and data not shown).
Contributions of fungal invasins, induced endocytosis and active penetration to C. albicans oral epithelial invasion.
Blocking induced endocytosis via cytD treatment (Wt, cytochalasin D, ) also reduced the invasion potential of C. albicans; however, by 2 and 3 h (24.8 and 50.8%, invasion respectively) the invasion inhibition elicited by blocking induced endocytosis was not as striking as for blocking active penetration. Together, these data demonstrate that C. albicans relies on both induced endocytosis and active penetration routes for optimal invasion during the early stages of interaction with epithelial cells, but that by 3 h, active penetration represents the dominant invasion mechanism.
Following epithelial invasion, viable C. albicans hyphae rarely remained in the primarily invaded cell, but rather continued to penetrate through the basal or lateral membranes and into the next neighbouring cell (, upper panel). Typically, within 3 h, viable C. albicans hyphae penetrated from the primarily invaded cell and through several adjacent epithelial cells or exited the invaded cell and migrated through the extracellular space before invading subsequent cells (not shown).
Induced endocytosis of C. albicans
has been shown to be mediated by Als3- and Ssa1- E-cadherin interactions 
. We therefore sought to quantitatively determine the contribution of both Als3 and Ssa1 to the two invasion routes. Viable cells or killed hyphae of als3
Δ and ssa1
strains (kindly provided by Scott Filler) were used to infect oral epithelial cells and invasion kinetics monitored. At both investigated time-points, invasion of untreated epithelial monolayers by viable als3
Δ was significantly reduced compared to the wild type under the same conditions and invasive potential was restored in the als3Δ
strain. This confirms previous studies that Als3 is important for invasion 
. Deletion of SSA1
also reduced C. albicans
invasion into epithelial cells (24% less invasion than wild type), but this difference was not statistically significant.
At 2 h and 3 h post-infection, the invasion potential of als3Δ cells into untreated and cytD treated monolayers was virtually identical (als3Δ, control vs. als3Δ, cytochalasin D; ). This result, that blocking induced endocytosis in the absence of Als3 had no further effect on fungal invasion, confirms previous findings that Als3 mediates induced endocytosis. However, because the invasion rate of als3Δ into cytD-treated monolayers was significantly lower than for wild type cells (Wt, cytochalasin D vs. als3Δ, cytochalasin D; ), Als3 likely also plays a role in active penetration, possibly via anchoring C. albicans to the epithelial substrate. In contrast, deletion of SSA1 did not result in further reduction in invasion of cytD-treated epithelial monolayers.
Interestingly, under conditions which permit induced endocytosis only (thimerosal-treatment of hyphae – als3Δ, Thimerosal; B), invasion of als3Δ was not completely blocked, although significantly lower than that of thimerosal-treated wild type cells at 3 h post-infection. This remaining, albeit low level, invasion potential of killed als3Δ hyphae provides evidence that C. albicans possesses other factors which mediate induced endocytosis. However, because killed als3Δ hyphae were internalized around 65% less than killed wild type hyphae, we conclude that Als3 is one of the major invasins of C. albicans.
Based on these observations, we conclude that Als3 plays major roles in both induced endocytosis and active penetration.
Since fungal-driven active penetration appeared to represent the dominant route of invasion into oral epithelial cells, we next investigated whether invasion requires viable host cells. Therefore, TR-146 oral epithelial cells were killed by fixing with paraformaldehyde and invasion of C. albicans was quantified by differential staining. Killing of host cells did not prevent invasion. However, after 3 h, we observed approximately 30% reduced invasion into inactivated epithelial cells as compared to viable cells (data not shown), indicating that viability of host cells and native host cell properties are not essential for, but enhance invasion.
The Invasion Efficiency of C. Albicans into Host Cells is Epithelial Cell Type Dependent
Induced endocytosis of non-professional phagocytic host cells occurs predominantly via cell surface associated protein interactions (e.g. Als3-E-cadherin), whereas active penetration relies on a combination of physical forces and directed hyphal growth 
To further examine the relative importance of the two invasion mechanisms and to determine whether invasion is epithelial cell type-dependent, we next investigated the invasion potential of C. albicans
interacting with HeLa epithelial cells as compared to TR–146 oral cells. HeLa cells can be invaded by C. albicans
, but do not express E–cadherin on the cell surface 
. We first confirmed the presence of E–cadherin on the surface of TR-146, but not HeLa cells after infection with C. albicans
via immuno-fluorescent microscopy (data not shown).
We reasoned that if induced endocytosis was exclusively reliant on C. albicans- E–cadherin interactions on epithelial cells, then blocking active penetration should completely block invasion of HeLa cells. Indeed, during the first 2 h, killed hyphae invaded HeLa cells very poorly (Wt+Thim (HeLa), ). However, by 3 h, the number of killed hyphae endocytosed by TR-146 and HeLa cells was comparable. This data suggests the existence of a secondary, E-cadherin-independent endocytic pathway.
On the fungal side, Als3 and Ssa1 are required for E-cadherin-mediated induced endocytosis. Viable als3Δ exhibited significantly reduced HeLa invasion compared to the wild type and invasion was restored by ALS3 complementation (). However, the relative reduction in invasiveness of als3Δ into HeLa cells was not as strong as compared to TR-146 oral epithelial cells (/B and ). Indeed, whilst wild type C. albicans invaded TR-146 more efficiently than HeLa cells, als3Δ cells exhibited a much greater defect in TR146 invasion. This reinforces the concept that Als3 plays a major role in E-cadherin-dependent epithelial invasion, but also functions independently of E-cadherin. Killed als3Δ cells (induced endocytosis only) were again internalized by HeLa cells, although to a lesser degree than the wild type and this defect was reversed in the als3Δ+ALS3 strain. ssa1Δ also exhibited reduced invasion of HeLa cells. However, internalization of killed ssa1Δ cells by HeLa cells was comparable to the wild type.
Together these data suggest that, although Als3-E–cadherin interaction represents a major mechanism of induced endocytosis, other endocytic mechanisms exist, however, the specific mechanisms and receptors remain unknown.
Invasion potential is calculated based on the percentage of invading cells which remain attached to the epithelium following the differential staining procedure. However, the different treatments and genetic backgrounds analyzed may also influence the absolute number of cells which remain attached. We therefore determined the percentage of C. albicans
cells which remained attached to the epithelium. Adhesion rates are summarized in Supplementary Table S1
. Compared to living C. albicans
cells, killed hyphae adhered poorly to both epithelial cell lines. Notably, even viable als3Δ
cells adhered poorly to epithelial monolayers, indicating that the absolute (invasion events per inoculum) invasion defect of this strain is even greater than the “specific” invasion potential of attached cells.
Host Factors Influence Adhesion, Invasion and Damage
Symptomatic stages of superficial C. albicans
infections are characterized by damage and destruction of epithelial layers. Disruption of these barriers may result in increased exposure of extracellular matrix (ECM) molecules and the release of blood and blood components into the mucosal layer. Such extracellular components can be used by pathogenic microorganisms to facilitate infection or colonization of epithelial tissues by bridging between adhesins of the pathogen and epithelial receptors 
To investigate whether C. albicans can utilise ECM proteins during invasion, fungal cells were incubated with fibronectin, vitronectin, collagen, laminin or E–cadherin and both adhesion and induced endocytosis (invasion of thimerosal-killed hyphae) were independently assessed. Pre-incubation of either epithelial cells or C. albicans cells with fibronectin, vitronectin, laminin or collagen did not influence adhesion or induced endocytosis of C. albicans (not shown). On the other hand, pre-incubation of C. albicans with E–cadherin prior to killing significantly reduced fungal uptake by 48% (not shown), indicating that invasin molecule(s) on the surface of hyphae may have been blocked, thus preventing induced endocytosis.
Next, we tested the influence of serum on C. albicans adhesion, invasion and damage. C. albicans was pre- and/or co-incubated with serum during interaction with epithelial cells. Both native or heat-treated (complement-inactivated) human or foetal bovine serum were used. Interestingly, despite having a positive effect on hyphal formation (not shown), treatment with either human or bovine serum strongly reduced adhesion to epithelial cells (). Serum heat-treatment did not further affect adhesion, suggesting that complement, or other heat-labile components were not responsible for the observed serum-mediated inhibition of adhesion.
Adhesion, invasion and damage of oral epithelial cells by C. albicans in the presence of serum.
Invasion of thimerosal inactivated hyphae (induced endocytosis only) was not significantly affected by the presence of FBS (). In contrast, induced endocytosis was dramatically and significantly reduced in the presence of human serum () and this effect was also observed upon pre-treatment of fungal cells. Heat-treatment of human serum did not further affect epithelial invasion, indicating that complement or other heat-labile components, do not influence the invasion process under these conditions. Finally, epithelial cell damage was strongly and significantly reduced when C. albicans was pre- and co-incubated with human, but not bovine serum (). Again, heat-treatment did not influence the extent of epithelial damage. In summary, these data show that both bovine and human serum block C. albicans adhesion to epithelial cells, whilst human serum strongly reduced both epithelial invasion and damage.