Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections

Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

cific nicotinic receptor agonist, SIB1508Y maleate (SIBm), and a nonspecific muscarinic receptor agonist, pilocarpine hydrochloride (PHCl), on C. albicans biofilm formation was investigated to determine whether this was due to activation of a specific subtype of cholinergic receptor.
Biomass quantification assays revealed that SIBm had no effect on C. albicans biofilm formation in vitro (Fig. 1A). In contrast, PHCl caused a dose-dependent decrease in biofilm biomass, with statistically significant reductions observed with concentrations ranging between 0.39 and 50 mM (all P Ͻ 0.001) (Fig. 1B). The XTT metabolic assay revealed that SIBm had no effect on C. albicans metabolic activity (Fig. 1C). However, a slight but significant reduction in C. albicans metabolic activity compared to the untreated control (0 mM) was observed when treated with PHCl concentrations ranging between 3.125 and 50 mM (all P Ͻ 0.01) (Fig. 1D).
Reductions in metabolic activity are associated with reduced biofilm formation but can also be attributed to cell death. A planktonic MFC was performed according to the CLSI M-27A broth microdilution methodology (28) and revealed that none of concentrations of SIBm or PHCl investigated possessed fungicidal activity (data not shown). In addition, PHCl had no destabilizing effects on the C. albicans cell wall as observed by measuring both PI uptake (Fig. 1E) and ATP release (Fig. 1F). Microscopy was also employed to further ensure that PHCl was specifically inhibiting filamentation and not affecting cell viability. Light microscopy (LM) ( Fig. 2A to E) revealed that PHCl inhibited filamentation and biofilm formation in a dose-dependent manner. Furthermore, visually, in the presence of increasing concentrations of PHCl, more C. albicans cells maintained a yeast morphology, suggesting that PHCl was inhibiting the yeast-tohypha transition. Scanning electron microscopy (SEM) analysis ( Fig. 2F to J) further confirmed the fact that PHCl inhibited biofilm formation due to inhibition of the yeast-to-hypha transition in a dose-dependent manner. Fluorescence microscopy (FM) (Fig. 2K to O) with CFW (blue) and PI staining (red) revealed no visible differences in cell viability between any of the concentrations of PHCl investigated and therefore confirmed that PHCl was not toxic to C. albicans at the concentrations used in this study.
To ensure that PHCl was specifically targeting a receptor with homology to human muscarinic receptors, biofilm biomass and metabolic activity assays were repeated in the presence of different concentrations of the nonspecific muscarinic receptor antagonist scopolamine (SCP). SCP inhibits the PHCl-induced reduction in biofilm formation in a dose-dependent manner with concentrations ranging from 64 to 128 m, revealing no significant differences in biofilm biomass in the presence of 25 mM PHCl compared to the control (Fig. 3A). Furthermore, all concentrations of SCP abolished any PHClinduced reductions in metabolic activity (Fig. 3B).
As hydrophobicity is a characteristic related to C. albicans biofilm formation (29), the effect of PHCl on C. albicans cell surface hydrophobicity was investigated using the microbial adhesion to hydrocarbon (MATH) assay. A statistically significant increase in cell surface hydrophobicity of C. albicans cells cultured as a biofilm compared to planktonic cells was observed (P Ͻ 0.05) (Fig. 3C). Interestingly, when C. albicans biofilms were cultured in the presence of 25, 12.5 (both P Ͻ 0.01), and 6.25 (P Ͻ 0.05) mM PHCl, there was a statistically significant decrease in cell surface hydrophobicity (Fig. 3C).
Pilocarpine hydrochloride specifically modulates the pathogenesis of Candida albicans infection in a Galleria mellonella model. Biofilm formation is associated with C. albicans pathogenicity, and ACh has previously been shown to protect G. mellonella larvae from C. albicans-induced mortality (17). Therefore, the effect of PHCl on C. albicans pathogenicity in vivo was investigated using a G. mellonella killing assay. PHCl alone (10.5 mM) had no adverse effects on survival of the larvae (Fig. 4A). Indeed, PHCl protects G. mellonella larvae from C. albicans-induced mortality in a dose-dependent manner compared to larvae inoculated with C. albicans alone observed using 10.5 and 6.25 mM PHCl (P Ͻ 0.001 and P Ͻ 0.05, respectively; as determined using the log rank test) (Fig. 4A). Furthermore, SCP (6.25 mM) alone had no adverse effects on the survival of the larvae. In fact, in the presence of PHCl and SCP, the survival of the larvae was [error bars]) from triplicate wells of six independent experiments (n ϭ 6). (C and D) Biofilm metabolic activity was assessed using the XTT assay after C. albicans was cultured for 24 h in RPMI 1640 containing different concentrations of SIBm (0 to 10 mM) (C) and PHCl (0 to 50 mM) (D). Data are expressed as percent metabolic activity compared to untreated controls (0 mM PHCl), and the bars represent the mean values (ϩSD) from triplicate wells of six independent experiments (n ϭ 6). For panels A to D, values that are significantly different compared to the control values (0 mM SIBm or PHCl) are indicated by asterisks as follows: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. (E and F) The effect of PHCl on the permeability of the C. albicans cell wall was investigated using a propidium iodide (PI) uptake (E) and ATP release assay (F). For the PI uptake assay, data are shown as fluorescence intensity units, and the bars represent the mean values (ϩSD) from triplicate wells of three independent experiments (n ϭ 3). Heat-killed (HK) and chlorhexidine (CHX) (0.2%)-treated C. albicans were included as positive controls, and cells in RPMI 1640 alone were included as a negative control. For the ATP release assays, data are shown as nanomolar concentrations of ATP release, and the bars represent the mean values (ϩSD) from triplicate wells of three independent experiments (n ϭ 3). Chlorhexidine (0.2%)-and fulvic acid (2.0%)-treated C. albicans cells were included as positive controls, and cells in RPMI 1640 alone were included as a negative control. *, P Ͻ 0.05; **, P Ͻ 0.01 compared to the control (cells in RPMI 1640 alone). comparable to those inoculated with C. albicans alone, suggesting that SCP inhibited the PHCl-induced protection against C. albicans infection (Fig. 4B).
To visualize the effects of PHCl alone and in combination with SCP on C. albicans pathogenicity and G. mellonella hemocyte responses to infection, in vivo histological analysis was performed. Control sham-injected larvae after both 24 and 48 h demonstrated the presence of small hemocyte aggregates, adjacent to the gut and the tracheal system, resembling human MALT (mucosa-associated lymphoid tissue) and BALT (bronchus-associated lymphoid tissue). Furthermore, some hemocytes were also detected close to the fat body, near hemocoel cavities and distributed as a monolayer in the subcuticular areas (Fig. 4C, panels i and vi).
Twenty-four hours postinoculation, the hemocyte response in PHCl-injected larvae was characterized by uniformly dispersed small melanized nodules and an increase in circulating hemocytes (Fig. 4Cii). SCP alone resulted in poor immune activation and failed to induce melanization (data not shown). In larvae inoculated with C. albicans alone, the hemocyte response was characterized by melanized nodules of intermediate size, mainly located in the subcuticular area and in the fat body, with nodules found only rarely in paratracheal areas (Fig. 4Ciii). In contrast, the hemocyte response in larvae inoculated with C. albicans plus PHCl was characterized by the presence of single melanized hemocytes or very small aggregates of hemocytes with melanin deposition surrounding yeast cells. These aggregates were uniformly distributed in the hemolymph, close to the fat body and in peritracheal tissues. In addition, no filamentous growth was detected (Fig. 4Civ). In contrast, larvae inoculated with C. albicans SC5314 and treated with PHCl and SCP showed the presence of medium-sized nodules with scanty melanization and poor hemocyte recruitment into invaded tissues. Furthermore, poorly melanized yeast cells and hyphae were seldom detected in the nodules (Fig. 4Cv), similar to larvae infected with C. albicans alone (Fig. 4Ciii).
Forty-eight hours postinoculation, PHCl-injected larvae showed very few and small nodules with faint melanization (Fig. 4Cvii). Larvae infected with C. albicans alone showed increased hemocytes in the subcuticular, intestinal, and paratracheal areas with large nodules and multifocal melanization and heavy damage of the fat body. Hyphal invasion of the intestinal walls, and to a lesser extent, of the bronchial system was also detected (Fig. 4Cviii). In contrast, larvae infected with C. albicans plus PHCl exhibited decreased inflammation and less aggressive fungal infiltration of vital larval tissues, with only small melanized nodules mainly distributed in subcuticular areas. In addition, C. albicans hypha formation was not observed, and there was less microvacuolization of the fat body (Fig. 4Cix). When treated with both PHCl and SCP, C. albicans infection resulted in hyphal invasion of the intestinal walls (Fig. 4Cx), with a histological picture similar to larvae infected with C. albicans alone (Fig. 4Cviii).
Pilocarpine hydrochloride and acetylcholine differentially modulate hemocyte responses to C. albicans both in vitro and in vivo. It has previously been demonstrated that ACh promotes G. mellonella hemocyte function (17). Therefore, the effects of PHCl on hemocyte cellularity, subtypes, and nodule formation during the pathogen- . Candida albicans was cultured in the absence of any compound, in the presence of PHCl alone, and in the presence of SCP alone as controls. #, significantly different from cells cultured in the absence of any compound; *, significantly different from cells cultured in PHCl alone; * or #, P Ͻ 0.05; ** or ##, ϭ P Ͻ 0.01; *** or ###, P Ͻ 0.001. (B) Biofilm metabolic activity was assessed using the XTT assay after C. albicans was cultured for 24 h in RPMI 1640 containing 25 mM PHCl and different concentrations of the nonspecific muscarinic receptor antagonist scopolamine (SCP) (0 to 128 M). Data are expressed as percent metabolic activity compared to untreated controls (0 mM PHCl or SCP), and the bars represent the mean values (ϩSD) from triplicate wells of three independent experiments (n ϭ 3). Candida albicans cultured in the presence of PHCl and SCP alone acted as controls. *, significantly different from cells cultured in SCP alone; ***, P Ͻ 0.001. (C) Cell wall hydrophobicity was assessed using the MATH assay (29). The bars represent the mean values (ϩSD) from duplicate samples of five independent experiments (n ϭ 5). #, significantly different from cells cultured planktonically; *, significantly different from cells cultured as a biofilm in the absence of PHCl. esis of C. albicans infection were assessed and compared to the effects of ACh ( Fig. 5 and Table 1).
An increase in hemocyte cellularity with a predominance of plasmatocytes, granulocytes, and spherulocytes was observed 24 h after exposure to C. albicans alone in vitro. The hemocytes formed small, mainly two-dimensional, melanized nodules. Furthermore, aggregates of yeast cells and hyphae were observed, as well as coagulation fibers similar to neutrophil extracellular traps (NETs) (Fig. 5Aii and Table 1). ACh alone induced an increase in cellularity, with a predominance of plasmatocytes and granulocytes, but no spherulocytes. Cell aggregation was more evident as well as multidimensional nodule formation with no visible melanization ( Fig. 5Aiii and Table 1). Similarly, PHCl induced hemocyte aggregation, leading to multidimensional nodule formation. However, nodules were smaller and consisted mainly of plasmatocytes, a few granulocytes, and no spherulocytes ( Fig. 5Aiv and Table 1). In the presence of C. albicans plus ACh, there was an increase in cellularity with a predominance of plasmatocytes, granulocytes, and spherulocytes. A strong induction of aggregation and multidimensional nodule formation with no melanization was also clearly visible ( Fig. 5Av and Table 1). In contrast, although an increase in cellularity was also evident in C. albicans-plus-PHCl treated hemocytes, granulocyte and spherulocyte numbers were reduced compared with cells treated with C. albicans plus ACh. There was also . ***, P Ͻ 0.001; *, P Ͻ 0.05, as determined by the log rank test in comparison to larvae inoculated with C. albicans alone. PBS alone and PHCl alone-injected larvae were used as controls and had no effect on larvae survival. (B) To verify specificity, C. albicans-infected larvae were also inoculated with PHCl and SCP in combination. The data are derived from three independent experiments with groups of 16 larvae (n ϭ 48). *, P Ͻ 0.05, as determined by the log rank test in comparison to larvae inoculated with C. albicans. Larvae injected with PBS alone and SCP alone were used as controls, and PBS and SCP alone had no effect on larva survival. limited cell aggregation and the formation of small two-dimensional nodules with no melanization (Fig. 5Avi and Table 1).
To determine whether the effects observed in vitro could be differently affected in vivo due to tissue secretion of regulatory molecules, the same analysis was performed on hemocytes isolated from G. mellonella larvae 24 h after infection with C. albicans in the presence and absence of ACh or PHCl. Sham-infected larvae were used as a control ( Fig. 5Bi and Table 1). Comparison with the control cells in vitro ( Fig. 5Ai and Table 1)   revealed a greater number of cells with finely vacuolated cytoplasm, resembling spherulocytes and adipocytes. For all experimental conditions, the in vivo observations substantially overlapped the in vitro observations. However, overall cellularity was slightly reduced in vivo compared to in vitro due to tissue sequestration of hemocytes. With respect to specific treatments, hemocytes from C. albicans-infected larvae revealed a predominance of granulocytes and spherulocytes ( Fig. 5Bii and Table 1) compared to hemocytes stimulated with C. albicans alone in vitro. In addition, the granulocytes were larger and contained large vacuoles within melanized cytoplasm compared with the small two-dimensional melanized nodules observed in the cells in vitro (Fig. 5Aii and Table 1). Hemocytes from larvae inoculated with ACh or PHCl alone and with C. albicans plus PHCl (Fig. 5Biii, iv, and vi, respectively; Table 1) showed similar cellularity and differentiation properties to hemocytes exposed to the same compound in vitro (Fig. 5Aiii, iv, and vi, respectively; Table 1). In contrast, hemocytes isolated from larvae infected with C. albicans plus ACh (Fig. 5Bv and Table 1) showed increased melanization compared to hemocytes cultured in vitro and infected with C. albicans alone (Fig. 5Av and Table 1).

DISCUSSION
Acetylcholine has previously been found to inhibit C. albicans virulence, both in vitro and in vivo, leading to the hypothesis that C. albicans possesses putative cholinergic receptors (17,30). ACh is a general cholinergic receptor agonist with activity against both nicotinic ACh receptors (nAChR) and muscarinic ACh receptors (mAChR). In this study, the general nicotinic agonist SIB1508Y maleate had no effect on C. albicans biofilm formation in vitro. In contrast, PHCl, a nonspecific muscarinic agonist, inhibited filamentation and biofilm formation. PHCl had no fungicidal activity, and the pharmacological specificity of the response was confirmed both in vitro and in vivo using a general muscarinic receptor antagonist, scopolamine. Therefore, C. albicans possesses an uncharacterized cholinergic receptor involved in regulating filamentation similar in phenotype to human muscarinic receptors.
PHCl is used clinically to treat xerostomia and glaucoma (31,32). Pharmacological studies using in vitro and in vivo models indicate that, despite being a general muscarinic agonist, PHCl has a predominance for M3 and M1 (M3ϽM1) muscarinic receptors in mammals (33). Muscarinic receptors are G protein-coupled receptors specialized in responding to ligands involved in cell-cell communication (34). Fungi express a number of G protein-coupled receptors that play vital roles in sensing extracellular signals (34). In C. albicans, G protein-coupled receptors have been shown to regulate filamentation. However, current evidence suggests that G protein-coupled receptors promote filamentation and biofilm formation (34,35). In addition, the general muscarinic receptor antagonist dicyclomine attenuates C. albicans hypha formation by upregulating tup1 expression, the master negative regulator of hypha formation (36). However, in this study, dicycloamine was found to be toxic to C. albicans, and the authors did not discriminate between direct fungicidal activity and specific on target effects of the compound (36).
The data herein suggest that C. albicans possesses a G protein-coupled receptor that can negatively regulate filamentation and biofilm formation. To date, our knowledge of fungal G protein receptors and their roles in regulation of cellular phenotype is not complete, and a number of orphan receptors have still to be functionally characterized (34). However, human studies have shown that different muscarinic receptor subtypes can have opposing functions (37,38). It is therefore feasible that C. albicans possesses more than one G protein-coupled receptor that can differentially modulate filamentation, biofilm formation, and virulence. Further research is required to confirm this hypothesis.
Both M1 and M3 receptors utilize intracellular calcium as a second messenger (39). There is evidence that suggests that unrestricted calcium uptake can inhibit C. albicans mycelial growth, which indicates a critical role for calcium in the regulation of C. albicans morphogenesis (40). Calcineurin is a major player in eukaryotic calcium-dependent signal transduction pathways. In C. albicans, the calcineurin pathway has been shown to be involved in tolerance to antifungal agents, cation homeostasis, and virulence. However, studies of the role of the calcineurin/calmodulin pathway in C. albicans hyphal growth and biofilm formation have revealed contradictory results (41). Calcineurin is a calcium-dependent serine/threonine-specific protein phosphatase, and studies have identified at least 15 novel downstream signaling targets in yeast (42). These downstream targets have been found to play roles in modulating the cell cycle, membrane structure, and cell wall integrity (42). In this study, PHCl was found to decrease C. albicans cell surface hydrophobicity. Cell surface hydrophobicity has been shown to be related to cell wall composition and is also a predictor of biofilm-forming capability in Candida species (43). Cell adhesion to host surfaces is regarded as a major virulence factor for C. albicans (44). Therefore, these findings suggest that cholinergic signaling inhibits C. albicans virulence by additional mechanisms, possibly regulated by intracellular calcium via the calcineurin/calmodulin pathway. However, further research is required to confirm this hypothesis.
C. albicans-triggered diseases represent an intriguing immunological paradigm as they result from a disrupted balance between tolerance and resistance by the immune system (45). The host immune response must eliminate the fungus while limiting collateral damage to tissues and restoring a homeostatic environment. The invasive growth of C. albicans, however, triggers a strong host inflammatory response that can damage infected organs (46). Acetylcholine has been found to promote rapid clearance of C. albicans in a G. mellonella infection model, while at the same time protecting against inflammation-induced tissue damage (17). The data in this article show that PHCl can also modulate host immunity to C. albicans infection with similar outcomes. Indeed, PHCl not only inhibited filamentation in vivo but could also promote rapid and effective clearance of the pathogen while limiting bystander vital tissue damage. Therefore, PHCl can also modulate the pathogenesis of C. albicans infection via muscarinic receptors on G. mellonella immune cell subsets.
Despite similarities between ACh and PHCl in terms of pathogenesis of a C. albicans infection in G. mellonella, distinct differences in the subtypes of hemocytes involved were noted. G. mellonella possesses at least six immune cell subsets, granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes (47). Granulocytes, the very first cells recruited to counteract pathogen invasion, were strongly activated in vivo in the presence of ACh, but not PHCl. Similarly, spherulocytes, whose function is still to be defined, were specifically recruited into tissues in AChinoculated larvae, but not in PHCl-inoculated larvae. As spherulocyte recruitment is known to be dependent on granulocyte recruitment, this finding is perhaps unsurprising. However, a direct inhibitory effect of PHCl on spherulocyte recruitment cannot be ruled out. In addition to hemocyte recruitment, PHCl was found to promote less hemocyte aggregation in vivo than ACh, and the resulting nodules were mainly characterized by the presence of plasmatocytes, which are usually necessary during the later phases of nodulation and encapsulation.
In this study, differences between in vitro-and in vivo-stimulated hemocytes, both in terms of morphology and quantity, were observed. In particular, under all conditions investigated, the in vitro spherulocyte content was reduced compared with the in vivo spherulocyte content, as spherulocytes do not originate from division of circulating hemocytes but are derived from the granulocyte lineage of hematopoietic organs (48). We hypothesize therefore that the biological functions of ACh and PHCl may also be modulated by larval tissue or plasma factors that are missing in cultured cells. Indeed, the fat body of G. mellonella plays a crucial metabolic function, producing antimicrobial peptides and proteins that are involved in dictating immune responses.
The data in this article suggest that cholinergic receptors can modulate G. mellonella hematopoiesis and differentiation. Interestingly, in humans, cholinergic receptors have been shown to regulate hematopoiesis (49). Platelet and megakaryocyte precursors (50), as well as myeloid and erythroid progenitors in the bone marrow (51), all express the ␣7nAChR which is suggested to play a vital role in regulating their differentiation and maturation. Therefore, it is interesting to speculate that similar systems operate in G. mellonella. However, further research using specific cholinergic receptor agonist and antagonists is required to begin to delineate the type of cholinergic receptors that regulate G. mellonella cellular immunity. The fact that ACh, and not PHCl, induced hemocyte recruitment and aggregation suggests that nicotinic receptors rather than muscarinic receptors may play a more important role in immune regulation in this model host.
In conclusion, the present study suggests that different cholinergic receptors may be involved in the promotion of favorable outcome to C. albicans systemic infection. A muscarinic-type receptor seems to modulate C. albicans filamentation and biofilm formation. In addition, the data suggest that hemocyte subsets of G. mellonella possess different repertoires of cholinergic receptors that can modulate their differentiation and function. Therefore, this article provides evidence that targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may be a direct or adjunctive therapeutic strategy to prevent or treat potentially fatal fungal infections.

MATERIALS AND METHODS
Candida albicans yeast culture and biofilm formation. C. albicans SC5314 was subcultured and propagated as described previously (17). C. albicans SC5314 was then standardized to 1 ϫ 10 6 cells/ml in Roswell Park Memorial Institute 1640 medium (RPMI 1640) in the presence or absence of various concentrations of pilocarpine hydrochloride (PHCl) (Tocris, UK) or SIB1508Y maleate (Tocris, UK).
To determine specificity of action, experiments were also performed with PHCl in the presence or absence of various concentrations of scolopamine (SCP) (Tocris, UK). Standardized cells were allowed to form biofilms in flat-bottomed 96-well microtiter plates at 37°C for 24 h. Following incubation, metabolic activity was assessed using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide) (XTT) assay and biofilm biomass determined using the crystal violet assay as described previously (52). Experimental conditions were run in triplicate. Results are presented as mean values from at least four independent experiments.
Propidium iodide uptake and ATP release assays. To evaluate whether any indirect antifungal activity through the disruption of the cell membrane was induced by PHCl, we used a propidium iodide (PI) uptake and ATP release assay, as previously described (29). Briefly, C. albicans SC5314 was standardized to 5 ϫ 10 7 cells/ml in RPMI 1640 and treated with 50 mM PHCl for 60 min. After treatment, the supernatant was harvested, and the cells were washed with PBS and treated with 2 M PI (in PBS). After incubation at 37˚C for 15 min, fluorescence was measured at excitation and emission wavelengths of 485 and 620 nm, respectively. The ATP release assay was performed according to the manufacturer's instructions on supernatants harvested after 60 min using an ATP bioluminescent assay kit (Sigma-Aldrich, UK). Experimental conditions were run in triplicate and repeated on three independent occasions.
Biofilm viability and cell morphology. Standardized C. albicans SC5314 (1 ϫ 10 6 cells/ml) were inoculated in RPMI with or without PHCl on Thermanox coverslips (13 mm) within a 24-well tissue culture plate and then incubated for 24 h at 37°C. For light microscopy, coverslips were washed gently with PBS and stained with crystal violet (0.05% [vol/vol]). For fluorescence microscopy, the coverslips were washed gently with PBS and stained according to the manufacturers' instructions with 5 M calcofluor white (CFW) (Invitrogen, UK) and 20 M propidium iodide (PI) (Sigma, UK). For scanning electron microscopy (SEM), the specimens were prepared as previously described (53). Biofilms were visualized under a fluorescence microscope (Motic BA400 Colorview system) in normal light mode for crystal violet-stained coverslips or at Ex 350 /Em 400 for calcofluor white-stained coverslips and Ex 540 /Em 525 for propidium iodide-stained coverslips. For SEM, biofilms were visualized by using a JEOL JSM-6400 scanning electron microscope. Representative images from 10 fields were taken. Experiments were repeated in duplicate on at least three independent occasions, and representative images are shown.
Microbial adhesion to hydrocarbon assay. The ability of yeast cells to adhere to a hydrocarbon source (Octane; Sigma, Italy) was used to measure cell surface hydrophobicity (CSH), as previously described (43). Briefly, C. albicans SC5314 was cultured in YPD (planktonic cells), RPMI 1640 alone (biofilm), or RPMI 1640 with 6.25, 12.5, and 25 mM PHCl for 24 h. The cells were then standardized to 10 8 cells/ml and mixed with octane for 2 min. The aqueous and organic phases were allowed to separate for 10 min at 30°C, and the optical density of the aqueous phase was determined at OD 600 . The CSH index was calculated as follows: percent CSH ϭ [1 Ϫ (B Ϫ A)] ϫ 100 (where A is the initial absorbance of the aqueous phase, and B is the absorbance of the aqueous phase after partitioning). Samples were run in duplicate. Results are presented as mean values from five independent experiments.
Galleria mellonella killing assay. Pathogenicity of C. albicans SC5314 with or without PHCl was assessed using the G. mellonella killing assay as described previously (17). Sixteen randomly selected sixth-instar G. mellonella larvae (Allevamento Cirà, Como, Italy) with a body weight of between 200 to 300 mg were employed for each experimental group. Overnight YPD cultures of C. albicans SC5314 were standardized in PBS to the desired cell density. Larvae were inoculated into the hemocoel with 5 ϫ 10 5 cells/larva with or without PHCl at different concentrations using a 10-l Hamilton syringe with a 26-gauge needle. In addition, larvae inoculated with PBS and PHCl alone were included for control purposes. A group injected simultaneously with PHCl with or without SCP was further added to verify the specificity of PHCl action. The inoculated larvae were incubated at 37°C, and the number of dead larvae was scored daily. All experiments were repeated on three independent occasions.
Hemocyte characteristics. For the in vitro experiments, the hemolymph of five G. mellonella larvae was collected into ice-cold Grace's medium (Sigma-Aldrich, Italy) by lateral bleeding corresponding to the last right proleg. Hemocytes were counted and plated onto coverslips in a 24-well plate. The plates were incubated for 2 h to allow hemocytes to adhere prior to stimulation with C. albicans SC5314 (10 2 yeast cells/well) for 24 h in the presence and absence of PHCl or ACh. After treatment, hemocytes were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin. Hemocytes incubated in media alone acted as a control.
For the in vivo experiments, G. mellonella larvae were inoculated using the same conditions described for the killing assay. Twenty-four hours after treatment, hemocytes were isolated from the larvae as described above. Hemocytes from three larvae for each experimental condition were then pooled and plated onto coverslips in a 24-well plate. The coverslips were centrifuged for 3 min at 300 ϫ g at room temperature and fixed in 4% paraformaldehyde and stained with hematoxylin and eosin.
In both in vitro and in vivo experiments, hemocyte proliferation (cellularity), subtype differentiation and nodule formation were assessed in comparison to hemocytes incubated in media only (in vitro) or hemocytes from sham-inoculated larvae (in vivo) A single trained and experienced pathologist (M. Falleni) carried out all characterization and quantification.
Larval histology. After hemolymph extraction, the same larvae were processed for histology as previously described (54). Briefly, the larvae were inoculated with buffered formalin and processed by means of transverse cut serial sections. Tissue sections were embedded in paraffin and routinely processed for conventional histopathology. Serial 4-m tissue sections were stained with hematoxylin and eosin (HE) or periodic acid-Schiff stain (PAS). Image acquisition was performed by the NanoZoomer-XR C12000 series (Hamamatsu Photonics). To investigate C. albicans filamentation and the effect on cellular immunity in vivo, two time points were used, 24 and 48 h. Data were confirmed in three independent experiments, and representative images are shown.
Statistical analysis. Graph production, data distribution, and statistical analysis were performed using GraphPad Prism (version 4; La Jolla, CA). Crystal violet and XTT data were found to be abnormally distributed; therefore, all concentrations were compared to the control using a Kruskal-Wallis nonparametric test with a Dunns post-test. The ATP assay data were found to be normally distributed; therefore, a Tukey's multiple-comparison test was used for statistical analysis. The G. mellonella survival curves were analyzed using a log rank test. Statistical significance was achieved if P Ͻ 0.05.