Cell Rupture and Morphogenesis Control of the Dimorphic Yeast Candida albicans by Nanostructured Surfaces.

Nanostructured surfaces control microbial biofilm formation by killing mechanically via surface architecture. However, the interactions between nanostructured surfaces (NSS) and cellular fungi have not been thoroughly investigated and the application of NSS as a means of controlling fungal biofilms is uncertain. Cellular yeast such as Candida albicans are structurally and biologically distinct from prokaryotic microbes and therefore are predicted to react differently to nanostructured surfaces. The dimorphic opportunistic fungal pathogen, C. albicans, is responsible for most cases of invasive candidiasis and is a serious health concern due to the rapid increase of drug resistance strains. In this paper, we show that the nanostructured surfaces from a cicada wing alter C. albicans' viability, biofilm formation, adhesion, and morphogenesis through physical contact. However, the fungal cell response to the NSS suggests that nanoscale mechanical interactions impact C. albicans differently than prokaryotic microbes. This study informs on the use of nanoscale architecture for the control of eukaryotic biofilm formation and illustrates some potential caveats with the application of NSS as an antimicrobial means.

Introduction

Candida albicans (C. albicans) is a dimorphic opportunistic fungal pathogen and a commensal member of the human microbiome.[1−6]C. albicans infections in tissue or on biomedical devices are a serious problem in immunodeficient patients and implant recipients.[7−10] Candida infections result in invasive candidiasis, which often leads to fungal sepsis and 100 000 deaths worldwide per year.[1,11−15] Moreover, mature Candida biofilms are structurally complex and often serve as a reservoir for more serious bacterial infections via biofilm encapsulation.[12,16−21] Candida biofilms often host pathogenic microbes like Methicillin-resistant Staphylococcus aureus and as such are a major source of secondary bacterial infections.[22−26] Candida biofilm controls are diverse and have involved a wide range of material interventions including polymeric composites that contain metal and metal oxide nanomaterials, biopolymer thin films, and even structural low aspect ratio colloidal monolayers;[27−32] however, little work has been done on nanoscale two-dimensional (2D) materials like the high aspect ratio nanostructured surfaces (NSS) derived from insect wings.

The structure and composition of the Candida biofilm depend on the properties of the contact surface, environmental factors, fungal cell morphology, and the fungal species.[12,28,33−35] Antimicrobial NSS have been proposed as a stand-alone treatment or as a potential additive treatment with existing antimicrobial agents.[36−41] NSS were initially identified as structurally defining components of insect wings, but more recently these have been fabricated in materials like polymers or metal oxides.[42−49] NSS inhibit microbial growth and viability via mechanical interrogation of a microbial cell.[38,50−54] Physical parameters like cell–substrate adhesion and cell wall rigidity appear to play a role in NSS-induced cell rupture.[55−58] Biophysical models suggest that the bactericidal mechanism is a result of physical incompatibility between the cell membrane and the surface.[41,59−65]

In this manuscript, we characterized the interaction of the cellular fungi C. albicans with NSS found on the wing of the dog day cicada, Neotibicen tibicen. We define critical timing events associated with specific physiological and morphological responses of the C. albicans cell to the NSS. C. albicans reacts to the NSS differently than other microbes exhibiting a slower rupture rate and evidence of mechanically induced cell wall stress. We also have identified a novel example of thigmotropism, a mechanosensory response of a cell or organism to a surface, regarding hyphal morphogenesis of C. albicans, which may provide a novel means of controlling C. albicans pathogenesis.

Results and Discussion

Surface Characterization

We examined C. albicans/surface interactions on a NSS derived from the wings of the dog day cicada (N. tibicen); this NSS contains an ordered array of nanoscale cones (Figure ), 200 nm tall, 200 nm wide with 30 nm tips, and spaced 200 nm apart.[38] We used a flat glass coverslip for the controls. To control for surface composition effects, all experiments used uncoated surfaces and surfaces glow-coated with a 7 nm layer of gold. We observed a slight increase in hydrophobicity with the deposition of 7 nm gold later to the flat glass and a light reduction in contact of the Au-coated NSS when compared to the native NSS (Table ). However, all of the surfaces used in the experiments displayed a similar range of contact angles/surface energy.

Figure 1

SEM micrographs of surfaces used in these experiments. (A) SEM of the flat, glass coverslip that was used as a control. (B) Image of the glass coverslip. (C) SEM micrograph of the NSS from the dog day cicada wing, N. tibicen. (D) Graphical representation of the NSS.

Table 1

Contact Angle for Control and NSS Used in This Study

surface	native/uncoated	gold coated
glass coverslip	79.8 ± 01°	93.1 ± 0.04°
NSS/cicada wing	114.6 ± 0.1°	87.0 ± 0.1°

C. albicans Adhesion to NSS

The formation of a stable adhesion between a microorganism and a surface is the first step of biofilm formation.[66−70] To determine the role that NSS roughness had on C. albicans cell–surface adhesion, we cultured both cellular and hyphal forms of C. albicans on flat and NSS and counted the number of cells bound to the surface. On control surfaces, the number of C. albicans cells/FOV increased as a function of the time of incubation on flat surfaces, both Au coated and uncoated (Figures and S1), and during the first 8 h, the number of cells/FOV on the NSS mirrored those on control surfaces (Figures and S1). After 16 h the rate of cell deposition on NSS slowed relative to control surfaces, but eventually reached the same surface density after 24 h.[71,72]

Figure 2

Surface–cell adhesion of C. albicans with control and NSS. (A) Confocal micrographs of CFW labeled over time. First column shows C. albicans cells cultured on flat control surfaces; the second column shows C. albicans cells cultured on NSS. (B) Average number of cells per field of view with time for each condition.

C. albicans Cell Wall Composition Response to NSS Interaction

The polysaccharide chitin provides mechanical stability to the yeast cell wall, and its expression level is controlled by mechanical stress via the cell wall integrity signaling pathway.[73−75] During the first 4 h of contact with an NSS We observe a significant increase in the fluorescence of chitin binding dye Calcofluor white (CFW) in C. albicans cells (Figures and S2). In contrast, C. albicans cells cultured on control surfaces show no change in levels of CFW fluorescence over the time course (Figures and S2). However, after 4 h, the CFW fluorescent levels of C. albicanson NSS dropped to the level observed on control surfaces. The transient chitin increase/stress response suggests that either the yeast cells recover from NSS mechanical stress or the NSS stops presenting a mechanical challenge to the C. albicans cell. NSS activity is complex; some model NSS as a passive mechanical device, in which microbes impale themselves onto the nanostructures, while other models evoke a more active, “nano-snare” device in which cellular interactions with the surface trigger lateral mechanical reactions.[41,76−79] In either case, it is unclear how a cell would compensate for either activity. However, microbial activity such as the deposition of extracellular matrix and dead cells has been shown to alter surface properties such as super hydrophobicity,[80] and it is possible that C. albicans biofilm foul the NSS during culture.

Figure 3

NSS induces an increase in cell wall chitin. The CFW intensity in the C. albicans cell walls is represented as a grayscale value (y-axis) and is examined over six time points (x-axis) on gold-coated flat and NSS substrates.

C. albicans Viability and Rupture on NSS

NSS with a high aspect ratio nanoscale architecture rupture Gram-negative bacteria within moments after contact.[53,54,79,81,82] We examined changes to C. albicans cell morphology on control and NSS materials using scanning electron microscopy. In these experiments, we observed ruptured C. albicans cells on the NSS after 8 h of incubation on the NSS. These ruptured cells resembled the ruptured Saccharomyces cerevisiae yeast cells from previous work and appeared as deflated cells and the deposition of cell wall remnants and other materials (Figure , second row, second and third columns, arrows).[38]

Figure 4

Scanning electron micrographs of the interaction of C. albicans with flat control surfaces and NSS. First column C. albicans cells cultured on a flat surface: top row, 0 h, an ovoid/intact cell; middle row at 8 h of contact, a cluster of ovoid/intact cells; bottom row 16 h of contact, a large cluster of intact cells. Second column, top row, two ovoid intact cells, the arrow denotes a common feature of NSS interactions with yeast cells, a trough of crushed nanoscale cones; the middle row 8 h of contact, a deflated yeast cell, arrow; and the bottom row at 16 h of contact, a field of ruptured cells and cellular debris that is identified by altered contrast (arrows).

To complement these experiments, we labeled C. albicans cells with the vital dye FUN-1 and monitored FUN-1 fluorescence on control and experimental surfaces with confocal microscopy. FUN-1 is a ratio metric dye that is taken up and processed by living, metabolically active fungal cells; upon cellular uptake, the FUN-1 dye fluoresces red and in metabolically active cells the FUN-1 dye will be enzymatically processed to a product with green fluorescence.[83] We observed a reduction in viability in C. albicans cells that have been exposed to NSS, as indicated by loss/reduction of green FUN-1 fluorescence (Figures A, arrows, and S3). The maximal loss of green FUN-1 fluorescence in cells that have been exposed to the NSS for 8 h and increased as incubation time increased (Figure B).

Figure 5

Cell viability of C. albicans stained with fluorescent dye FUN-1. Merged images showing cells labeled with CFW (blue) and cells labeled with FUN-1. Cells labeling with green fluorescence live cells and those only containing red cells (arrows) are the dead or dying cells. (A) Representative confocal micrographs of cells on either Flat control surface (first column) or NSS (second column). (B) Graphical summary of the percentage of dead/dying cells on each surface over the time recorded. **p values < 0.001.

To confirm these results, we measured lactate dehydrogenase (LDH) release and metabolic activity in C. albicans cells cultured on control and NSS. Release of lactate dehydrogenase (LDH) is indicative of cell rupture.[84]C. albicans cells exposed to NSS exhibited increased LDH release relative to controls (Figure A). We also observed a reduction of total cellular metabolic activity of C. albicans cells exposed to NSS, when compared to the control surfaces using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Figure B). The timing of C. albicans loss of viability/rupture was preceded by NSS-induced increase in cell/wall chitin, suggesting that NSS rupture may result from an early NSS-induced cell wall remodeling response.

Figure 6

LDH and MTT assays of C. albicans exposed to control and NSS. (A) Normalized LDH release by C. albicans on NSS; (B) MTT assay of C. albicans on flat/control surfaces (gray columns), NSS (white columns), and a negative control treatment with antifungal drug voriconazole (black columns); y-axis shows absorbance at 570 nm and x-axis shows time.

When compared to the rates that have been demonstrated for bacterial cell rupture via NSS, C. albicans ruptures on NSS was slower, measured in hours versus minutes, and to a lesser extent than bacterial cells.[85] Cellular fungi such as C. albicans are larger (i.e., 5 μm versus 1–2 μm), have a different shape (i.e., ovoid versus cylindrical), and are structurally different from bacterial cells with cell walls that are an order of magnitude stronger than most bacterial cell walls.[86,87] The lower amount of NSS-induced rupture of C. albicans cells when compared to what has been described in the literature for bacteria may reflect these structural/physical differences.

Surface Structure and Biofilm Formation

Many microbes including cellular yeast respond to contact with surfaces through the deposition of extracellular matrix.[34,88,89] We examined C. albicans biofilm production in response to control and NSS; we examined biofilm production using a crystal violet assay.[90] In control experiments, we observed an increase in biofilm deposition as the culture ages, and after 24 h, we observed a thick and mature biofilm (Figure A); however, C. albicans cells cultured on NSS showed a significantly reduced biofilm when compared to the control surfaces (Figure B).

Figure 7

Biofilm formation of C. albicans on control and NSS. (A) Scanning electron micrograph of biofilm formation of C. albicans on a flat surface. (B) Crystal violet biofilm assay of C. albicans cultured on NSS and other surfaces. Quantification of crystal violet at 550 nm absorbance collected in a microtiter plate at different incubation time periods.

NSS Interactions with C. albicans Hyphae

C. albicans is a polymorphic fungus with hyphal morph that is associated with pathogenesis and virulence.[11,17] Hyphal C. albicans exhibited no alteration in adhesion, viability, or loss of cell integrity on NSS when compared to control surfaces, which suggests that this morphological form of C. albicans responded differently to the NSS. However, NSS contact inhibits C. albicans ability to form a hyphal morphology. In control experiments, we observed 80 ± 0.81% (n = 30) hyphal differentiation of the C. albicans microcolonies when cultured with a hyphal-induction medium. However, microcolonies of C. albicans cultured with this same medium on NSS never resulted in hyphal formation (Figure B, second column). Other materials and conditions have been shown to inhibit C. albicans hyphal formation including natural and synthetic peptides, plant-derived compounds, polymeric/oligomeric materials, and metal oxide-based nanomaterials.[91−94] In some of these cases, a reduction of biofilm was associated with the inhibition of hyphal formation. Many of these conditions or treatments appear to interfere with the chemical signals and quorum sensing pathways. The NSS results may be the first example of a purely structural inhibition of hyphal morphogenesis.

Figure 8

NSS controls C. albicans morphogenesis. (A) Schematic of the microcolony assay; (B) first column, C. albicans cultured on flat control surfaces in differentiation media. Top image, confocal micrograph showing hyphal formation (arrow); bottom image, SEM showing hyphal growth; second column, C. albicans cultured in differentiation medium on NSS. (Top) A representative confocal micrograph shows cellular forms of C. albicans (arrow); bottom image, an SEM showing a cluster of C. albicans cells, note the lack of any hyphae.

Contact-dependent/mechanical response or thigmotropism has been observed in plant root tips and fungal hyphal growth.[95−100] Perturbation of these structures results in a stretching of the cell wall and/or plasma membrane, thereby activating an intercellular signaling cascade.[95] While microscale structures have been shown to redirect the growth of fungal hyphae, this is among the first nanoscale thigmotropic response that controls a genetic process, i.e., fungal cell morphology switch. Many different environmental conditions result in the switch from a cellular of C. albicans to a hyphal morphology including nutrient levels, pH, temperature, and cell density[92,97,98] and is inhibited by the quorum sensing through the activation of the cAMP-PKA pathway.[92,101,102] It is unclear how an NSS, an external mechanical challenge, inhibits hyphal morphogenesis; however, in some fungal species such as the fission yeast Schizosaccharomyces pombe there is some cross-talk between the cell wall integrity and cAMP-PKA pathways,[103] which suggests that mechanical cues may impinge pathways needed for hyphal morphogenesis. Future work using genetic knockouts of components of these pathways will be necessary to determine how interaction with NSS controls intracellular signaling pathways to inhibit hyphal morphogenesis.

Conclusions

The application of NSS for the control of bacterial biofilms has made them an attractive alternative to or complement to traditional antibiotics. The rupture of bacterial cells on native and synthetic NSS is well documented and has been ascribed to physical incompatibility between the bacterial cell and the surface that depends on cell wall mechanics and NSS structure. However, a role of NSS for controlling pathogenic fungal biofilms is not as clear. As an antifungal surface, NSS lags significantly in both the timing and the amount of C. albicans rupture when compared to NSS bactericidal effects. In medically relevant biofilms where fungal and bacterial microbes coexist, this may lead to preferential selection for fungal growth. The interaction between the cellular yeast cell and NSS may be a more complex than that between bacteria and the NSS, perhaps involving other surface properties and/or evoking different biological responses. Understanding and controlling these differences will be critical for NSS controls of microbial biofilm formation in medically relevant situations.

Experimental Section

Yeast Strains and Cultures

A wild type strain of C. albicans (ATCC 90028) was grown in Sabouraud dextrose broth (SDB) at 25 °C in 50 mL conical flasks. Depending on the experimental needs, cultures were grown to the mid-log phase at an optical density at 600 nm (OD600) ∼0.6. OD600 measurements were made using a Thermo Scientific Nanodrop 2000C spectrophotometer. To induce hyphal growth, C. albicans was cultured in a modified SDB media containing 10% bovine serum albumin (81066, Sigma-Aldrich) in 50 mL conical flasks incubated overnight in a shaking incubator at 37 °C and 200 rpm. Hyphal growth was indicated by the formation of granular sediments in the growth media.

Surface Preparation and Characterization

We investigated the interaction of C. albicans cells with flat control coverslips and compared to NSS from the wing of the cicada, N. tibicen (BioQuip Products, Inc., CA). Wings from whole cicadas were dissected from the organism. Isolated wings were sonicated in 70% ethanol for 10 min to remove any contaminants, air-dried at room temperature, and carefully cut into smaller pieces of size 5 mm × 5 mm. Glass coverslips were cleaned using 70% ethanol. The cleaned sections of the cicada wings were mounted on cleaned coverslips using silicone glue (Silicone VC6-1/2). All of the surfaces (wings and coverslips) used in the experiments were glow-coated with a 7 nm layer of gold using a Leica EM ACE200; this was an additional control for differences in surface composition. The surface energy was measured via contact angle goniometry using a Rame-Hart 260-F4 goniometer and DROPIMAGE advanced software. A volume of 3 μL of deionized water was used on all surfaces, and the averages of four repetitions on similar surfaces were calculated.

Culturing Conditions for Imaging C. albicans Response to NSS

To examine the interactions of C. albicans cells with a different surface, the surfaces with cells were placed in a poly(ethylene glycol) (PEG) 8000 treated (Sigma, Cat # 1546605) well of a 24-well plate.; PEG treatment prevented nonspecific interactions of yeast cells with nonexperimental surfaces. Cells were imaged using a Zeiss Z1 Spinning disk confocal with a setting appropriate for each dye set at six time points: 0, 2, 4, 8, 16, and 24 h.

Determining Cell Viability

The vital dye, FUN-1 (ex ∼470/560–610 em1, em2 510–560), measures cell viability. Cells were labeled with a 5 mM solution of FUN-1 stain (Molecular Probes F-7030). FUN-1-labeled C. albicans cells were imaged using a Zeiss Z1 spinning disk confocal with excitation with the 488 laser and imaging with simultaneously with the 515 nm filter and 561 nm filters settings and the 100× objective. A CyQUANT MTT assay kit (Life Technologies, Catalog#: V13154) was used to measure viabilty and a CyQUANT LDH Cytotoxicity 2001 assay kit (Life Technologies, Catalog#: C20300) to measure LDH. For these experiments, C. albicans yeast cultures were grown to an OD600 of 0.6/∼1 × 106 cells/mL. Negative controls for these experiments were replicated C. albicans grown parallelly and treated with an antifungal drug (7 μg/mL) voriconazole.

Densitometric Analysis of C. albicans Cell Wall Chitin

The fluorescent dye Calcofluor white (CFW; ex, 370 nm/em 440 nm) binds chitin. C. albicans cells labeled with 1 μg/mL solution of calcofluor white (CFW) (Sigma-Aldrich, 18909). The intensity values were captured using Zen Blue Software from 100 images per sample/condition per time point. All data were statistically analyzed using the ANOVA: single-factor analysis, and statistical significance is denoted by (*) for p ≤ 0.05 and (**) for p ≤ 0.001.

Biofilm Quantification

Biofilms formed on different nanostructured surfaces were quantified by a crystal violet microtiter dish biofilm formation assay as previously described.[90] The absorbance intensity of the crystal violet dye is measured using a BioTek Synergy Mx plate reader at 550 nm.

Scanning Electron Microscopy (SEM)

C. albicans cells samples were prepared on both the flat surfaces and NSS; cells were fixed overnight with 2.5% glutaraldehyde/2% formaldehyde solution in 0.1 M cacodylate buffer. The samples were dehydrated by an ethanol series (e.g., 30, 50, 70, 95, and 100%) with 10 min washes/exchanges at each concentration. The samples were air-dried, mounted onto aluminum SEM stubs, and coated with a 5 nm gold layer using Leica EM ACE200. The samples were then observed using a Zeiss Auriga scanning electron microscope.

Microcolony Assay

To determine the effect of NSS on the morphogenesis of C. albicans from its cellular to its hyphal morphology, we performed a microcolony assay; 100 μL of C. albicans yeast was cultured in a modified SBD medium that contained 10% bovine serum albumin and was placed on control and experimental surfaces and incubated at 37 °C overnight. C. albicans cellular forms that had been cultured in SBD media and C. albicans hyphal forms that had been induced on glass coverslips served as negative and positive controls. To evaluate the microcolony assay, microcolony C. albicans cultures on control and NSS were washed twice with PBS, labeled with CFW, and imaged using a confocal microscope as described above.