Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms

Abstract

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.

FIG. 1.

Metal ions may promote or inhibit cellular differentiation during biofilm growth of C. albicans 3153A and C. tropicalis 99916. Biofilms were grown in RPMI 1640 plus l-glutamine plus 0.165 M MOPS in microtiter plates for 48 h at 35°C on a gyratory shaker. (A) The untreated C. albicans 3153A biofilms consisted of yeast cells interspersed with many hyphae. (B to J) C. albicans 3153A biofilms were grown in medium containing the indicated metal ion, and with the exception of Pb²⁺, all of the metal ions inhibited hyphal formation. (K) The untreated C. tropicalis 99916 biofilms consisted of yeast cells intertwined with hyphae. In comparison to C. albicans, C. tropicalis produced fewer hyphae in the community. (L to T) C. tropicalis 99916 biofilms were grown in medium containing the indicated metal ion. For C. tropicalis grown in these conditions, CrO₄²⁻ and Zn²⁺ triggered the formation of hyphal cells, whereas the remaining metal ions inhibited the formation of hyphal cells. For the sake of comparison, an inhibitory concentration of Cd²⁺ has been shown for C. albicans and C. tropicalis. These digital photos were captured at ×100 magnification, and the images were contrast and brightness enhanced using Adobe Photoshop 7.0. Arrows indicate the hyphal cells in the biofilm populations.

FIG. 2.

C. albicans 3153A and C. tropicalis 99916 undergo multiple shifts in growth rate and biofilm community structure when cultivated in a concentration gradient of divalent mercury cations (Hg²⁺). Biofilms were grown in RPMI 1640 plus l-glutamine plus 0.165 M MOPS in microtiter plates for 48 h at 35°C on a gyratory shaker. Pictured at the top of each column is a row from a microtiter plate that contained a log₂ concentration gradient of Hg²⁺ (ranging from 20 mM to 0.04 mM). (A and D) Untreated C. albicans 3153A and C. tropicalis 99916 biofilms contained yeast cells interspersed with hyphae. (B and E) C. albicans 3153A or C. tropicalis 99916 biofilms cultivated in as little as 0.04 mM Hg²⁺ consisted of yeast cells only. (C and F) As the concentration of Hg²⁺ increased, C. albicans 3153A and C. tropicalis 99916 gradually abandoned solid-surface attachment, and at ≥10 mM Hg²⁺, these microorganisms formed a pellicle of yeast cells at the air-liquid interface.

FIG. 3.

C. tropicalis 99916 forms biofilm communities with characteristic 3D structure that may be influenced by metal ions. Here, the heavy metal ions Zn²⁺ and CrO₄²⁻ influenced the maturation of C. tropicalis communities at an intermediate stage of biofilm development. In these experiments, C. tropicalis was grown on pegs in the CBD and was then exposed to Zn²⁺ and CrO₄²⁻ for 24 h. The exposed biofilms were stained with the Live/Dead BacLight kit, imaged by CLSM, and then visualized using amira 3.1. The mean and standard deviation of biofilm cell densities were evaluated by viable cell counting (log₁₀ CFU peg⁻¹), and this is indicated where appropriate. (A and B) The 2D average projection and isosurface rendering of a C. tropicalis biofilm grown for 48 h on the CBD. This “domed” biofilm structure type was named for the formation of small microcolonies of yeast cells in surface-adherent communities. (C and F) The 2D average projection and isosurface rendering of an untreated C. tropicalis biofilm grown for a total of 72 h. This structure type was named “layer cake” for the biphasic arrangement of yeast and hyphal cells in the community. (D and G) The 2D average projection and isosurface rendering of a C. tropicalis biofilm exposed to 0.96 mM Zn²⁺ for 24 h. These “flat” biofilms had few hyphae and lacked microcolony structures. (E and H) The 2D average projection and isosurface rendering of a C. tropicalis biofilm exposed to 0.27 mM CrO₄²⁻ for 24 h. “Mycelial” biofilms consisted of masses of hyphal cells attached to the polystyrene surface with few yeast cells remaining in the community. Each panel represents an area of 238 by 238 μm. Green cells are alive, and red cells are dead.

FIG. 4.

Many metal ions (Co²⁺, Cu²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺, AsO₂⁻, and SeO₃²⁻) inhibited hyphal formation by C. tropicalis 99916 at an intermediate stage of biofilm development. In every case, treating biofilms with these compounds resulted in the “flat” biofilm community structure type. Biofilm populations were grown, imaged, and enumerated as described in the legend to Fig. 1. Each panel represents an area of 238 by 238 μm. Green cells are alive, and red cells are dead.

FIG. 5.

Cultivation of C. tropicalis 99916 biofilms in SeO₃²⁻ and CrO₄²⁻ decreases the resistance of the fungal community to amphotericin B and Cu²⁺, respectively. In these experiments, C. tropicalis biofilms were grown on pegs in the CBD for 48 h in TSB and then transferred into fresh medium containing no additive, SeO₃²⁻, or CrO₄²⁻ for an additional 24 h. Biofilms cultivated in this manner were either exposed to antifungals (Cu²⁺ and amphotericin B) or fixed and then stained with Syto-9 and TRITC-ConA (for CLSM and 3D visualization). The mean and standard deviation of biofilm cell densities were evaluated by viable cell counting (log₁₀ CFU peg⁻¹), and this is indicated where appropriate. (A) Reduction of MTS, a tetrazolium salt, by C. tropicalis “layer cake,” “flat,” and “mycelial” biofilms after a 24-h exposure to Cu²⁺. (B) Reduction of MTS by C. tropicalis “layer cake,” “flat,” and “mycelial” biofilms after a 24-h exposure to amphotericin B. (C to E) The 2D average projections of CLSM image stacks for biofilms grown on the CBD pegs. (F to Q) Volume rendering of the Syto-9- and TRITC-ConA-labeled 3D volume data sets extrapolated from the image z-stacks used to create the images of the “layer cake,” “flat,” and “mycelial” biofilms in panels C to E. Each panel represents an area of 238 by 238 μm. Green fluorescence corresponds to cellular biomass, whereas red fluorescence corresponds to extracellular polymers.