Dispersion as an important step in the Candida albicans biofilm developmental cycle

Abstract

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of "virulence factors" important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

Figure 1. Quantification of numbers of dispersed cells from C. albicans biofilms grown in three different media.

C. albicans biofilms were developed in RPMI, YNB and YPD media, and the number of dispersed cells recovered from the biofilms was counted over time. Results shown are expressed as mean and standard deviation from three independent experiments for each condition.

Figure 2. Effect of carbon source on C. albicans biofilm dispersion.

Biofilms were developed for 24 h in YNB medium with 50 mM glucose. The media was then changed to YNB containing varying concentrations of glucose or alternative carbon sources (maltose and galactose). The impact of varying carbon sources and/or concentrations on the level of dispersion was quantified at various time points. Results shown are expressed as mean and standard deviation from two independent experiments for each condition tested.

Figure 3. Evaluation of the adhesive, biofilm development and invasive properties of dispersed cells.

The adhesive and biofilm-forming properties of planktonic C. albicans yeast cells grown at 37°C, and biofilm dispersed cells were compared in microtiter plate-based adhesion (A) and biofilm development (B) assays using XTT-reduction. Measurement of LDH released from endothelial cells damaged by C. albicans grown under planktonic conditions and by biofilm dispersed cells were expressed as percent cytotoxicity (C).

Figure 4. Dispersed cells display increased virulence in vivo.

Groups of mice were injected with the same dose (2.8×10⁵ CFU) of either dispersed cells from biofilms (black squares) or cells obtained from matched planktonic cultures (open circles), and their survival was monitored over the course of infection in this murine model of hematogenously disseminated canididiasis. Statistically significant differences were observed between the corresponding survival curves generated for the two groups of mice (P<0.05).

Figure 5. Regulation of C. albicans biofilm dispersion by UME6.

Biofilms developed by the C. albicans tetO-UME6 strain in the presence (A) and absence (B) of DOX were imaged by SEM. Number of dispersed cells released from biofilms formed by the tetO-UME6 strain in the presence and absence of DOX were enumerated (C) and so was the impact of media switch from + DOX to - DOX and vice versa, on biofilm dispersion (D).

Figure 6. Regulation of C. albicans biofilm dispersion by PES1.

Biofilms (formed by the C. albicans PES1/PES1 and tetO-PES1 strains) were developed for 17 hours in YNB with DOX, and the number of dispersed cells counted. DOX was withdrawn from the media and number of dispersed cells counted at various time points after antibiotic removal (A). Biofilms formed by the C. albicans PES1/PES1 and tetO-PES1 strains were developed in the presence of DOX (B and C respectively). Impact of change DOX withdrawal on the tetO-PES1 biofilm is shown in panel D. Panel E shows the morphology of the cells in a crack in the tetO-PES1 biofilm developed after removal of the antibiotic from the medium. Since PES1/PES1 showed similar biofilm phenotype before and after media change, only one representative image for both conditions is shown. Scale bar for SEM corresponds to 10 µm for all four images.