Candida albicans forms biofilms on the vaginal mucosa

Abstract

Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).

Fig. 1.

Establishment of vaginal mucosal biofilm. (a, d) For the in vivo model, mice were administered oestrogen and intravaginally inoculated with C. albicans 3153A (5×10⁴ blastoconidia). At 48 h post-inoculation, vaginae were excised and the tissue was processed for SEM or CM. For CM, vaginae were stained with calcoflour white (blue) to visualize yeast/hyphae, and with ConA-TR to visualize biofilm ECM. (b, e) For the ex vivo model, mice were administered oestrogen and vaginae were harvested at 48 h post-inoculation. Tissues were inoculated with C. albicans 3153A (10⁶ blastoconidia). At 48 h post-inoculation, vaginae were bisected and processed for SEM or CM and confirmation of fungal burden. (c, f) In vivo control tissues were uninoculated. SEM images were taken at 1000× magnification, and CM images at 600× magnification. The figure shows representative images of areas of biofilm growth from three independent repeats; n=4 mice or vaginal explants per time point.

Fig. 2.

Microscopic analysis of vaginal mucosal biofilm formation in vivo and ex vivo. For the in vivo model, mice were administered oestrogen and intravaginally inoculated with C. albicans 3153A (5×10⁴ blastoconidia), and vaginae were harvested at 8, 24, 48 and 72 h. For the ex vivo model, mice were administered oestrogen and vaginae were harvested and inoculated with C. albicans 3153A (10⁶ blastoconidia) and incubated for 8, 24, 48, or 72 h. Tissues were bisected and processed for SEM, CM or fungal burden (Fig. 3). SEM images were taken at 1000× magnification, and CM images at 600× magnification. The figure shows representative images of areas of biofilm growth from three independent repeats, n=4 mice or vaginal explants per time point.

Fig. 3.

In vivo and ex vivo kinetics of vaginal fungal burden. For the in vivo model (a), mice were administered oestrogen and intravaginally inoculated with C. albicans 3153A (5×10⁴ blastoconidia), and vaginae were harvested at 8, 24, 48 and 74 h. For the ex vivo model (b), mice were administered oestrogen and vaginae were harvested and inoculated with C. albicans 3153A (10⁶ blastoconidia) and incubated for 8, 24, 48, or 72 h. Tissues were bisected and processed for quantitative plate counts. The same mice were used for microscopy (Fig. 2) and c.f.u. analysis. Results represent means (±se) of three independent repeats; n=4 mice or vaginal explants per time point.

Fig. 4.

Role of EFG1 and BCR1 during in vivo vaginal biofilm formation. Mice were administered oestrogen and intravaginally inoculated with C. albicans strain DAY185, efg1/efg1, efg1/efg1+pEFG1, bcr1/bcr1 or bcr1/bcr1+pBCR1 (5×10⁶ blastoconidia), and vaginae were harvested at 72 h. Tissues were bisected and processed for (a) SEM or CM, or (b) quantitative plate counts. The figure shows representative images or means (±se) of three independent repeats; n=4 mice or vaginal explants per time point.

Fig. 5.

Role of EFG1 and BCR1 during ex vivo vaginal biofilm formation. Mice were administered oestrogen and vaginae were harvested and inoculated with C. albicans strains DAY185, efg1/efg1, efg1/efg1+pEFG1, bcr1/bcr1, bcr1/bcr1+pBCR1 (10⁶ blastoconidia) and incubated for 48 h. Tissues were bisected and processed for (a) SEM or CM, or (b) quantitative plate counts. The figure shows representative images or means (±se) of three independent repeats; n=4 mice or vaginal explants per time point.