Fig 1.
C. glabrata and C. albicans dual species biofilms showed increased biofilm mass that reflects C. glabrata adherence to C. albicans hyphae.
(A) C. albicans CAI4 and C. glabrata strains CgBG2 and CgVSY55 were grown as biofilms for 24h in plastic wells. Both CgBG2 and CgVSY562 strains showed significantly (*P<0.02, ***P<0.001 by student’s t test) increased biomass in a dual species biofilm (black bars) as compared to single species biofilm (white and grey bars). Fluorescent quantitation of co-culture of C. albicans CAF2-ycherry with C. glabrata CgVSY55 showed enhanced growth of both species compared with single species (Fig 1B). When both species were co-cultured under dynamic flow conditions, C. glabrata (green) was able to bind C. albicans (red) biofilms and hyphae under conditions of flow (Fig 1C). Fluorescent images were merged with DIC images to enhance hyphal visibility. Hyphal binding is indicated by arrows. Scale bars represent 50 μm.
Fig 2.
C. glabrata adheres to C. albicans hyphae.
C. albicans germinated or yeast cells were incubated with C. glabrata cells at 1:1 ratio for 60 min. While little adherence of C. glabrata was found with C. albicans yeast, large numbers of C. glabrata CgVSY55 or CgBG2 were found adherent along the length of C. albicans hyphae using fluorescence microscopy (A left panel). Adhesion of along the length of C. albicans hyphae was also found in scanning electron micrograph images of CgVSY55 and CAF2-yCherry (A right panel). Magnifications are 500X (scale bar: 5μm, 20μm). C. glabrata cells adhesion to C. albicans hyphae (10μm length) was quantitated microscopically. Seven wild type strains of C. glabrata were screened for adherence to C. albicans hyphae and grouped as high (CgDSY562), medium (CgBG2, Cg60032, Cg931010) and low (Cg932474, Cg148042, Cg90030) binders. (B).
Fig 3.
C. albicans Als3 and Als1 hyphal cell wall adhesins are involved in C. glabrata adherence to C. albicans hyphae.
(A) C. glabrata (CgVSY55) adherence to C. albicans Als1, Als3 and Als1/3 deficient strains was measured using fluorescent microscopy. C. glabrata showed a significant (**P<0.001,***P<0.0006) decrease in adherence to C. albicans hyphae of als3Δ/Δ, als1Δ/Δ, and als1/als3Δ/Δ mutants; while C. albicans Als1 and Als3 complementation strains had restored adherence. (B) S. cerevisiae strains expressing C. albicans Als1 and Als3 adhesins and an empty vector (control) were incubated with CgVSY55 and their adherence was quantifed by direct visualization. Both Als1 and Als3 expressing S. cerevisiae strains had a significant (***P<0.001) increase in Binding Index compared to control empty vector. Differences between groups were analyzed by a student’s t test.
Fig 4.
C. albicans enhances C. glabrata colonization in murine oropharyngeal candidiasis model.
(A) Colony Forming Units (CFU) per gram of tongue tissues were recovered from infections with C. albicans alone, C. glabrata alone or C. albicans and C. glabrata co-infected mice after 5 days. C. glabrata colonization increased by one log-fold upon co-infection with C. albicans. (B) Pre-establishing C. albicans infection (24 h or 48 h before C. glabrata infection) further increased subsequent C. glabrata colonization by two log-fold in mice tongue tissues. Differences between groups were analyzed by a student’s t test (**P<0.003, **P<0.001, ***P<0.0001). (C) C. glabrata infection alone did not result in mice weight loss, however the rate of weight loss was accelerated in the mixed infection (red arrow indicates initiation of C. glabrata infection). Animal weights (mean and SD of each group, n = 7) are shown for each group (black, C. glabrata infection only; blue C. albicans infection only; red, C. albicans infection for 48 h followed by C. glabrata infection).
Fig 5.
C. albicans and C. glabrata are co-localized and both invade murine tongue tissues in a mixed infection.
(A, B and C) PAS stained formalin-fixed and paraffin embedded sections (5 μm) from tongues of mixed C. albicans and C. glabrata infection at day 5 showed widespread fungal plaques and hyphal invasion (yeast are stained magenta) of superficial epithelium (box) and underlying epithelium (arrows). Dark blue cells are neutrophils, lighter blue cells are tongue epithelia. Magnification is 10x. (B) 40x and (C) 100x magnification show C. albicans hyphae with associated yeast cells within the mucosa. (D, E and F) Immunofluorescent and DIC merged images of C. albicans (red) and C. glabrata (green) from tongues at day 5 post-infection. (D) Arrowheads show hyphae penetration into the epithelium. (E and F) Arrowheads at left indicate C. glabrata in contact with hyphae, while other C. glabrata are within epithelium unassociated with C. albicans hyphae (E, lower arrow). Scale bars represent, in order (A-F), 50, 10, 5, 50, 10, and 10 μm.
Fig 6.
C. glabrata requires C. albicans for early infection.
Mixed species oral infection with fluconazole resistant (CaFluR, CgFluR) and fluconazole sensitive (CaFluS) strains showed a significant reduction in number C. glabrata fluconazole resistant CgFluR cells (solid circles) following fluconazole treatment (despite being fluconazole resistant) that was proportional with reduction in fluconazole sensitive C. albicans CaFluS (open circles) CFUs (right). Tongue tissues were stained with Periodic acid-Schiff stain and viewed at 10X magnification (left). CaFluS and CgFluR infected mice showed normal tongue histology with a reduced fungal burden following fluconazole treatment, while CaFluR and CgFluR (yeast cells are shown in pink) infected mice showed typical hyphal invasion of the superficial epithelium with high fungal burden.
Fig 7.
C. glabrata colonization requires C. albicans Als1 and Als3 in murine oropharyngeal candidiasis.
Infection levels of Als1 and Als3 deficient strains of C. albicans did not differ significantly from WT; however, C. glabrata infection levels were decreased significantly in mice upon co-infection with Ca als1Δ/Δ, als3Δ/Δ mutants compared to WT. Mixed infection of C. glabrata with C. albicans Als1 and Als3 complementation strains showed restoration of C. glabrata colonization. Differences between groups were analyzed by a student’s t test (*P<0.02, **P<0.001).
Fig 8.
C. glabrata cell wall adhesins are induced in the presence of C. albicans.
(A) Adherence of S. cerevisiae strains expressing C. glabrata cell wall adhesins to C. albicans hyphae was measured. Among 44 strains tested, only five strains were adherent to C. albicans hyphae, and adhesion levels were about half that of CgDSY562 (Cg) (B) Mixed species infection with CgDSY562 (High adherence), CgBG2 (Medium adherence) and Cg90030 (Low adherence) and C. albicans CAI4 showed significant reduction in infection levels (*P<0.01 student’s t test) between the High (CgDSY562), and Low (Cg90030) adherence C. glabrata strains. (C) Expression levels of C. glabrata EPA8, EPA19, AWP2, AWP7 and CAGL0F00181 genes in three wild type strains CgDSY562 (High adherence), CgBG2 (Medium adherence) and Cg90030 (Low adherence) were measured by qRT-PCR following incubation with germinated C. albicans. All five genes identified by screening (A) were induced in the presence of C. albicans hyphae proportionally with strain adherence and infection levels (B). In contrast, control C. glabrata EPA1 and EPA6 genes (far right) were not induced by C. albicans hyphae. The values plotted are means ± SEMs of n = 3 or 4 independent experiments.
Table 1.
List of strains used in this study.