Fig 1.
Structure and expression of C. albicans TLO genes.
(A) Diagram comparing the structure of the Tlo proteins analysed in this study, based on the models of Anderson et al. [11]. The green box represents the conserved Med2-like domain. The blue box represents the clade-specific c-terminus. The γ- and β-specific regions are indicated by yellow and red boxes, respectively. The table on the right indicates which genes have been expressed in wild-type C. dubliniensis Wü284 and the TLO null derivative ΔΔtlo. ND = not done. (B) RT-PCR expression data of C. albicans TLOs expressed in the C. dubliniensis WT Wü284 strain. RT-PCR expression graphs represent the results of three independent experiments.
Fig 2.
C. albicans TLOβ2 confers filamentous growth in C. dubliniensis.
Colony (A) and cellular (B) morphology of C. dubliniensis WT Wü284 and derivatives harboring TLOβ2 expressed from the TLO1 promoter (PTLO1-TLOβ2) and the ACT1 promoter (PACT1-TLOβ2). Colonies were grown for 48 h on solid YEPD agar. The morphology of the cells in representative colonies of each derivative was visualised using a x40 objective lens.
Fig 3.
The effect of C. albicans TLO genes on growth rates in YEP-Glucose and -Galactose broth.
Doubling times of WT Wü284 and derivatives expressing the indicated C. albicans TLO genes in YEP-Glucose (A) and -Galactose (B). Asterisks indicate significant difference from Wü284. Data were generated in three replicate experiments.
Fig 4.
The effect of C. albicans TLO genes on susceptibility to H2O2 and cell wall damaging agents.
(A) Minimum inhibitory concentration of H2O2 was determined by broth dilution. The IC80 is indicated and shows the concentration of H2O2 that reduced growth of the derivatives tested below 80% of the inhibitor-free control. (B) Ten-fold serial dilutions (left to right) of 2 x 104 cells were spotted on to plates containing 3 μg/ml Congo Red and 20 μg/ml Calcofluor White.
Fig 5.
The effect of C. albicans TLO genes on biofilm formation on plastic surfaces.
Each strain was grown in Spider medium in a 96-well plate for 48 h and biomass measured using a Crystal Violet assay. An asterisk indicates significant differences with * at 24 h and red * at 48 h.
Fig 6.
The effect of C. albicans TLO genes on the virulence of wild-type Wü284.
Ten G. mellonella larvae were inoculated with 1 X 106 cells of each indicated strain (performed blind) and viability was monitored over 3 days. Results presented represent three independent infection experiments. P values indicate results of a Log-Rank (Mantel-Cox) test against the wild type Wü284 survival curve.
Fig 7.
Morphology of C. dubliniensis ΔΔtlo expressing CaTLO genes.
A. Photomicrographs of C. dubliniensis ΔΔtlo and derivatives harboring the indicated C. albicans TLO genes following 4 h growth in water supplemented with 10% (v/v) foetal bovine serum at 37°C. B. Chlamydospore formation of C. dubliniensis ΔΔtlo and derivatives harboring the indicated C. albicans TLO genes on cornmeal agar supplemented with tween. Chlamydospores are indicated by arrows. Identical results were observed in replicate experiments.
Fig 8.
Growth of C. dubliniensis ΔΔtlo and derivatives harboring C. albicans TLO genes.
A and B show doubling times of WT Wü284, the ΔΔtlo double mutant and derivatives expressing indicated C. albicans TLO genes in YEP-Glucose (A) and -Galactose (B). Stars indicate strains exhibiting doubling times significantly different from ΔΔtlo (p ≤ 0.05). Panel C shows biofilm formation on plastic surfaces. Each ΔΔtlo::TLO strain was grown in the presence of YEPD in a 96-well plate for 48 h. Biomass was measured using a crystal violet assay in three replicate experiments. Asterisks indicate significant differences from ΔΔtlo at 24 h (*) and 48 h (red *), respectively. Data are the result of three independent experiments.
Fig 9.
C. albicans TLO genes differentially affect tolerance of environmental stress conditions.
Growth of each ΔΔtlo::CaTLO strain in the presence (A) H2O2, (B) NaCl, (C) Congo Red and (D) Calcofluor White. Ten-fold serial dilutions (left to right) of 2 x 104 cells were spotted onto YEPD agar and YEPD agar containing the indicated agents. Plates were incubated for 48 h at 37°C.
Fig 10.
The effect of C. albicans TLO genes on the virulence of the ΔΔtlo mutant.
Ten G. mellonella larvae were inoculated with 1 X 106 cells of each indicated strain (performed blind) and viability was monitored over 3 days. Results presented represent three independent infection experiments. P values indicate results of a Log-Rank (Mantel-Cox) test against the ΔΔtlo mutant survival curve.
Fig 11.
Heat maps summarizing phenotypic effects of CaTLO genes.
The phenotypic effects of expressing each CaTLO gene in the C. dubliniensis wild-type (panel A) and in C. dubliniensis ΔΔtlo (panel B) are colour coded; yellow indicates the same phenotype as the mutant, green indicates a gain of function and red indicates a loss of function.