Fig 4. Copper limitation and differentiation into pathogenic yeast phase induce CTR3 expression.

Expression of CTR genes based on qRT-PCR (A) or a CTR3 promoter fusion to gfp (B). Expression was calculated relative to the constitutive expression of the TEF1 gene. (A) H. capsulatum cells in the host-infecting form (yeasts, 37°C) and environmental form (mycelia, 25°C) were grown in 3M media with low (10 nM) or high (10 μM) CuSO₄ added and the CTR1 (blue), CTR2 (green), and CTR3 (red) transcript levels were quantified by qRT-PCR and calculated relative to ACT1 transcript levels. Data represent the average expression ± standard deviation of results from biological replicates (n = 3). (B) CTR3 promoter activity (P_CTR3) of yeasts grown in 3M medium with different copper concentrations was measured by fluorescence and normalized to the fluorescence of a strain with a TEF1 promoter-gfp fusion (P_TEF1). Fluorescence intensity was measured under different CuSO₄ concentrations with concentrations lower than 60 nM (the trace amount present in 3M medium) achieved by addition of the copper chelator BCS. A non-linear regression standard curve (black line) was fit to the data with known copper concentrations. Data represent average CTR3 promoter activity of replicates (n = 3).