(A) As depicted in the schematics, mice were fed a normal Fe (NFD) or HFD for 3 d prior to C. albicans inoculation. The mice continuously received the same diet during the course of experiments. (B) Colonic reactive oxygen species (ROS) accumulation in mice receiving an NFD or HFD diet for 3 d. Cryostat colonic sections were incubated with dihydroethidium (DHE) and DAPI. Scale bar, 100 μm. (C) Quantitative analysis using fluorescence intensity of DHE (a) in the colon. (D) Colonic samples were collected from mice fed either an NFD or HFD, formalin-fixed, paraffin-embedded, sectioned, and stained with Prussian blue for iron. Representative Prussian blue-stained colonic samples confirmed higher iron deposits in mice receiving HFD (iron blue, nucleus red). Scale bar, 200 μm; inset, 50 μm. (E) The expression of DUOX2 mRNA in the colonic tissue of mice receiving NFD or HFD. Values were normalized to the expression levels of GAPDH. (F) The effect of iron on hydrogen peroxide (H2O2) levels in NFD or HFD-treated mice (n = 4 mice per group). (G) Mutant lacking HAP43 exhibits enhanced commensal fitness in HFD-treated mice. Mice (n = 8 mice per group) fed an HFD were inoculated by gavage with 1:1 mixtures of the wild-type (WT) and either hap43Δ/Δ mutant or HAP43 reintegrant (HAP43 AB) cells (1 × 108 CFU per mice). The fitness value for each strain was calculated as the log2 ratio of its relative abundance in the recovered pool from the host (R) to the initial inoculum (I), and was determined by qPCR using strain-specific primers that could distinguish one from another. (H) Treatment of the antioxidant N-acetyl-L-cysteine (NAC) is able to partially but significantly abrogate the commensal fitness of Hap43 mutant in HFD-fed mice. During the course of experiments, mice (n = 6 mice per group) fed HFD were received drinking water supplemented with or without NAC (1.5 g/L) and then inoculated by gavage with 1:1 mixtures of the wild-type (WT) and hap43Δ/Δ mutant cells (1 × 108 CFU per mice). Results from three independent experiments are shown. All data shown are means ± SD. ns, no significance; *p<0.05, **p<0.01, ****p<0.0001; by unpaired Student’s t-test (C, E, F, G, H).