Bcr1 functions downstream of Ssd1 to mediate antimicrobial peptide resistance in Candida albicans

Abstract

In order to colonize the host and cause disease, Candida albicans must avoid being killed by host defense peptides. Previously, we determined that the regulatory protein Ssd1 governs antimicrobial peptide resistance in C. albicans. Here, we sought to identify additional genes whose products govern susceptibility to antimicrobial peptides. We discovered that a bcr1Δ/Δ mutant, like the ssd1Δ/Δ mutant, had increased susceptibility to the antimicrobial peptides, protamine, RP-1, and human β defensin-2. Homozygous deletion of BCR1 in the ssd1Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1Δ/Δ and ssd1Δ/Δ mutants to RP-1 induced greater loss of mitochondrial membrane potential and increased plasma membrane permeability than with the control strains. Therefore, Bcr1 and Ssd1 govern antimicrobial peptide susceptibility and likely function in the same pathway. Furthermore, BCR1 mRNA expression was downregulated in the ssd1Δ/Δ mutant, and the forced expression of BCR1 in the ssd1Δ/Δ mutant partially restored antimicrobial peptide resistance. These results suggest that Bcr1 functions downstream of Ssd1. Interestingly, overexpression of 11 known Bcr1 target genes in the bcr1Δ/Δ mutant failed to restore antimicrobial peptide resistance, suggesting that other Bcr1 target genes are likely responsible for antimicrobial peptide resistance. Collectively, these results demonstrate that Bcr1 functions downstream of Ssd1 to govern antimicrobial peptide resistance by maintaining mitochondrial energetics and reducing membrane permeabilization.

Fig 1

Comparative antimicrobial peptide susceptibilities and time courses of RTA2 and BCR1 mRNA levels in two C. albicans bloodstream isolates (CA024 [Amp^s] and CA080 [Amp^r]) with differing levels of antimicrobial peptide susceptibility and in the DAY185 reference strain. (A) Susceptibilities of the three C. albicans strains to the indicated antimicrobial peptides were determined by a radial diffusion assay at pH 7.5. Antimicrobial susceptibility was measured as the zone of inhibition (ZOI) after incubation at 30°C for 24 h. Results are means ± standard deviations (SD) from three independent experiments. (B and C) RTA2 (B) and BCR1 (C) transcript levels in the Amp^s, Amp^r, and DAY185 strains after incubation for the indicated time in the presence of a sublethal concentration of RP-1 (2.5 μg/ml for Amp^s, 100 μg/ml for Amp^r, and 5 μg/ml for DAY185). Transcript levels were measured by real-time PCR using ACT1 as the endogenous control gene and normalized to organisms incubated for 60 min in medium without RP-1 (untreated). Results are means ± SD for three biological replicates, each measured in duplicate. *, P < 0.05 compared to cells grown in the absence of RP-1. HNP-1, human neutrophil peptide 1; hBD-2, human β-defensin 2.

Fig 2

Influence of RTA2 and BCR1 on C. albicans antimicrobial peptide susceptibility. The susceptibilities of the indicated strains of C. albicans to RP-1 (A and C) and hBD-2 (B and D) were determined by a radial diffusion assay after incubation at 30°C for 24 h. Results are the means ± SD from two independent experiments. *, P < 0.05 compared to the wild-type strain (WT).

Fig 3

Susceptibilities of the ssd1Δ/Δ and bcr1Δ/Δ mutants to protamine and nonpeptide stressors. Images of serial 10-fold dilutions of the indicated strains that were plated onto YPD agar containing 2 mg/ml protamine sulfate, 0.1% SDS, or 300 μg/ml Congo red and incubated at 30°C for 2 days are shown.

Fig 4

Epistasis analysis of BCR1 and SSD1. (A) Susceptibilities of independent ssd1Δ/Δ bcr1Δ/Δ double deletion mutants to protamine (1.8 mg/ml). (B) Effects of deletion of SSD1 and BCR1 on BCR1 and SSD1 mRNA expression. Total RNA was isolated from the indicated strains grown in YPD at 30°C to early log phase, after which expression of BCR1 and SSD1 was determined by real-time PCR and normalized ACT1. Results are means ± SD for three biological replicates, each measured in duplicate. *, P < 0.05 compared to the wild type (DAY185). (C and D) Effects of overexpression of BCR1 in the ssd1Δ/Δ mutant (C) and overexpression of SSD1 in the bcr1Δ/Δ mutant (D) on susceptibility to protamine (2 mg/ml).

Fig 5

Effects of SSD1 and BCR1 deletion or overexpression on susceptibility of C. albicans to diverse antimicrobial peptides. The susceptibilities of the indicated strains of C. albicans to HNP-1, hBD-2, LL-37, and RP-1 were measured using a radial diffusion assay. The zones of growth inhibition were imaged after incubation at 30°C for 24 h.

Fig 6

Effects of RP-1 on C. albicans plasma membrane permeability and mitochondrial membrane potential. The indicated strains of C. albicans were exposed to RP-1 at pH 7.5 for 1 h and then analyzed by flow cytometry. (A and C) Histograms of propidium iodide fluorescence, a measure of membrane permeabilization, of the Amp^s and Amp^r strains exposed to 5 μg/ml RP-1 (A) and of strain DAY185 exposed to 5 to 20 μg/ml RP-1 (C). The fluorescence of untreated control cells is indicated by the black lines, and the fluorescence of cells exposed to RP-1 is indicated by the red lines. (B and D) Histogram of DiOC₅ fluorescence, a measure of mitochondrial membrane potential, of the Amp^s and Amp^r strains exposed to 5 μg/ml RP-1 (B) and of strain DAY185 exposed to 5 to 20 μg/ml RP-1 (D). The fluorescence of untreated control cells is indicated by the black lines, and the fluorescence of cells exposed to RP-1 is indicated by the green lines.

Fig 7

Effects of RP-1 on plasma membrane permeability and mitochondrial membrane potential of the ssd1Δ/Δ and bcr1Δ/Δ mutants. The indicated strains of C. albicans were exposed to 5 μg/ml RP-1 at pH 7.5 for 1 h and then analyzed by flow cytometry. (A) Histogram of propidium iodide fluorescence, a measure of membrane permeabilization. The fluorescence of untreated control cells is indicated by the black lines, and the fluorescence of cells exposed to RP-1 is indicated by the red lines. (B) Histogram of DiOC₅ fluorescence, a measure of mitochondrial membrane potential. The fluorescence of untreated control cells is indicated by the black lines, and the fluorescence of cells exposed to RP-1 is indicated by the green lines.

Fig 8

Proposed model of the interactions of Ssd1 and Bcr1 in the regulation of C. albicans susceptibility to different antimicrobial peptides.