Candida albicans utilizes a modified β-oxidation pathway for the degradation of toxic propionyl-CoA

Abstract

Propionyl-CoA arises as a metabolic intermediate from the degradation of propionate, odd-chain fatty acids, and some amino acids. Thus, pathways for catabolism of this intermediate have evolved in all kingdoms of life, preventing the accumulation of toxic propionyl-CoA concentrations. Previous studies have shown that fungi generally use the methyl citrate cycle for propionyl-CoA degradation. Here, we show that this is not the case for the pathogenic fungus Candida albicans despite its ability to use propionate and valerate as carbon sources. Comparative proteome analyses suggested the presence of a modified β-oxidation pathway with the key intermediate 3-hydroxypropionate. Gene deletion analyses confirmed that the enoyl-CoA hydratase/dehydrogenase Fox2p, the putative 3-hydroxypropionyl-CoA hydrolase Ehd3p, the 3-hydroxypropionate dehydrogenase Hpd1p, and the putative malonate semialdehyde dehydrogenase Ald6p essentially contribute to propionyl-CoA degradation and its conversion to acetyl-CoA. The function of Hpd1p was further supported by the detection of accumulating 3-hydroxypropionate in the hpd1 mutant on propionyl-CoA-generating nutrients. Substrate specificity of Hpd1p was determined from recombinant purified enzyme, which revealed a preference for 3-hydroxypropionate, although serine and 3-hydroxyisobutyrate could also serve as substrates. Finally, virulence studies in a murine sepsis model revealed attenuated virulence of the hpd1 mutant, which indicates generation of propionyl-CoA from host-provided nutrients during infection.

Keywords: Amino Acid; CUG clade; Candida albicans; Enzyme Kinetics; Fatty Acid Metabolism; Fatty Acid Oxidation; Odd-chain Fatty Acids; Pathogenesis; β-Hydroxypropionate.

FIGURE 1.

Propionate utilization by C. albicans. A, growth analysis of C. albicans wild type on 5 mm glucose, 5 mm glucose + 50 mm acetate, and 5 mm glucose + 50 mm propionate media. An arrow indicates the time point of total glucose consumption in all cultures. In medium supplemented with acetate, growth continues after glucose consumption. On medium supplemented with propionate, a 20-h lag phase follows glucose consumption after which biomass starts to increase. No further increase is observed from cultures growing without acetate or propionate supplementation. Data points show mean values with standard deviations from three independent cultures. B, growth of C. albicans on 20 mm propionate as sole carbon source. Cells were inoculated at high density, and aliquots were removed for propionate detection by HPLC analysis. C, HPLC-based determination of propionate consumption from cultures shown in B. After 30 h, no residual propionate is detected from the culture medium.

FIGURE 2.

Proteomic analysis of protein extracts from C. albicans wild type cultivated on glucose, acetate, or propionate medium. Protein extracts were prepared from cells in the exponential growth phase and separated by two-dimensional gel electrophoresis. Major or unique spots from different conditions were analyzed by MALDI-TOF-MS analysis. Proteins assigned to catabolic processes by GO term analyses (see also Table 2) are highlighted and marked by numbered 1–33. Details on all proteins identified can be found in supplemental Table S1.

FIGURE 3.

Scheme of the modified β-oxidation pathway via 3-hydroxypropionate. Candidate genes predicted for the plant A. thaliana (31) are shown on the left side and candidate genes for C. albicans on the right side of the pathway. Propionyl-CoA enters the β-oxidation pathway and is oxidized to 3-hydroxypropionyl-CoA. This intermediate exits the β-oxidation pathway by hydrolysis of the CoA ester and is further oxidized to either acetate or acetyl-CoA.

FIGURE 4.

Growth analysis of C. albicans wild type and deletion mutants on solid media containing different carbon sources. Serial dilutions (10⁶ to 10¹ cells/spot) were used for inoculation, and plates were incubated at either 30 or 37 °C. The scheme on top denotes the order of strains on all plates. Lane 1, SC5314 wild-type strain; lane 2, homozygous fox2 deletion strain; lane 3, homozygous ehd3 deletion strain; lane 4, homozygous hpd1 (orf19.5565) deletion strain; lane 5, homozygous ald5 deletion strain; lane 6, homozygous ald6 deletion strain; lane 7, homozygous mutant with ald5 and ald6 deletions; lane 8, homozygous triple mutant with deletion of the ketoacyl-CoA thiolases pot1, fox3, and pot13. Strains complemented on one allele showed no altered phenotype in comparison with the wild type (data not shown).

FIGURE 5.

Growth analysis of wild type, Δ/Δhpd1 (hpd1) mutant, and Δ/Δehd3 (ehd3) mutant on liquid media and on valine-containing solid media. A, growth of wild type, hpd1 deletion mutant, and ehd3 deletion mutant on propionate and valerate medium. The hpd1 mutant shows some initial biomass formation on propionate, but growth is completely abolished at later time points. On valerate, the mutant shows a decreased growth rate and reduced final biomass. The ehd3 deletion mutant only reveals very limited growth after a 15–20-h lag phase and is unable to grow on valerate. Mean values from three independent parallel cultures are shown. B, growth of the ehd3 deletion mutant and its complemented strain in comparison with the wild type on mixed carbon sources. Although the mutant is able to grow at a reduced rate in the presence of acetate with propionate, no growth is observed when butyrate is supplemented with propionate. Mean values from three independent parallel cultures are shown. C, growth analysis on valine-containing solid media. The homozygous ehd3 and hpd1 deletion mutants show no altered growth phenotype in comparison with the wild type.

FIGURE 6.

Analysis of HPD1-GFP and EHD3-GFP fusion strains. A, fluorescence intensity of the HPD1-GFP fusion under control of its natural HPD1 promoter (pHPD1). Background fluorescence from the wild type has been subtracted, and values are given as relative fluorescence units/μg of total protein (RFU/μg). Data represent mean values from two independent transformants measured in at least three serial dilutions. Error bars indicate standard deviations. EHD3-GFP fusion strains showed no fluorescence above background levels and are not depicted. B, fluorescence microscopy of a representative pHPD1-HPD1-GFP fusion strain grown in the presence of propionate. Mitochondria were stained with MitoTracker Red. C, subcellular localization of GFP fused with an N-terminal fragment of Ehd3p under the control of the constitutively expressed actin promoter pACT1 (pACT1-EHD3_N-GFP). Mitochondria were stained with MitoTracker Red.

FIGURE 7.

Identification and quantification of 3-hydroxypropionic acid by HPLC and GC-MS analysis. Silylation of 3-hydroxypropionate leads to the 3-[(trimethylsilyl)oxy]-trimethylsilyl ester of propionic acid. A, GC-MS spectrum of silylated 3-hydroxypropionate from cell-free extracts of propionate/acetate-cultivated hpd1 mutant cells (upper panel), authentic 3-hydroxypropionate (middle panel), and a reference spectrum from a GC-MS database (lower panel). B, HPLC quantification of 3-hydroxypropionate from cell-free extracts of the hpd1 mutant (left) and wild type (right) cultivated on acetate + propionate medium for the indicated time points. C, GC-MS analysis of 3-hydroxypropionate accumulation from cell-free extracts of the hpd1 mutant (left) and wild type (right) cultivated for the indicated time points on valerate.

FIGURE 8.

NMR analysis of 3-hydroxypropionate isolated from homozygous hpd1 mutant cultivated for 6 h in the presence of acetate and 2-[¹³C]propionate. A, ¹H NMR spectrum of 3-[2-¹³C]hydroxypropionic acid recorded at 500 Hz in methanol-d₄. The signal of the methylene protons resonating at 2.50 ppm is split into two discrete triplets because of ¹J coupling to ¹³C. B, ¹H-decoupled ¹³C NMR spectrum of 3-[2-¹³C]hydroxypropionic acid recorded at 125 MHz in methanol-d₄. The α-carbon in 3-hydroxypropionic acid is strongly enriched in the ¹H-decoupled ¹³C NMR spectrum, supporting the metabolic origin from 2-[¹³C]propionate.

FIGURE 9.

Purification and pH dependence of recombinant purified Hpd1p from C. albicans. A, SDS-PAGE analysis. Lane M, molecular mass marker; lane 1, cell-free extract; lane 2, column flow-through; lane 3, wash fraction; lanes 4–6, elution fractions. B, buffer and pH dependence of recombinant Hpd1p. Highest activity is observed in a range between pH 9.5 and 10.0 with CHES buffer.

FIGURE 10.

Murine infection model of disseminated candidiasis. BALB/C mice (n = 10 mice per strain) were infected intravenously with 2.5 × 10⁴ cfu/g of body weight of the hpd1 mutant, the wild type (WT), or the reconstituted mutant (HPD1C). Survival was monitored over a period of 21 days, and data are shown as Kaplan-Meyer plots. The hpd1 mutant reveals a significantly attenuated virulence (p < 0.01) compared with the wild type and the reconstituted mutant as calculated by the Log-rank (Mantel-Cox) test of the GraphPad Prism 5 software.