Folate-mediated one-carbon metabolism as a potential antifungal target for the sustainable cultivation of microalga Haematococcus pluvialis

Abstract

Background: Microalgae are widely considered as multifunctional cell factories that are able to transform the photo-synthetically fixed CO₂ to numerous high-value compounds, including lipids, carbohydrates, proteins and pigments. However, contamination of the algal mass culture with fungal parasites continues to threaten the production of algal biomass, which dramatically highlights the importance of developing effective measures to control the fungal infection. One viable solution is to identify potential metabolic pathways that are essential for fungal pathogenicity but are not obligate for algal growth, and to use inhibitors targeting such pathways to restrain the infection. However, such targets remain largely unknown, making it challenging to develop effective measures to mitigate the infection in algal mass culture.

Results: In the present study, we conducted RNA-Seq analysis for the fungus Paraphysoderma sedebokerense, which can infect the astaxanthin-producing microalga Haematococcus pluvialis. It was found that many differentially expressed genes (DEGs) related to folate-mediated one-carbon metabolism (FOCM) were enriched in P. sedebokerense, which was assumed to produce metabolites required for the fungal parasitism. To verify this hypothesis, antifolate that hampered FOCM was applied to the culture systems. Results showed that when 20 ppm of the antifolate co-trimoxazole were added, the infection ratio decreased to ~ 10% after 9 days inoculation (for the control, the infection ratio was 100% after 5 days inoculation). Moreover, application of co-trimoxazole to H. pluvialis mono-culture showed no obvious differences in the biomass and pigment accumulation compared with the control, suggesting that this is a potentially algae-safe, fungi-targeted treatment.

Conclusions: This study demonstrated that applying antifolate to H. pluvialis culturing systems can abolish the infection of the fungus P. sedebokerense and the treatment shows no obvious disturbance to the algal culture, suggesting FOCM is a potential target for antifungal drug design in the microalgal mass culture industry.

Keywords: Antifolate; Drug design; Fungal contamination; Haematococcus pluvialis; Microalgae cultivation; One-carbon metabolism; Paraphysoderma sedebokerense.

Fig. 1

Characterization of the infection features of the parasitic fungus P. sedebokerense on its microalgal host H. pluvialis at various infection stages. Typical cell features at various infection stages are shown under optical microscope (top and middle) and transmission electron microscope (bottom). White arrows indicate the cell interaction regions of interest; arrowheads indicate lipid droplets inside the fungus cell. DPI days post-inoculation. F P. sedebokerense. Hp H. pluvialis. Bars = 10 μm

Fig. 2

BODIPY staining of algal–fungal co-culture samples collected at various infection stages. A Samples observed under optical microscope (top) and fluorescent microscope (bottom). DIC, differential interference contrast. Bars = 20 μm. B Quantitative analysis of the fluorescent intensity detected using the cytometer in samples collected at various infection stages. Insert, the BODIPY stained the fungal cells but not the algal cells. C, D Flow cytometer dot plots showing the correlations between the fluorescence intensity (FL1, x-axis) and side scatter (SS, y-axis, C) or forward scatter (FS, y-axis, D). DPI, days post-inoculation

Fig. 3

Overview of the transcriptomic results of P. sedebokerense upon infection. A Venn diagram showing the differentially expressed genes in P. sedebokerense at the various infection stages versus the CK. B The annotated up- and down- regulated genes in P. sedebokerense at the various infection stages versus the CK. C The Euclidean distance coefficients of sample-to-sample analysis. CK fungal spores before infection; DPI days post-inoculation

Fig. 4

GO analysis and KEGG functional enrichment of differentially expressed genes in P. sedebokerense at various stages of infection. The top 10 GO/KEGG terms for the up- and down- regulated differentially expressed genes are shown in A 1 DPI vs CK, B 3 DPI vs CK, C 5 DPI vs CK. Vertical dashed lines correspond to p = 0.05. BP biological process; CC cellular component; MF molecular function; GO Gene Ontology; KEGG Kyoto Encyclopedia of Genes and Genomes; CK fungal spores before infection; DPI days post-inoculation

Fig. 5

Analysis of the differentially expressed genes associated with the pathway of folate-mediated one-carbon metabolism. A Heatmap analysis of the pathways, including carbon metabolism, glycerophospholipid metabolism, one-carbon pool by folate, cell cycle, DNA replication, cysteine and methionine metabolism, purine/pyrimidine metabolism and glutathione metabolism. The full names of the proteins are provided in Additional file 1: Dataset S1. Numbers in the heatmap were mean FPKM value of each gene, color in the box was indicated by z-score. B Main processes of folate-mediated one-carbon metabolism. Gray ellipses represent potential metabolism required for the fungal. THF tetrahydrofolate; 5-mTHF 5-methyl-tetrahydrofolate; 5,10-mTHF 5,10-methylene-tetrahydrofolate; SAM S-adenosylmethionine; SAH S-adenosylhomocysteine; GSSG glutathione (oxidized); DPI days post-inoculation

Fig. 6

Result of applying co-trimoxazole to the algal–fungal co-culture and the algal mono-culture. A Microscopic images showing the inhibitory effect of applying various concentrations of co-trimoxazole to P. sedebokerense-infected H. pluvialis cultures. The images were taken on the 5th day post-inoculation. B The infection ratio in P. sedebokerense-infected H. pluvialis supplied with various concentrations of co-trimoxazole. C Changes in the H. pluvialis biomass dry weight (DW) and pigment content, including carotenoids (Car.) and chlorophyll (Chl.), upon co-trimoxazole application. DPI days post-inoculation. Bars = 20 μm

Fig. 7

Hypothetical working model showing folate-mediated one-carbon metabolism is required for the parasitism of P. sedebokerense. Folate and methionine participate in the folate cycle and the methionine cycle of one-carbon metabolism, respectively. These pathways produce nucleotides, NADPH, glutathione and methyl groups, which are crucial products associated with cell proliferation, redox balancing, ROS scavenging and methylation reaction in the fungus P. sedebokerense, allowing it to successfully parasitize its algal host H. pluvialis. However, such metabolic processes were arrested by applying antifolates (this study) or inhibitors targeting methionine biosynthesis [40], resulting in restrained infections of P. sedebokerense on H. pluvialis, collectively suggesting that folate-mediated one-carbon metabolism is required for the parasitism of P. sedebokerense