The pre-mRNA splicing modulator pladienolide B inhibits Cryptococcus neoformans germination and growth

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections, particularly in immunocompromised individuals. The limitations of current antifungal therapies due to toxicity and the emergence of resistance highlight the need for novel treatment strategies and targets. C. neoformans has an intron-rich genome, and pre-mRNA splicing is required for expression of the vast majority of its genes. In this study, we investigated the efficacy of a human splicing inhibitor, pladienolide B (PladB), as an antifungal against C. neoformans. PladB inhibited the growth of C. neoformans in liquid culture and spore germination. The potency of PladB could be increased by simultaneous treatment with either FK506 or clorgyline. This combination treatment resulted in significant reductions in fungal growth and prevented spore germination. Transcriptomic analysis revealed that PladB inhibits splicing in C. neoformans and results in widespread intron retention. In combination with FK506, this resulted in downregulation of or intron retention in transcripts from processes vital for cellular growth, including translation, transcription, and RNA processing. Together, these results suggest that targeting RNA splicing pathways could be a promising antifungal strategy and that the effectiveness of splicing inhibitors as antifungals can be increased by co-administering drugs such as FK506.IMPORTANCEFungal infections, like those caused by Cryptococcus neoformans, can turn deadly for many patients. New treatments and therapeutic targets are needed to combat these pathogens. One potential target is the pre-mRNA processing pathway, which is required for expression of nearly all protein-coding genes in C. neoformans. We have determined that a pre-mRNA splicing inhibitor can inhibit both C. neoformans growth and germination and that the potency of this drug can be increased when used in combination with other molecules. This work provides evidence that targeting steps in pre-mRNA processing may be an effective antifungal strategy and avenue for the development of new medicines.

Keywords: C. neoformans; antifungal; inhibitor; pre-mRNA; splicing.

Fig 1

Pladienolide B (PladB) inhibits C. neoformans yeast growth and spore germination. (A) (Left) Structures of the human B complex spliceosome with SF3B1 are highlighted in blue. The region of SF3B1 corresponding to the view in the image to the right is shown in a red box. (Right) The drug- and branch point-binding pocket of SF3B1 with key amino acids for resistance in S. cerevisiae is shown in spacefill, and PladB is shown in stick representation. Protein sequences for C. albicans (strain SC5314) and C. neoformans (strain H99) were obtained from UniProt. (B) Chemical structure of PladB. (C) Normalized growth of C. neoformans strain KN99α in liquid culture relative to a DMSO control in the presence of increasing concentrations of PladB (±SD from n = 3 biological replicates). (D) Percent spore escape during germination of C. neoformans serotype D spores (JEC20 × JEC21) in the presence of DMSO or 30 µM PladB. Lines were fit to the mean values per time point from n = 9 technical replicates. Structures shown in panel A were generated using ChimeraX and Pymol (25, 26).

Fig 2

PladB potency increases in combination with FK506 or clorgyline. (A) Chemical structures of FK506 and clorgyline. (B and C) Normalized growth of C. neoformans strain KN99α in the presence of increasing concentrations (0.01–100 µM) of FK506 (B) or clorgyline (C) (±SD from n = 3 biological replicates). (D and E) Growth curves of C. neoformans strain KN99α in the presence of DMSO, PladB alone, FK506 alone, clorgyline alone, or combinations of PladB with FK506 (D) or clorgyline (E) (error bars represent the ±SD from n = 3 replicates). (F and G) two-dimensional contour maps depicting the combinatorial effects of PladB with FK506 (F) and clorgyline (G) in C. neoformans strain KN99α, where red and blue represent areas of high or low synergy values, respectively, calculated using the Bliss synergy scoring method. In panels B–E, data points were connected by straight lines for clarity.

Fig 3

PladB in combination with FK506 or clorgyline inhibits C. neoformans spore germination. (A) Germination profiles of C. neoformans serotype D spores (JEC20 × JEC21) in the presence of DMSO, FK506 (50 µM), clorgyline (50 µM), PladB (30 µM), or their combinations. Each two-dimensional histogram was obtained by quantitative analysis of microscopy data where each x-axis represents cell area (µm²) and each y-axis represents cellular aspect ratio (width/length). Pixel intensities represent the number of cells with particular values of area and aspect ratio. Spores have smaller areas and aspect ratios (lower left) relative to yeast (upper right). (B) Stacked bar plots quantifying cell populations at each time point from the histograms shown in panel A. Error bars represent ±SD from n = 9 technical replicates. Portions of the data shown for the PladB condition here were also used to generate the plot in Fig. 1D.

Fig 4

FK506, PladB, and their combination have unique impacts on gene expression. (A) Principal component analysis of RNA-seq data illustrating variance among conditions and clustering of replicates (n = 3) for cells treated with either DMSO or 10 µM of either PladB or FK506 or their combination (combo). (B and C) Venn diagrams illustrating common and unique upregulated and downregulated (B and C, respectively) differentially expressed genes (DEGs) in each condition. (D–F) Volcano plots displaying DEGs in C. neoformans strain KN99 treated with PladB (D), FK506 (E), or the combination of PladB and FK506 (F). DEGs were defined as those with log₂ fold change of <−1 or >1 and a −log₁₀ P value of >0.05 (red dots). A total of 6,979 genes (not including any non-coding RNAs) were analyzed in each plot. (G) Gene ontology (GO) enrichment analysis of downregulated DEGs in the combination treatment of PladB and FK506.

Fig 5

PladB inhibits pre-mRNA splicing in C. neoformans. (A) Example of read coverage tracks over the gene CNAG_02234 (encoding the large subunit ribosomal protein L6e) in the presence of DMSO, PladB, FK506, or the combination of PladB with FK506. Note the presence of intronic reads in PladB-treated conditions (arrows). (B) Stacked bar graph depicting the distribution of alternative splicing events across each condition. A3SS and A5SS represent alternative 3′ and 5′ splice site selections, respectively. IR, intron retention; MXE, mutually exclusive exon usage; SE, skipped exon. (C) Venn diagram showing the numbers of unique and common intron retention events under each condition relative to the DMSO controls. (D) Gene ontology enrichment analysis of genes with retained introns in PladB-containing conditions.