Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans

Abstract

Cryptococcus neoformans is a leading cause of life-threatening fungal infection in immunocompromised patients. Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungus-specific enzyme, encoded by the essential IPC1 gene, that catalyzes the formation of complex sphingolipids and may also regulate the levels of phytoceramide and diacylglycerol. Here, we investigated the functions of this essential gene by modulating its expression in C. neoformans using a galactose-inducible promoter. Down-regulation of IPC1 significantly lowers the expression of certain virulence traits such as melanin pigmentation and, remarkably, impairs pathogenicity of C. neoformans in an established rabbit model. Interestingly, we found that IPC1 down-regulation significantly decreases the intracellular growth of C. neoformans in the J774.16 murine macrophage-like cells. Finally, we studied the effect of IPC1 expression under different stress conditions and found that down-regulation of IPC1 confers a defect on in vitro growth at low pH. Because this environment is similar to that in the phagolysosome of J774.16 macrophage-like cells, our findings indicate that down-regulation of IPC1 confers a growth defect in vivo through a pH-dependent mechanism. In conclusion, our study is the first to define a novel and crucial function of Ipc1 in fungal pathogenesis.

Figure 1

Replacement of the endogenous wild-type IPC1 with pGAL7::IPC1/ADE2. Southern analysis of wild-type strain H99 and transformants with genomic DNA digested with PstI (PI), EcoRV (RV), HindIII (H3), and EcoRI (RI), as indicated. Transformant #2 showed a single crossover event at the upstream region of the IPC1 locus. Transformant #10 showed a double crossover event with insert of plasmid loop (or single crossover followed by a double crossover event). Transformant #13 showed a double crossover event. Confirmation of the GAL7::IPC1 gene replacement was also confirmed by PCR analysis using different primer combinations (data not shown). (SK) pBluescript SK plasmid.

Figure 2

(A) In vitro quantitative analysis of C. neoformans IPC1 mRNA in H99 and GAL7::IPC1 strains. The isogenic wild-type strain H99 (WT) and the GAL7::IPC1 strains were grown on galactose (inducing conditions). RNA was extracted at different time points (30 min; 1, 3, 6, and 24 h), converted to cDNA, and amplified with primers directed to IPC1 or actin genes. RT-PCR products were separated by electrophoresis in a 1% agarose gel and stained with ethidium bromide. The IPC1 and actin RT-PCR products are 926 and 543 bp, as indicated. (B) In vitro quantitative analysis of Ipc1 activity in C. neoformans wild-type (H99) and GAL7::IPC1 strains grown on glucose (repressing conditions) and galactose (inducing conditions). (Left) The Ipc1 synthase in vitro activity was determined as conversion of NBD-C6-ceramide (NBD-C6-CER) into NBC-C6-IPC by thin layer chromatography (TLC) analysis. (Right) Numerical analysis of Ipc1 activity from the TLC is reported.

Figure 3

(A) IPC1 regulates melanin pigmentation in C. neoformans. The wild-type (WT) and GAL7::IPC1 strains were grown for 16 h at 30°C under inducing and repressing conditions. Cells were permeabilized with toluene:ethanol, incubated for 16 h in presence of L-dopamine, and the cellular phenoloxidase activity was assayed spectrophotometrically by measuring the appearance of melanin in the supernatant by the change in absorbance at 480 nm. The A₄₈₀ of samples to which L-dopamine had been added was compared with the blank sample in which no L-dopamine had been added. Data represent geometric means ± standard deviations of three separate experiments. (B) IPC1 regulates virulence of C. neoformans in a rabbit animal model of cryptococcal meningitis. Rabbits (three for each strain) were immunosuppressed with steroids and inoculated with 3 × 10⁸ cells. CSF was removed on days 4, 7, 11, and 14 following inoculation, and the number of surviving organisms was determined by serial dilutions and plating on YPD medium. Each data point represents the geometric means of all cultures for each strain, and the standard deviations of the mean are indicated.

Figure 4

IPC1 regulates intracellular growth in the J774.16 macrophage-like cell line. (A) Five hours postinoculation, buds were counted only in yeast cells inside the macrophages (% budding/phagocytic index). Counts are geometric means ± standard deviations of four different fields. (B) Lysis of macrophage infected by the GAL7::IPC1 strain on galactose medium at 18 h postinoculation. (Frame 1) Intact macrophage. Arrowheads indicate the phagolysosome. (Frames 2–4) Lysis of macrophage. (C) Phagolysosomes size. Arrowheads indicate yeast cells inside the phagolysosome, whereas long arrows in the last right panel indicate the larger phagolysosome. (N) nucleus. Cells were photographed at ∼18 h postinoculation.

Figure 5

IPC1 expression modulates in vitro growth under low-pH stress condition. C. neoformans wild-type (WT) and GAL7::IPC1 strains grown on YP medium (pH 4.0), supplemented with 2% glucose or 2% galactose. Colony forming units/mL (CFU/mL) are geometric means ± SD of three separate experiments.

Figure 6

A model for the role of Inositol-phosphoryl ceramide synthase 1 (Ipc1) in regulation of virulence traits and pathogenesis of C. neoformans. Ipc1 transfers the phosphoryl-inositol moiety from phosphatidylinositol (PI) to phytoceramide, forming inositol-phosphoryl ceramide (IPC) and diacylglycerol (DAG).