The environmental yeast Cryptococcus liquefaciens produces capsular and secreted polysaccharides with similar pathogenic properties to those of C. neoformans

Abstract

Invasive fungal infections, including cryptococcosis, are a growing threat to immunocompromised patients. Although Cryptococcus neoformans and Cryptococcus gattii are the main agents of human cryptococcosis, opportunistic infections by environmental species, such as C. liquefaciens, have been observed recently. The main Cryptococcus virulence factor is the production and secretion of polysaccharides (PS). Previously, we showed that both species produce PS of similar composition. Here, we examined the ultrastructure and biological activity of capsular and secreted PS from C. liquefaciens, and yeast pathogenicity to an invertebrate host, in comparison with C. neoformans. Ultrastructural analysis by high-resolution microscopy showed that both species produce large and complex capsules. PS from both species had indistinguishable effects on phagocytosis levels, NO production and the secretion of a variety of immune mediators. Challenge with C. liquefaciens or C. neoformans led to complete lethality of G. mellonella larvae. Treatment with C. liquefaciens PS could not protect mice against infection with C. neoformans. We conclude that polysaccharides of the environmental yeast C. liquefaciens have strikingly similar ultrastructural and biological properties to those of C. neoformans, highlighting the importance of monitoring the emergence of new fungal pathogens for which thermotolerance may be an important transitional step towards pathogenesis in humans.

Figure 1. Strutural characterization of Cryptococcus neoformans and C. liquefaciens capsule.

(A,B) Light microscopy of C. neoformans (A) and C. liquefaciens (B) cells after negative staining with India ink, showing the capsule as a light halo of approximately 5.1 ± 2.2 and 4.5 ± 2.8 μm, in C. neoformans and C. liquefaciens, respectively (p = 0.094, N = 100 cells). (C–H) High resolution scanning electron microscopy (HRSEM) of carbon-coated PS capsule formed by C. neoformans (C,E and G) and C. liquefaciens (D,F and H). (I,J) Helium ion microscopy (HIM) of the surface of C. neoformans (I) and C. liquefaciens (J) cells (with no metal coating). Arrowheads indicate triskelion structures. Scale bars: 10 μm (A and B); 5 μm (C and D); 500 nm (E and F); 200 nm (G and H); and 100 nm (I and J).

Figure 2. Atomic force microscopy of Cryptococcus secreted polysaccharides (secreted-PS).

Topographical images obtained at PeakForce tapping mode (in air) of C. neoformans (A and C) and C. liquefaciens (B and D) secreted-PS. Arrowheads indicate handle-shaped structures branching off polysaccharide fibers. Scale bars: 300 nm (A,B) and 100 nm (C,D).

Figure 3. Phagocytosis by murine macrophages of acapsular Cryptococcus neoformans cells (cap59 mutant) or beads coated with secreted PS.

(A,B) Macrophages were allowed to interact, for 72 h, with FITC-labelled Cap59 (A) or polystyrene beads (B) (coated with secreted-PS from C. neoformans (Cn-PS) or C. liquefaciens (Cl-PS), and then analyzed by flow cytometry. Non-infected macrophages were used as controls (CT). (C) Cell viability (by the XTT assay) and nitric oxide production (D) of murine macrophages exposed to secreted-PS from C. neoformans and C. liquefaciens for 24, 48 and 72 h. Treatment with Triton X-100 and LPS were used as positive controls for cell viability loss and macrophage activation, respectively. In (C and D) data represent mean ± SD of 3 independent experiments.

Figure 4. Production of secreted mediators by TPH-1 human macrophages incubated for 24 h with 1, 10 or 100 μg/ml of secreted polysaccharides (secreted-PS) from C. neoformans or C. liquefaciens.

Secreted mediator levels in culture supernatants were determined using the Magpix xMAP multiplex system (Biorad Laboratories Inc., Hercules, CA, USA). Control, untreated cells; + Control, cells incubated with 20 ng/mL TNFα. Data represent mean ± SEM of 4 independent experiments. **p < 0.01 relative to cells challenged by 100 μg/ml C. neoformans vs. C. liquefaciens (Student’s t-test).

Figure 5

Survival of Galleria mellonella larvae after challenge with C. neoformans (blue) or C. liquefaciens (red) whole cells (A) or secreted PS (coated beads (B). There were no significant differences between groups infected with cells (p-value = 0.2446) or treated with PS from different Cryptococcus species (p-value = 0.2446). n = 20 larvae/group, in two independent experiments. Black- untreated larvae; purple – PBS-treated animals; green - uncoated beads.

Figure 6. Effect of secreted PS from C. liquefaciens on mouse survival following C. neoformans infection.

C57/BL6 mice were administered PBS (black circles) or secreted-PS from C. liquefaciens (gray squares), by intraperitoneal injection, 15 and 7 days pre-infection and 1, 7 and 15 days post-infection with C. neoformans (C.n.).