Phospholipids Trigger Cryptococcus neoformans Capsular Enlargement during Interactions with Amoebae and Macrophages

Abstract

A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

“…6C). Next, a variety of small molecules were tested as candidates for the growth factor, including phosphatidylcholine, which was recently found to induce capsule formation, as well as inositol and farnesol, which act as signaling molecules in fungi (8,37,83). However, no influence on mutant growth was found (data not shown).…”

“…In addition to possible modulation of the host immune response, lipids are likely to have complex influences on C. neoformans. For example, it was recently found that phospholipids influence capsule size during cryptococcal interactions with macrophages and amoebae (8). Fatty acids or related molecules such as phospholipids and oxylipins are important signaling molecules in other fungi (3).…”

“…C. neoformans mutants derived from the wild-type strain H99 (see Table S1 in the supplemental material) were grown in yeast peptone dextrose (YPD) broth or agar, yeast nitrogen base (YNB) broth or agar, and minimal medium (MM; 15 mM glucose, 10 mM MgSO 4 , 29.4 mM KH 2 PO 4 , 13 mM glycine, and 3.0 M thiamine) (8). Low-iron medium (LIM) was also employed to induce capsule, and this medium contains 27.75 mM glucose, 20 mM HEPES, 2.3 mM K 2 HPO 4 , 1.7 mM CaCl 2 , 0.325 mM MgSO 4 , 22 mM NaHCO 3 , and 37.8 mM asparagine, as well as the microelements copper sulfate, zinc sulfate, manganese chloride, magnesium sulfate, sodium molybdate, boric acid, and thiamine (76).…”

The Cryptococcus neoformans Capsule: a Sword and a Shield

“…Recent work has suggested an association between Plb1 activity and titan cell formation, and this is discussed later in this review. Plb1 may be necessary to cleave certain host phospholipids to allow for activation of specific signaling pathways (39). Plb1 is secreted using the same vesicle-dependent pathway as that used by the capsule monomers.…”

“…Investigators have speculated that this capsule may protect the fungus from environmental desiccation and/or natural predators, such as nematodes or amoebae (39,70,150,191,192,226). In the host, the capsule serves many protective functions, including reducing host immune responses by downregulating inflammatory cytokines, depleting complement components, and inhibiting the antigenpresenting capacity of monocytes (174,200,201).…”