Cryptococcus neoformans Escape From Dictyostelium Amoeba by Both WASH-Mediated Constitutive Exocytosis and Vomocytosis

Abstract

Cryptococcus neoformans is an environmental yeast that can cause opportunistic infections in humans. As infecting animals does not form part of its normal life-cycle, it has been proposed that the virulence traits that allow cryptococci to resist immune cells were selected through interactions with environmental phagocytes such as amoebae. Here, we investigate the interactions between C. neoformans and the social amoeba Dictyostelium discoideum. We show that like macrophages, D. discoideum is unable to kill C. neoformans upon phagocytosis. Despite this, we find that the yeast pass through the amoebae with an apparently normal phagocytic transit and are released alive by constitutive exocytosis after ~80 min. This is the canonical pathway in amoebae, used to dispose of indigestible material after nutrient extraction. Surprisingly however, we show that upon either genetic or pharmacological blockage of constitutive exocytosis, C. neoformans still escape from D. discoideum by a secondary mechanism. We demonstrate that constitutive exocytosis-independent egress is stochastic and actin-independent. This strongly resembles the non-lytic release of cryptococci by vomocytosis from macrophages, which do not perform constitutive exocytosis and normally retain phagocytosed material. Our data indicate that vomocytosis is functionally redundant for escape from amoebae, which thus may not be the primary driver for its evolutionary selection. Nonetheless, we show that vomocytosis of C. neoformans is mechanistically conserved in hosts ranging from amoebae to man, providing new avenues to understand this poorly-understood but important virulence mechanism.

Keywords: Dictyostelium; WASH; amoeba; cryptococcosis; cryptococcus; exocytosis; pathogen; vomocytosis.

Figure 1

Cryptococcus neoformans is constitutively exocytosed from Dictyostelium discoideum amoeba. (A–C) Example exocytosis of (A) 4.5 um green fluorescent latex beads (images start at 40 min after phagocytosis) (B) heat killed Saccharomyces cerevisiae (images start at 60 min after phagocytosis) and (C) C. neoformans strain Kn99mCherry from wild type D. discoideum strain Ax2 (images start at 60 min after phagocytosis). Time 0 s indicates point of exocytosis. Scale bars 5 μm. (D–F) Frequency histograms of combined transit times measured from three independent 12 h time lapses. (D) Latex beads 126 transit times. (E) Heat killed S. cerevisiae 66 transit times. (F) C. neoformans 57 transit times. (G) Comparison of transit times for latex beads, heat killed S. cerevisiae and C. neoformans. (H) Quantification of phagocytic events for live, heat killed and UV killed cryptococci in Ax2 and WASH null cells. P-values are Mann-Whitney test.

Figure 2

Phagosomes containing Cryptococcus neoformans acquire V-ATPase following phagocytosis that is lost prior to exocytosis. (A) Confocal time lapse microscopy of C. neoformans phagocytosis by wild type D. discoideum strain Ax2. Representative time lapse from three independent experiments. Images were captured every 10 s. VatM is a subunit of the D. discoideum V-ATPase complex. Arrow indicates phagocytosed cryptococcal cell. Inset box is a magnification of phagosome containing cryptococcal cell demonstrating acquisition of V-ATPase. (B) Confocal time lapse microscopy of C. neoformans exocytosis by wild type D. discoideum strain Ax2. Representative time lapse from three independent experiments. Images were captured every 5 s. Arrow indicates exocytosed cryptococcal cell. Inset box is a magnification of exocytosed cryptococcal cell demonstrating absence of V-ATPase prior to exocytosis. Scale bars 10 μm.

Figure 3

Exocytosis of Cryptococcus neoformans from Dictyostelium is dependent on WASH and the actin cytoskeleton. (A) Heat killed S. cerevisiae are not exocytosed from WASH null D. discoideum. Example from 12 h time lapse imaging of heat killed S. cerevisiae in WASH null D. discoideum representative of 60 phagosomes containing heat killed S. cerevisiae from three independent experiments (images start at 500 min after phagocytosis). (B) C. neoformans are exocytosed from WASH null D. discoideum. Example from 12 h time lapse imaging representative of 62 phagosomes containing C. neoformans from three independent experiments (images start at 200 min after phagocytosis). Scale bars 5 μm. (C) Quantification of the percentage of exocytosis of C. neoformans and heat killed S. cerevisiae from WASH null D. discoideum from three independent 12 h time lapses. (D) Frequency histogram of combined 35 transit times measured from three independent 12 h time lapses. (E) Exocytosis of heat killed S. cerevisiae from wild type Ax2 but not WASH null or latrunculin A treated Ax2 D. discoideum. Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P-values are Fishers test. (F) Exocytosis of C. neoformans from wild type Ax2, WASH null, and latrunculin A treated D. discoideum. Quantification of the percentage of exocytosis from three independent 12 h time lapses. Total of 60 phagosomes were analyzed from each condition. P-values are Fishers test. (G) Transit times of C. neoformans through WASH null and latrunculin A treated Ax2 D. discoideum are not significantly different. P-values are Mann-Whitney test.

Figure 4

Exocytosis and vomocytosis of C. neoformans cap59 and plb1 mutants are indistinguishable from wild type H99. (A) Quantification of the percentage of non-constitutive exocytosis of C. neoformans mutants from wild-type Ax2 D. discoideum treated with latrunculin A from three independent 12 h time lapses. Total of 45 phagosomes were analyzed for wild type H99, 90 for cap59 and 90 for plb1. P-values are Fisher's exact test. (B) Transit times of C. neoformans H99 wild type, mutant cap59 and plb1. Phagosomes analyzed the same as (A). P-values for between cryptococcal strain significance tests are non-significant in all cases. P-values are Mann-Whitney test.

Figure 5

Outcomes of interaction between cryptococcal cells and amoebae with inhibition of constitutive exocytosis. Quantification of outcome from three independent 12 h time lapses. Numbers of phagosomes analyzed the same as Figure 4A. (A) Percentage of fungal cells digested by amoebae over 12 h. (B) Percentage of fungal cells that budded while intracellular in amoebae over 12 h. P-values are Fisher's exact test.