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.

The endocytic pathways of Dictyostelium discoideum

“…It should be noted that in general, biochemical analysis of purified phagosomes often indicates slower maturation than that observed by live-cell imaging, with maturation occuring over an almost 3 hour period (Buckley et al, 2016;Gopaldass et al, 2012;Gotthardt et al, 2002a). Microscopy studies in contrast, indicate exocytosis of yeast and bead-containing phagosomes, as well as macropinosomes occurs at around 60-90 minutes after engulfment (Clarke et al, 2010;Lima et al, 2012;Ravanel et al, 2001;Watkins et al, 2018). We have consolidated and corollated data from both techniques to an "average" 60-minute transit.…”

“…Although environmental isolates of Dictyostelium are strictly bacterivorous and cannot generate phagocytic cups large enough to engulf fungi, axenic laboratory strains can ingest larger organisms (Bloomfield et al, 2015). These strains have therefore also been used to study fungal infections (Hillmann et al, 2015;Koller et al, 2016;Watkins et al, 2018). As many pathogens also have an environmental niche, avoiding predation by amoebae such as Dictyostelium or the more common water amoeba Acanthamoeba castellanii has been proposed to be an important evolutionary driver of virulence traits -further validating the use of these organisms to study host-pathogen interactions (Casadevall et al, 2019;Molmeret et al, 2005).…”

Dinoflagellate symbionts escape vomocytosis by host cell immune suppression

“…In this context, it is noteworthy that unicellular, phagotrophic amoeba have two redundant, yet mechanistically distinct expulsion mechanisms: constitutive exocytosis and vomocytosis (Watkins et al, 2018). Constitutive exocytosis results in the expulsion of undigested remnants of food;…”

Vomocytosis: What we know so far

“…Amoebae are also able to vomocytose C. neoformans (Chrisman, Alvarez, & Casadevall, 2010), although it should be noted that amoebae, unlike macrophages, also have a constitutive exocytosis pathway to release indigestible material (Watkins et al, 2018). This has led to the proposal that the capacity C. neoformans has for mammalian infection evolved in response to selective pressure exerted during interactions with such phagocytic predators.…”

“…This has led to the proposal that the capacity C. neoformans has for mammalian infection evolved in response to selective pressure exerted during interactions with such phagocytic predators. Amoebae are also able to vomocytose C. neoformans (Chrisman, Alvarez, & Casadevall, 2010), although it should be noted that amoebae, unlike macrophages, also have a constitutive exocytosis pathway to release indigestible material (Watkins et al, 2018). To date, vomocytosis has not been directly evidenced in a mammalian host.…”