Surface Localization of Glucosylceramide during Cryptococcus neoformans Infection Allows Targeting as a Potential Antifungal

Abstract

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO2). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.

Sphingolipids as Targets for Treatment of Fungal Infections

“…However, whether this effect is due directly to the inhibition of fungal GCS1 or to an off-target effect is not known. In C. neoformans, for instance, these inhibitors have no significant effect against C. neoformans GCS1 [61].…”

“…Interestingly, deletion of gcs1 in C. neoformans results in a dramatic growth defect at neutral/alkaline pH, 5% CO 2 and 37°C [15], suggesting that GlcCer may be important in fungi to promote alkaline tolerance [60]. When exposed to a neutral/alkaline environment, C. neoformans cells do concentrate GlcCer at the plasma membrane/cell wall/bud necks without upregulating its synthesis [61]. This phenomenon was observed not only during in vitro growth, but also in cells recovered ex vivo from murine infection model [61].…”

“…When exposed to a neutral/alkaline environment, C. neoformans cells do concentrate GlcCer at the plasma membrane/cell wall/bud necks without upregulating its synthesis [61]. This phenomenon was observed not only during in vitro growth, but also in cells recovered ex vivo from murine infection model [61]. This redistribution of GlcCer at fungal membranes under these physiological environments (neutral/alkaline pH) may be important for proper function of specific transmembrane transporters [62], which may be particularly important for pro moting fungal cell replication at neutral and alkaline pH.…”

Vesicular Transport Systems in Fungi

“…GlcCer is an important regulator of cryptococcal pathogenicity that is also targeted by antimicrobial antibodies [29–33]. Remarkably, this lipid appeared to be distributed into C. neoformans cell wall compartments resembling vesicular structures [29].…”

Role of Glucose in the Expression of Cryptococcus neoformans Antiphagocytic Protein 1, App1

“…For example, melanin production is induced during glucose starvation due to the lowglucose lung environment, and capsule production is stimulated by iron deprivation and by a physiological concentration of CO 2 (5%). Mating is induced by nitrogen starvation (1,48), and the sphingolipid glucosylceramide is localized mainly on the cell surface at neutral/alkaline pH (38). Thus, fungi can interpret and respond to temperature, pH, gases, and nutrients and adapt to new environments through modified biological responses (3).…”