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Surface localization of glucosylceramide during Cryptococcus neoformans infection allows targeting as a potential
Ryan Rhome1, Arpita Singh, Talar Kechichian
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
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 CO(2)). 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.
Insights
Targeting glucosylceramide (GlcCer) synthesis in Cryptococcus neoformans (Cn) offers a novel therapeutic strategy. Inhibiting GlcCer with Cerezyme (Cz) reduced fungal growth and improved survival in infected mice, highlighting GlcCer as a potential drug target.
Area of Science:
- Mycology
- Infectious Diseases
- Drug Discovery
Background:
- Cryptococcus neoformans (Cn) causes significant morbidity, particularly in sub-Saharan Africa, necessitating novel therapeutic approaches.
- Current treatments for Cn infections are limited, and there is a clear need for more tolerable and specific therapies.
- The gene encoding glucosylceramide synthase (GCS1) deletion in Cn results in an avirulent strain (Δgcs1) lacking glucosylceramide (GlcCer), suggesting its role in virulence.
Purpose of the Study:
- To characterize GlcCer production and localization in Cn under conditions mimicking the lung environment during infection.
- To investigate the potential of targeting GlcCer metabolism with Cerezyme (Cz) as a therapeutic strategy against Cn infections.
Main Methods:
- Utilized immunofluorescence to assess GlcCer localization during growth in restrictive conditions (neutral pH, high CO2) and during infection.
- Administered Cerezyme (Cz) to in vitro cultures and infected mice to evaluate its effect on GlcCer levels, fungal growth, and host survival.
- Assessed membrane integrity defects in Cn cells following Cz treatment.
Main Results:
- GlcCer cell surface localization significantly increased in Cn grown under simulated lung conditions and during infection.
- Cerezyme (Cz) treatment effectively reduced GlcCer levels in vitro, in cultures, and in the lungs of infected mice.
- Cz treatment induced membrane integrity defects in wild-type Cn, inhibited fungal growth in a dose- and condition-dependent manner, and conferred a protective effect on survival in a mouse model of Cn infection.
Conclusions:
- Glucosylceramide (GlcCer) is crucial for Cryptococcus neoformans (Cn) virulence and its cell surface localization is enhanced under host-relevant conditions.
- Targeting the GlcCer pathway, for example, by using Cerezyme (Cz), represents a promising therapeutic strategy for treating Cn infections.
- Sphingolipid metabolism, particularly GlcCer, is a viable target for the development of novel anti-fungal therapeutics.
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