UMMS Affiliation

Department of Medicine, Division of Infectious Diseases and Immunology

Publication Date

2019-12-17

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Bacterial Infections and Mycoses | Biochemistry | Enzymes and Coenzymes | Immunology of Infectious Disease | Immunoprophylaxis and Therapy | Microbiology | Molecular Biology

Abstract

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformans

IMPORTANCE Cryptococcus neoformans is a fungal pathogen that kills approximately 200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.

Keywords

Cryptococcus neoformans, cAMP, chitosan, drug screening, flow cytometry

Rights and Permissions

Copyright© 2019 Maybruck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license

DOI of Published Version

10.1128/mBio.02264-19

Source

mBio. 2019 Dec 17;10(6). pii: mBio.02264-19. doi: 10.1128/mBio.02264-19. Link to article on publisher's site

Journal/Book/Conference Title

mBio

Related Resources

Link to Article in PubMed

PubMed ID

31848271

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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