The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>

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 co...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Brian T. Maybruck, Woei C. Lam, Charles A. Specht, Ma. Xenia G. Ilagan, Maureen J. Donlin, Jennifer K. Lodge
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Cryptococcus neoformans cAMP chitosan drug screening flow cytometry Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b3cde10d9e614e4c8f1c4ec327805652
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:b3cde10d9e614e4c8f1c4ec327805652
record_format	dspace
spelling	oai:doaj.org-article:b3cde10d9e614e4c8f1c4ec3278056522021-11-15T15:54:45ZThe Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>10.1128/mBio.02264-192150-7511https://doaj.org/article/b3cde10d9e614e4c8f1c4ec3278056522019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02264-19https://doaj.org/toc/2150-7511ABSTRACT 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 ∼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.Brian T. MaybruckWoei C. LamCharles A. SpechtMa. Xenia G. IlaganMaureen J. DonlinJennifer K. LodgeAmerican Society for MicrobiologyarticleCryptococcus neoformanscAMPchitosandrug screeningflow cytometryMicrobiologyQR1-502ENmBio, Vol 10, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cryptococcus neoformans cAMP chitosan drug screening flow cytometry Microbiology QR1-502
spellingShingle	Cryptococcus neoformans cAMP chitosan drug screening flow cytometry Microbiology QR1-502 Brian T. Maybruck Woei C. Lam Charles A. Specht Ma. Xenia G. Ilagan Maureen J. Donlin Jennifer K. Lodge The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
description	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 ∼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.
format	article
author	Brian T. Maybruck Woei C. Lam Charles A. Specht Ma. Xenia G. Ilagan Maureen J. Donlin Jennifer K. Lodge
author_facet	Brian T. Maybruck Woei C. Lam Charles A. Specht Ma. Xenia G. Ilagan Maureen J. Donlin Jennifer K. Lodge
author_sort	Brian T. Maybruck
title	The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
title_short	The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
title_full	The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
title_fullStr	The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
title_full_unstemmed	The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>
title_sort	aminoalkylindole bml-190 negatively regulates chitosan synthesis via the cyclic amp/protein kinase a1 pathway in <named-content content-type="genus-species">cryptococcus neoformans</named-content>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/b3cde10d9e614e4c8f1c4ec327805652
work_keys_str_mv	AT briantmaybruck theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT woeiclam theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT charlesaspecht theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT maxeniagilagan theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT maureenjdonlin theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT jenniferklodge theaminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT briantmaybruck aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT woeiclam aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT charlesaspecht aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT maxeniagilagan aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT maureenjdonlin aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent AT jenniferklodge aminoalkylindolebml190negativelyregulateschitosansynthesisviathecyclicampproteinkinasea1pathwayinnamedcontentcontenttypegenusspeciescryptococcusneoformansnamedcontent
_version_	1718427238305628160

The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in <named-content content-type="genus-species">Cryptococcus neoformans</named-content>

Ejemplares similares