Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State

Rêgo, Marco Túlio Aidar Mariano; Brandão, Fabiana; Pérez, Ana Laura A.; Marco, Janice Lisboa De; Castro, Raffael Júnio Araújo de; Nichols, Connie B.; Alspaugh, J. Andrew; Felipe, Maria Sueli Soares; Alanio, Alexandre; Bocca, Anamélia Lorenzetti; Fernandes, Larissa

doi:10.1128/spectrum.01504-22

Cited by 4 publications

(3 citation statements)

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“…neoformans undergo profound changes during adaptation to the intracellular environment of macrophages [ 46 , 133 ], which may predispose VBNC cells to reactivate. In addition, the phagolysosome may provide a gradual and less deleterious re-exposure of these severely stressed cells [ 47 ] to a richer environment, such as the experimental media. Rich media offer free radicals naturally or as byproducts of their metabolization, thus exacerbating oxidative stress [ 6 , 134 , 135 ].…”

Section: Discussionmentioning

confidence: 99%

“…These cells resemble the dormant yeast cell subpopulation induced during infection of mice and macrophage-like cells [ 46 ], paving the way for in-depth studies on the biology of VBNC cells and their interaction with host cells. VBNC cells exhibit stress-related transcriptional and posttranscriptional regulatory mechanisms, growth latency, and partial reactivation upon the addition of pantothenic acid (PA) [ 14 , 47 ], a vitamin involved in quorum sensing and synthesis of the central metabolic intermediate acetyl-CoA [ 48 , 49 ]. Nevertheless, research on the interplay between VBNC cells and macrophages is still warranted to better understand the mechanisms underlying the development of latent cryptococcal infection and its reactivation at the cellular level [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis

de Castro,

Marina,

Sturny-Leclère

et al. 2023

PLoS Pathog

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Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis

de Castro,

Marina,

Sturny-Leclère

et al. 2023

PLoS Pathog

View full text Add to dashboard Cite

show abstract

“…VBNC yeasts showed differential gene expression mainly in fatty acid metabolism pathways, signal transduction, and proton transport pathways [ 11 ]. Cellular stress pathways were up-regulated in VBNC yeasts, as seen by accumulated stress granules and poly(A) binding protein in the cytoplasm and nucleus, respectively [ 13 ]. Moreover, these yeasts showed a minimal immunostimulatory profile compared to proliferating or stationary phase cells in transcriptomics and cytokine profiling [ 14 ].…”

Section: Viable But Nonculturable State In C ...mentioning

confidence: 99%