Dataset Information

Cryptococcus neoformans resist to drastic conditions by switching to viable but non-culturable cell phenotype

ABSTRACT: Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. In specific infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, with at some point a phase of reactivation, leading to the symptoms of the infection. During murine cryptococcosis and macrophage uptake, stress and host immunity induce C. neoformans heterogeneity with generation of a sub-population of yeasts that has been shown to harbor a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity has been shown to be up regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro model and standardized the experimental conditions to generate dormancy in Cryptococcus neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia during 8 days (8D-HYPOx) were able to mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox with an increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, but proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and fatty acid pathway involving mitochondria is required for the generation and viability maintenance of VBNC. All together, these data show that we were able to generate for the first time VBNC phenotype in Cryptococcocus neoformans. This VBNC state is associated with a reduced and specific metabolism that should be further studied to understand dormancy/quiescence in this organism.

OTHER RELATED OMICS DATASETS IN: GSE118549

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Cryptococcus Neoformans

DISEASE(S): Cryptococcosis

SUBMITTER: Magalie Duchateau

LAB HEAD: Françoise Dromer

PROVIDER: PXD012570 | Pride | 2019-08-08

REPOSITORIES: Pride

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			Action	DRS
	20151203_TC_Q1_colQ1-14_Hommel_D4-Ae1.raw	Raw
	20151203_TC_Q1_colQ1-14_Hommel_D4-Ae2.raw	Raw
	20151203_TC_Q1_colQ1-14_Hommel_D4-Ae3.raw	Raw
	20151203_TC_Q1_colQ1-14_Hommel_D4-Ana1.raw	Raw
	20151203_TC_Q1_colQ1-14_Hommel_D4-Ana2.raw	Raw

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Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

Hommel Benjamin B Sturny-Leclère Aude A Volant Stevenn S Veluppillai Nathanaël N Duchateau Magalie M Yu Chen-Hsin CH Hourdel Véronique V Varet Hugo H Matondo Mariette M Perfect John R JR Casadevall Arturo A Dromer Françoise F Alanio Alexandre A

PLoS pathogens 20190729 7

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a pheno ...[more]

PMID: 31356623

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Project description:The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses, but also elucidated novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress-defense and HOG-dependent genes, which encodes a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system. There is more than 95% genome homology between JEC21 (Cryptococcus neoformans var. neoformans serotype D) and H99 (Cryptococcus neoformans var. grubii serotype A). Therefore, 100 slides of JEC21 70-mer oligo arrays are used in this analysis. 3 biological replicate experiments are performed. Total RNAs are extracted under 3 conditions (1M NaCl, 20ug/ul Fludioxonil, 2.5mM Hydrogen peroxide) at 3 time points (0time, 30min, 60min) with 4 strains from H99 (wild type, hog1Î , ssk1Î , skn7Î). We use a mix of all the total RNAs from this experiment as the control RNA. We use Cy5 as the sample dye and Cy3 as the control dye. Several samples are dye swapped.

Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. total RNAs are extracted 2 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

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