Cryptococcus neoformans resists 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. 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 phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is 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 and then standardize the experimental conditions to consistently generate this dormancy in C . neoformans . We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that 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 a 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 mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with 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, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C . neoformans . This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein

“…Then, proteins were digested for 5 h at 30 °C with 500 ng rLys-C Mass Spec Grade (PROMEGA, Madison, WI, USA). Samples were then treated as previously described 71 as well as tryptic peptides analysis 71 with minor changes.…”

“…All data were searched using Andromeda 72 with MaxQuant software 73 , 74 version 1.5.3.8 against Homo sapiens reference proteome from Uniprot (20,399 entries) concatenated with GST-PTPN3-PDZ protein sequence, usual known mass spectrometry contaminants and reversed sequences of all entries as previously described 71 . The “match between runs” feature was applied between replicates with a maximal retention time window of 0.7 min.…”

Dormancy in Cryptococcus neoformans: 60 years of accumulating evidence

“…Overtime, these yeast cells showed a decreased culturability on YPD agar plates, ending with about 1% of the cells still able to grow on agar at Day 8 of incubation. The phenotype observed in the in vivo subpopulation was resumed with delayed growth (increased latency) and low stress response (95). In total, the population obtained was homogenously composed of cells characterized as Viable but non-Culturable cells (VBNC) (Figure 1), phenotype well-known in many bacteria and first described in 1982 in Escherichia coli (105).…”

“…Therefore, the authors worked on an in vitro model to be able to generate a high number of dormant yeast cells. Recently, the authors released and studied the standardized conditions allowing the generation of yeast cells harboring a phenotype close to that dormant cells generated in the lungs of infected mice (95). These conditions are based on a combination of conditions (low oxygen and limited nutrients) inspired from the Wayne and the Loebel models (two well-documented conditions enabling the generation of quiescent M. tuberculosis) (104).…”

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts

“…Some studies have also evidenced this phenomenon in wine yeasts [ 24 , 72 ] and explored the different stress conditions that could lead to the VBNC state in non- Saccharomyces yeasts. Thermosonication in B. bruxellensis [ 73 ] and hypoxic growth conditions in Cryptococcus neoformans [ 74 ] have triggered this state. Moreover, Wang et al [ 24 ] showed that cell-free S. cerevisiae supernatant produced the loss of culturability of Hanseniaspora uvarum , M. pulcherrima and T. delbrueckii , suggesting that the presence of some antimicrobial metabolites could induce the VBNC state.…”