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Gene expression profiling of murine macrophages in response to infection with Cryptococcus neoformans yeast cells at distinct physiological states, including the dormant state known as viable but non-culturable (VBNC)

ABSTRACT: Macrophages play a pivotal role in determining the fate of invading Cryptococcus neoformans cells, and this outcome is highly dependent on how the pathogen modulates phagocyte activation and function. A common consequence of the C. neoformans-host interaction is the establishment of a latent infection, in which the yeast enters a dormant state. In this study, we investigated whether dormant cells could modulate murine bone marrow-derived macrophages (BMDMs) differently from non-dormant cells, thereby providing mechanistic insights into latent infection. To accomplish this, we examined the transcriptional responses of BMDMs upon in vitro infection with distinct physiological states of C. neoformans – exponential phase, stationary phase, or viable but non-culturable (VBNC; equivalent of dormant) cells – over a period of 6 hours. Our findings unveiled a relationship between the physiological state of the yeast and its impact on the transcriptome of BMDMs. Compared to non-dormant cells, particularly those in the logarithm phase, VBNC cells trigger a modest disruption in the host transcriptome, regulating a reduced number of genes, which clustered in far fewer Gene Ontology terms, many of which were unique to this specific condition. Nevertheless, VBNC cells retained the ability to regulate genes that are important for immune response, such as NLRP3 inflammasome-related genes. Our results suggest that VBNC cells retain the low immunostimulatory profile that is critical for the persistence of this fungal pathogen within the host.

ORGANISM(S): Mus musculus

PROVIDER: GSE243519 | GEO | 2023/11/23

REPOSITORIES: GEO

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Project description: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.

Project description:Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by ‘non-culturability’ – a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, ‘non-culturable’ M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

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Gene expression profiling of murine macrophages in response to infection with Cryptococcus neoformans yeast cells at distinct physiological states, including the dormant state known as viable but non-culturable (VBNC)

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