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Changes in global gene expression of Vibrio parahaemolyticus viable but non-culturable (VBNC) state

ABSTRACT: The phenomenon of viable but non-culturable (VBNC) referred as a dormant state of non-sporulating bacteria enhancing the survival in adverse environments. To our knowledge, only few studies have been performed on whole genomic expression of V. parahaemolyticus in VBNC state compared with cells in exponential and early stationary phases. Since many VBNC state studies found DNA, RNA and protein degradation, we hypothesise that gene regulation of VBNC cells is highly reduced, down-regulation of gene expression is dominant and only metabolic functions crucial for survival are kept on a sustained basis. In the VBNC state we found 509 significantly induced genes and 309 significantly repressed by more than twofold compared with unstressed phases among 4820 investigated genes (adjusted P-value < 0.05). Furthermore, up-regulation was dominant in most of the non-metabolism functional categories, while five metabolism-related functional categories revealed down-regulation in the VBNC state. To our knowledge, this is the first study of comprehensive transcriptomic analyses of three phases of V. parahaemolyticus RIMD2210633. Although the mechanism of VBNC state is not yet clear, massive regulation of gene expression occurs in the VBNC state compared with expression in unstressed phases and thus, VBNC cells are active cells.

ORGANISM(S): Vibrio parahaemolyticus RIMD 2210633

PROVIDER: GSE65340 | GEO | 2015/01/28

SECONDARY ACCESSION(S): PRJNA273733

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.

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Changes in global gene expression of Vibrio parahaemolyticus viable but non-culturable (VBNC) state

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