Project description:Rationale: Tuberculosis has a devastating impact on global health by claiming nearly 1.4 million lives each year. During infection Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, produces heterogenous populations some of which don’t produce colonies on agar but grow in liquid media and often require supplementation with culture supernatants or recombinant Resuscitation-promoting factor, thus defined as differentially culturable bacilli. Objectives: to evaluate whether exposure to nitric oxide (NO), a well-known host defence molecule, alters mycobacterial growth phenotypes and drives generation of Rpf-dependent differentially culturable bacilli. Methods: a novel NO donor was synthesised and tested against Mtb and Mycobacterium bovis BCG in vitro, followed by growth assays, flow cytometry analysis and assessment of transcriptomic responses. Resuscitation-promoting factor (Rpf) inhibitors were used to characterise the role of Rpf proteins in the reactivation of NO-treated mycobacteria. Mycobacterial phenotypes were also investigated during infection of THP-1 macrophages activated with retinoic acid and vitamin D3. Measurements and Main Results: differentially culturable mycobacteria were generated after exposure to the novel NO donor or during infection of activated THP-1 cells. Resuscitation of these differentially culturable bacilli was largely abolished by specific Rpf inhibitors. Transcriptomic analysis revealed redox-associated stress signatures mediated by SigH and SigF, with significant down-regulation of ribosome and cell wall architecture genes, including rpfA, rpfB and rpfE, and induction of genes involved in response to thiol stress, sulphur metabolism and iron acquisition. Conclusion: Our study provides mechanistic insights into the generation of Rpf-dependent Mtb during tuberculosis and outlines a critical role of NO in this process.
Project description:Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to phenotypic diversity, its interaction with genetic variation requires further investigation. MethylC-seq from naturally-occurring Arabidopsis accessions
Project description: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.
Project description: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. VBNC state gene expression was detected in total bacterial RNA of V. parahaemolyticus. Three phases (exponential phase, early stationary phase, and VBNC state) were used in 8 biological replicates. Gene expression in exponential phase and early stationary phase was used for normalization, respectively.
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.