RESUMO
BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.
Assuntos
Proteínas de Bactérias/genética , Biologia Computacional/métodos , Genes Essenciais , Peste/microbiologia , Yersinia pestis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Mutação , Yersinia pestis/crescimento & desenvolvimento , Yersinia pestis/metabolismoRESUMO
Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality.
Assuntos
DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Microbiologia Industrial/métodos , Biologia Molecular/métodos , RNA Fúngico/isolamento & purificação , Leveduras/química , Leveduras/genética , DNA Mitocondrial/genética , Citometria de Fluxo/métodos , Dosagem de Genes , Reprodutibilidade dos TestesRESUMO
All previous studies on the yeast metabolome have yielded a plethora of information on the components, function and organisation of low molecular mass and macromolecular components involved in the cellular metabolic network. Here we emphasise that an understanding of the global dynamics of the metabolome in vivo requires elucidation of the temporal dynamics of metabolic processes on many time-scales. We illustrate this using the 40 min oscillation in respiratory activity displayed in auto-synchronous continuously grown cultures of Saccharomyces cerevisiae, where respiration cycles between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Thereby an ultradian clock, i.e. a timekeeping device that runs through many cycles during one day, is involved in the co-ordination of the vast majority of events and processes in yeast. Through continuous online measurements, we first show that mitochondrial and redox physiology are intertwined to produce the temporal landscape on which cellular events occur. Next we look at the higher order processes of DNA duplication and mitochondrial structure to reveal that both events are choreographed during the respiratory cycles. Furthermore, spectral analysis using the discrete Fourier transformation of high-resolution (10 Hz) time-series of NAD(P)H confirms the existence of higher frequency components of biological origin and that these follow a scale-free architecture even in stable oscillating modes. A different signal-processing approach using discrete wavelet transformations (DWT) indicates that there is a significant contribution to the overall signal from ` ~5, ~ 10 and ~ 20-minutes cycles and the amplitudes of these cycles are phase-dependent. Further investigation (derivative of Gaussian continuous wavelet transformation) reveals that the observed 20-minutes cycles are actually confined to the reductive phase and consist of two ~15-minutes cycles. Moreover, the 5 and 10-minutes cycles are restricted to the oxidative phase of the cycle. The mitochondrial origin of these signals was confirmed by pulse-injection of the cytochrome c oxidase inhibitor H(2)S. We next discuss how these multi-oscillatory states can impinge on the apparently complex reactome (represented as a phase diagram of 1,650 chemical species that show oscillatory behaviour). We conclude that biological processes can be considerably more comprehensible when dynamic in vivo time-structure is taken into account.
Assuntos
Ciclo Celular/fisiologia , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/metabolismo , Citometria de Fluxo , Fase G1/fisiologia , Fase G2/fisiologia , Cinética , Microscopia Eletrônica , Modelos Biológicos , Oxirredução , Fase de Repouso do Ciclo Celular/fisiologia , Fase S/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
A comparative study of existing junction-primer-designing software revealed many limitations among them. Hence, we developed a new computational program, Ex-Ex Primer, which offers many improved, user-friendly features, and reliably creates junction primers and probes. This online suite can also be used to design primers/probes from other sites of nucleic acid recombination, insertion, deletion, or splicing, and regular probes/primers. The threshold for Tm difference between the complete junctional primer vs its partial sequence, which maps to one of the junctional regions, was changed based on an important observation made during the initial experimental validations. The tool is now thoroughly checked with RT-PCR and RT-qPCR experiments with more than 250 primer pairs over a few years. The junction-primer-designing features of the software are also better than other equivalent tools. Visualizing the exons and introns across transcripts, and enabling primer designing based on information from Ensembl, are some of the unique features of this tool. The primers suggested by the tool can be used to detect the expression of known transcripts, to test the existence of predicted DNA or RNA joints via hybridization-based techniques, or for validation and in silico analysis of RNA-Seq. URL: http://resource2.ibab.ac.in/exprimer/.
Assuntos
Ácidos Nucleicos , Oligonucleotídeos , Primers do DNA/genética , Reação em Cadeia da Polimerase , SoftwareRESUMO
Viruses rely on their host for reproduction. Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction. Some of the catalysing enzymes of such reactions have demonstrated interactions with existing drugs, which can be used for experimental testing of the presented predictions using gene knockouts and RNA interference techniques. In summary, the developed computational approach offers a platform for rapid, experimentally testable generation of drug predictions against existing and emerging viruses based on their biomass requirements.
Assuntos
Interações Hospedeiro-Patógeno , Pulmão , SARS-CoV-2 , Replicação Viral , Antivirais/farmacologia , Biomassa , COVID-19/prevenção & controle , COVID-19/virologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicólise/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Pulmão/citologia , Pulmão/metabolismo , Análise do Fluxo Metabólico , Modelos Biológicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Biologia de Sistemas , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.
Assuntos
Metabolismo Energético , Saccharomyces cerevisiae/crescimento & desenvolvimento , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Montagem e Desmontagem da Cromatina , Análise por Conglomerados , DNA/metabolismo , Dinitrocresóis/química , Eletroforese Capilar , Cromatografia Gasosa-Espectrometria de Massas , Mitocôndrias/química , Mitocôndrias/metabolismo , NAD/química , Oxirredução , Espécies Reativas de Oxigênio/química , Saccharomyces cerevisiae/metabolismo , TranscriptomaRESUMO
There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.