Proteome analysis of Klebsiella pneumoniae under infection-mimicking conditions
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ABSTRACT: Whole bacterial proteome and secretome analysis of Klebsiella pneumoniae under the infection-mimicking conditionsoxidative stress and heat stress in chemically defined medium
Project description:To understand how microRNAs are involved in stress response, we examined their expression changes in C. elegans animals that were exposed to stress conditions, including heat shock, oxidation, hypoxia and starvation. Total RNAs were purified from young adult animals that were exposed to each stress, and used for cDNA library preparation for small RNAs. In this experiment, spe-9(hc88), a temperature sensitive sterile mutant, that were cultured at 23dC, was used in order to avoid the effect from developing embryos. Stress conditions we examined include: Heat shock (32M-BM-0C, 6 hrs), Recovery from heat shock (6 hrs recovery at 23M-BM-0C after heat shock treatment at 32M-BM-0C for 6 hrs), Hypoxia (0.01%, 6 hrs), Oxidation (Juglone 750 M-NM-<M, 6 hrs), Starvation (complete food deprivation, 12 hrs). In addition to these stress conditions, RNAs were prepared from normally cultured, untreated animals at three time points, 0 hr (baseline), 6 hrs (as controls for heat shock, hypoxia and oxidation) and 12 hrs (as controls for heat shock recovery and starvation) after starting stress exposure. These cDNA libraries established were sequenced with Illumina Genome Analyzer II.
Project description:High-temperature stress is a critical environmental parameter for all organisms and induces a cellular heat shock response, characterized by an upregulation of heat shock proteins. For Crenarchaeota, it is unknown how this response is regulated, given the lack of conservation of known prokaryotic or eukaryotic heat shock-regulating transcription factors and how cellular processes are affected. To this end, we studied the heat shock response of the thermoacidophilic Sulfolobus acidocaldarius, thriving in volcanic hot springs at 75°C, upon shift to 86°C, at the transcriptome and proteome level. By pulse-labeling of neosynthesized RNA and proteins upon heat shock, we show a drastic reduction of the cell’s major transcriptional activity immediately after shift and neosynthesis of certain abundant (heat shock) proteins, including the thermosome. By combining RNA-sequencing and mass spectrometry, we show that RNA levels of half of the genes are affected immediately after temperature shift and that reprogramming of the protein landscape takes at least one hour and changes are more subtle. Correlation analysis provides evidence that considerable post-transcriptional and post-translational regulation is occurring upon heat shock. Functional enrichment analysis indicates that nearly all biological processes are affected by heat shock. An overall downregulation is observed for transcription, translation, DNA replication and cell division. However, quality control of translation and of the protein landscape is increased. Many DNA repair pathways are differentially expressed, accompanied with an increase in a DNA import system. Furthermore, expression of DNA topoisomerases is investigated, coinciding with a significant differential expression of nucleoid-associated proteins. Based on these findings and considering the absence of a conserved DNA-binding motif, we propose that regulation of the heat shock response in Sulfolobales is most likely not established by a classical transcription factor, but is rather caused by changes in chromosome organization or established at the post-transcriptional level.
Project description:We analyzed ~27nt small RNAs from Entamoeba histolytica trophozoites in basal conditions and after heat shock or oxidative stress E. histolytica trophozoites were treated with 1mM H2O2 for 1hr, or heat shocked at 42°C for 1hr and RNA was isolated and small RNA populations were compared to small RNA populations from untreated trophozoites
Project description:Within a GRO experiment, samples for mRNA amount (RA) were taken at 0, 4, 11, 16, 26 and 40 minutes after heat stress (from 25ºC to 37ºC). Analysis was done in filters also used for Transcription rate (TR) analysis of aliquots from the same cultures. The analysis includes 3 repeats of stress treatment of the W3030-1a strain. The same times were used for sampling in each repeat of the experiment. A single genomic DNA hybridization on every one of the filters is used for normalization between gene probes.
Project description:The fungal pathogen Histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. Here we provide the first genome-wide studies to experimentally validate its genome annotation. A functional interrogation of the Histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of H. capsulatum and related fungi. We employed a three-pronged approach to provide a functional annotation for the H. capsulatum G217B strain. First, we probed high-density tiling arrays with labeled cDNAs from cells grown under diverse conditions. These data defined 6,172 transcriptionally active regions (TARs), providing validation of 6,008 gene predictions. Interestingly, 22% of these predictions showed evidence of anti-sense transcription. Additionally, we detected transcription of 264 novel genes not present in the original gene predictions. To further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. These expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional 2,249 gene predictions. Finally, we compared the G217B gene predictions to other available fungal genomes, and observed that an additional 254 gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences. These analyses yielded a high confidence set of validated gene predictions for H. capsulatum. The transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. The data is available for interactive exploration at http://histo.ucsf.edu. The non-redundant genome sequence of Histoplasma capsulatum G217B was tiled over a set of 93 CombiMatrix arrays, which were then hybridized with labeled cDNA samples from yeast-form (red channel) or mycelial-form (green channel) Histoplasma. Due to low yields from the mycelial samples, only the red channel intensities were analyzed (and the red foreground intensities are, therefore, reported in the VALUE column for each sample). Rather than normalizing intensities across arrays, each probe was evaluated as detected or undetected relative to the negative control intensities on the corresponding array, and the density of detected probes as a function of genome position was used for the remaining analysis.
Project description:This SuperSeries is composed of the following subset Series: GSE24483: TR heat-shock GSE24484: RA heat-shock Refer to individual Series
Project description:15N-labeled N-acetylglucosamine (NAG) fed to B. infantis cells are incorporated into its proteome. This indicates that NAG, an amino sugar residue of human milk oligosaccharides (HMO) and other biopolymers, is used as a nitrogen source. Transcriptomics while subsisting on NAG nitrogen are consistent with the proteomics results. This further indicates that B. infantis utilizes NAG nitrogen in and shunts it towards a fundamental cellular processes.
Project description:C-Terminal cyclic imides are post-translational modifications (PTMs) that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that this class of aging-related modifications requires cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase (GSS) that are susceptible to aggregation if they are not removed by CRBN. This systematic investigation provides insight to the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on the protein stability, and the biological role of CRBN.
Project description:Phenotypic cell-to-cell variability is central for microbial populations and contributes to cell function, stress adaptation and drug resistance. Gene-expression heterogeneity underpins this variability, but has been challenging to study genome-wide. Here, we report a novel approach which combines imaging of individual fission yeast cells with single-cell RNA sequencing (scRNA-seq) and Bayesian normalisation. We analyse >2000 single cells and >700 matching RNA controls in various environmental conditions that include reposnse to various stresses as well as growth and entry into stationary phase.
Project description:Within a GRO experiment, samples for Transcription rate (TR) analysis were taken at 0, 4, 11, 16, 26 and 40 minutes after heat stress (from 25ºC to 37ºC) and subjected to run-on. Analysis was done in filters also used for RA analysis of aliquots from the same cultures. The analysis includes 3 repeats of stress treatment of the W3030-1a strain. The same times were used for sampling in each repeat of the experiment. A single genomic DNA hybridization on every one of the filters is used for normalization between gene probes.