ABSTRACT
Host-microbiome co-evolution drives homeostasis and disease susceptibility, yet regulatory principles governing the integrated intestinal host-commensal microenvironment remain obscure. While inflammasome signaling participates in these interactions, its activators and microbiome-modulating mechanisms are unknown. Here, we demonstrate that the microbiota-associated metabolites taurine, histamine, and spermine shape the host-microbiome interface by co-modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream anti-microbial peptide (AMP) profiles. Distortion of this balanced AMP landscape by inflammasome deficiency drives dysbiosis development. Upon fecal transfer, colitis-inducing microbiota hijacks this microenvironment-orchestrating machinery through metabolite-mediated inflammasome suppression, leading to distorted AMP balance favoring its preferential colonization. Restoration of the metabolite-inflammasome-AMP axis reinstates a normal microbiota and ameliorates colitis. Together, we identify microbial modulators of the NLRP6 inflammasome and highlight mechanisms by which microbiome-host interactions cooperatively drive microbial community stability through metabolite-mediated innate immune modulation. Therefore, targeted "postbiotic" metabolomic intervention may restore a normal microenvironment as treatment or prevention of dysbiosis-driven diseases.
Subject(s)
Colon/immunology , Colon/microbiology , Inflammasomes/immunology , Microbiota , Receptors, Cell Surface/metabolism , Signal Transduction , Animals , Antimicrobial Cationic Peptides , Colitis/chemically induced , Colitis/drug therapy , Colon/metabolism , Dysbiosis/metabolism , Germ-Free Life , Inflammatory Bowel Diseases/chemically induced , Inflammatory Bowel Diseases/drug therapy , Interleukin-18/immunology , Mice , Mice, Inbred C57BL , Receptors, Cell Surface/genetics , Taurine/administration & dosageABSTRACT
The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.
ABSTRACT
Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates.
Subject(s)
G-Quadruplexes , Stress Granules , Humans , DNA/chemistry , RecQ Helicases/metabolism , RNA/genetics , Stress Granules/metabolismABSTRACT
Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
Subject(s)
Aquatic Organisms , Bacteria , Environmental Pollutants , Organophosphorus Compounds , Phosphoric Triester Hydrolases , Aquatic Organisms/enzymology , Bacteria/enzymology , Biodegradation, Environmental , Environmental Pollutants/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Indian Ocean , Mediterranean Sea , Organophosphorus Compounds/metabolism , Phosphoric Triester Hydrolases/genetics , Phosphoric Triester Hydrolases/metabolism , Phosphorus/metabolism , Seawater/microbiologyABSTRACT
Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was KMT2D, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or reinitiation at the main coding sequence. Crosslinking protein-RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5's function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.
Subject(s)
Eukaryotic Initiation Factor-4G/metabolism , Human Embryonic Stem Cells , Histone Methyltransferases/genetics , Histone Methyltransferases/metabolism , Human Embryonic Stem Cells/metabolism , Humans , Open Reading Frames/genetics , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.
Subject(s)
Amino Acids, Diamino , Lathyrus/enzymology , Neurotoxins , Acetyltransferases , Amino Acids, Diamino/metabolism , Molecular Docking SimulationABSTRACT
Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.
Subject(s)
Bacteriolysis , Bacteriophages/physiology , Lysogeny , Amino Acid Sequence , Bacillus/cytology , Bacillus/virology , Bacteriolysis/drug effects , Bacteriophages/drug effects , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/pharmacology , DNA, Viral/metabolism , Lysogeny/drug effects , Models, Biological , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Protein Multimerization , Transcription, Genetic/drug effects , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Proteins/pharmacologyABSTRACT
BACKGROUND: Colonoscopy is the gold standard for evaluation of inflammation in inflammatory bowel diseases (IBDs), yet entails cumbersome preparations and risks of injury. Existing non-invasive prognostic tools are limited in their diagnostic power. Moreover, transcriptomics of colonic biopsies have been inconclusive in their association with clinical features. AIMS: To assess the utility of host transcriptomics of faecal wash samples of patients with IBD compared with controls. METHODS: In this prospective cohort study, we obtained biopsies and faecal-wash samples from patients with IBD and controls undergoing lower endoscopy. We performed RNAseq of biopsies and matching faecal-washes, and associated them with endoscopic and histological inflammation status. We also performed faecal mass-spectrometry proteomics on a subset of samples. We inferred cell compositions using computational deconvolution and used classification algorithms to identify informative genes. RESULTS: We analysed biopsies and faecal washes from 39 patients (20 IBD, 19 controls). Host faecal-transcriptome carried information that was distinct from biopsy RNAseq and faecal proteomics. Transcriptomics of faecal washes, yet not of biopsies, from patients with histological inflammation were significantly correlated to one another (p=5.3×10-12). Faecal-transcriptome had significantly higher statistical power in identifying histological inflammation compared with transctiptome of intestinal biopsies (150 genes with area under the curve >0.9 in faecal samples vs 10 genes in biopsy RNAseq). These results were replicated in a validation cohort of 22 patients (10 IBD, 12 controls). Faecal samples were enriched in inflammatory monocytes, regulatory T cells, natural killer-cells and innate lymphoid cells. CONCLUSIONS: Faecal wash host transcriptome is a statistically powerful biomarker reflecting histological inflammation. Furthermore, it opens the way to identifying important correlates and therapeutic targets that may be obscured using biopsy transcriptomics.
ABSTRACT
The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.
Subject(s)
Host-Pathogen Interactions , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Plant Diseases/microbiology , Plant Proteins/genetics , Protein Kinases/physiology , Xanthomonas , Agrobacterium tumefaciens , Gene Expression Regulation, Plant , MAP Kinase Signaling System/genetics , Organisms, Genetically Modified , Plant Proteins/metabolism , Xanthomonas/enzymology , Xanthomonas/metabolismABSTRACT
Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , MicroRNAs/biosynthesis , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/pathology , Animals , Base Sequence , Cytoplasmic Granules/metabolism , DEAD-box RNA Helicases/metabolism , Down-Regulation , Drug Evaluation, Preclinical , Enoxacin/pharmacology , Female , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , Motor Neurons/metabolism , RNA Interference , RNA Processing, Post-Transcriptional , Ribonuclease III/metabolism , Stress, Physiological , Superoxide Dismutase/genetics , Superoxide Dismutase-1ABSTRACT
During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.
Subject(s)
Bacterial Proteins/genetics , Carbon Dioxide/metabolism , Glucosyltransferases/genetics , Leuconostoc mesenteroides/genetics , Up-Regulation , Bacterial Proteins/metabolism , Daucus carota/microbiology , Dextrans/biosynthesis , Dextrans/genetics , Food Storage , Genes, Bacterial , Glucosyltransferases/metabolism , Leuconostoc mesenteroides/enzymology , PhylogenyABSTRACT
Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.
Subject(s)
Proteomics/methods , Sequence Analysis, Protein/methods , Antibodies/genetics , Chromatography, Liquid , Databases, Protein , Myoglobin/genetics , Sequence Analysis , Serum Albumin, Bovine/genetics , Tandem Mass Spectrometry , alpha-2-HS-Glycoprotein/geneticsABSTRACT
Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.
Subject(s)
Cytoplasmic Granules/metabolism , Intrinsically Disordered Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , ProteolysisABSTRACT
Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.
Subject(s)
Fruit/metabolism , Proteomics/methods , Solanum lycopersicum/metabolism , Fruit/genetics , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Solanum lycopersicum/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Proteome/genetics , Proteome/metabolismABSTRACT
In search of Botrytis cinerea cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the BcXYG1 gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a B. cinerea strain overexpressing BcXYG1 produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.
Subject(s)
Botrytis/enzymology , Glycoside Hydrolases/metabolism , Plant Diseases/microbiology , Plant Immunity , Signal Transduction , Arabidopsis/immunology , Arabidopsis/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Botrytis/genetics , Glycoside Hydrolases/genetics , Solanum lycopersicum/immunology , Solanum lycopersicum/microbiology , Phaseolus/immunology , Phaseolus/microbiology , Plant Diseases/immunology , Plant Leaves/immunology , Plant Leaves/microbiology , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/microbiology , Triticum/immunology , Triticum/microbiologyABSTRACT
The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.
ABSTRACT
Presented is a data set for benchmarking MS1-based label-free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1-intensity-based label-free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data were also used to compare different quantitative protein inference methods such as iBAQ and Hi-N. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385.
Subject(s)
Computational Biology/methods , Gene Expression Regulation/physiology , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Proteomics/methods , Benchmarking/methods , Escherichia coli , HeLa Cells , HumansABSTRACT
The plant pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterium responsible for wilt and canker disease of tomato. While disease development is well characterized and diagnosed, molecular mechanisms of Cmm virulence are poorly understood. Here, we identified and characterized two Cmm transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of Cmm. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. While both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type Cmm and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type Cmm and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for Cmm virulence.
ABSTRACT
The plant pathogen Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterium responsible for wilt and canker disease of tomato. Although disease development is well characterized and diagnosed, molecular mechanisms of C. michiganensis subsp. michiganensis virulence are poorly understood. Here, we identified and characterized two C. michiganensis subsp. michiganensis transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of C. michiganensis subsp. michiganensis. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. Although both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type C. michiganensis subsp. michiganensis and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type C. michiganensis subsp. michiganensis and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal-transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for C. michiganensis subsp. michiganensis virulence.
Subject(s)
Actinomycetales/genetics , Plant Diseases/microbiology , Solanum lycopersicum/microbiology , Transcription Factors/genetics , Actinomycetales/growth & development , Actinomycetales/pathogenicity , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Ethylenes/metabolism , Gene Expression Profiling , Gene Library , Models, Biological , Plant Growth Regulators/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Analysis, RNA , Sequence Deletion , Signal Transduction , Time Factors , Transcription Factors/metabolism , Virulence , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.