Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the ? subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).
Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to ?-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E.?coli?L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.
Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.
In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations.
Pseudomonas aeruginosa bacteriophage ?KZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ? 280-kb ?KZ genome to single-nucleotide resolution, we combine 369 ?KZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ?KZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ?KZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ?KZ genome is performed by the consecutive action of two ?KZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus.
The aim of this study was to assess the impact of nanocrystalline diamond (NCD) thin coatings on neural cell adhesion and proliferation. NCD was fabricated on fused silica substrates by microwave plasma chemical vapor deposition (MPCVD) method. Different surface terminations were performed through exposure to reactive hydrogen and by UV induced oxidation during ozone treatment. Boron doped NCD coatings were also prepared and investigated. NCD surface wettability was determined by contact angle measurement. To assess biocompatibility of the NCD coatings, the neuroblastoma SH-SY5Y cell line was used. Cells were plated directly onto diamond surfaces and cultured in medium with or without fetal bovine serum (FBS), in order to evaluate the ability of cells to adhere and to proliferate. The obtained results showed that these cells adhered and proliferated better on NCD surfaces than on the bare fused silica. The cell proliferation on NCD in medium with and without FBS after 48h from plating was on average, respectively, 20 and 58% higher than that on fused silica, irrespective of NCD surface modification. Our results showed that the hydrogenated, oxygenated and boron-doped NCD coatings can be used for biomedical purposes, especially where good optical transparency is required.
Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications.
This meta-analysis focuses on plant-proteome responses to cadmium (Cd) stress. Initially, some general topics related to a proteomics meta-analysis are discussed: (1) obstacles encountered during data analysis, (2) a consensus in proteomic research, (3) validation and good reporting practices for protein identification and (4) guidelines for statistical analysis of differentially abundant proteins. In a second part, the Cd responses in leaves and roots obtained from a proteomics meta-analysis are discussed in (1) a time comparison (short versus long term exposure), and (2) a culture comparison (hydroponics versus soil cultivation). Data of the meta-analysis confirmed the existence of an initial alarm phase upon Cd exposure. Whereas no metabolic equilibrium is established in hydroponically exposed plants, an equilibrium seems to be manifested in roots of plants grown in Cd-contaminated soil after long term exposure. In leaves, the carbohydrate metabolism is primarily affected independent of the exposure time and the cultivation method. In addition, a metabolic shift from CO2-fixation towards respiration is manifested, independent of the cultivation system. Finally, some ideas for the improvement of proteomics setups and for comparisons between studies are discussed.
Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.
White adipose tissue (WAT) regulates energy metabolism by secretion of proteins with endocrine and paracrine effects. Dysregulation of the secretome of obesity-associated enlarged WAT may lead to obesity-related disorders. This can be caused by hypoxia as a result of poorly vascularized WAT. The effect of hypoxia on the secretome of human (pre)adipocytes is largely unknown. Therefore, we investigated the effect of CoCl2, a hypoxia mimetic, on the secretome of human SGBS (pre)adipocytes by a proteomics approach combined with bioinformatic analysis. In addition, regulation of protein secretion was examined by protein turnover experiments. As such, secretome changes were particularly associated with protein down-regulation and extracellular matrix protein dysregulation. The observed up-regulation of collagens in adipocytes may be essential for cell survival while down-regulation of collagens in preadipocytes may indicate a disturbed differentiation process. These CoCl2-induced changes reflect WAT dysfunction that ultimately may lead to obesity-associated complications. In addition, 9 novel adipocyte secreted proteins were identified from which 6 were regulated by CoCl2. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000162.
Recently, we identified the mimotope UH-CIS6 as a novel candidate antibody target for clinically isolated syndrome (CIS) and relapsing-remitting (RR) multiple sclerosis (MS). The purpose of this study was to further validate UH-CIS6 as an antibody target for CIS and MS and to identify the in vivo antibody target of UH-CIS6. First, a UH-CIS6 peptide ELISA was optimized. Next, we investigated the antibody response toward UH-CIS6 in cerebrospinal fluid (CSF) from patients with CIS (n = 20), MS (n = 43) and other neurological diseases (n = 42). Immunoprecipitation of anti-UH-CIS6 antibodies on a normal human brain lysate was performed to identify the in vivo antibody target of UH-CIS6. The cellular expression of an in vivo candidate target was investigated by immunohistochemistry using MS brain tissue sections. Antibody reactivity toward UH-CIS6 was detected in a significantly increased proportion of CSF samples from CIS and RR-MS patients as compared with neurological controls (p = 0.046). We identified and confirmed coronin-1a as the in vivo antibody target for UH-CIS6. Furthermore, coronin-1a was expressed by T cells and macrophages in an active MS lesion. Together, these results demonstrate that coronin-1a is a novel antibody target for CIS and MS.
Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscles proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions.
Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as the gold standard. However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.
The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.
Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive.
The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the Twort-like viruses. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.
Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ?15, a T7-like virus with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ?15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ?15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ?15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.
In Pseudomonas aeruginosa the response to oxidative stress is orchestrated by the LysR regulator OxyR by activation of the transcription of two catalase genes (katA and katB), of the alkyl-hydroxyperoxidases ahpCF and ahpB. Next to the expected high sensitivity to oxidative stress generated by reactive oxygen species (ROS: H(2)O(2), O(2)(-)), the oxyR mutant shows a defective growth under conditions of iron limitation (Vinckx et al. 2008). Although production and uptake of the siderophore pyoverdine is not affected by the absence of oxyR, the mutant is unable to satisfy its need for iron when grown under iron limiting conditions. In order to get a better insight into the effects caused by iron limitation on the physiological response of the oxyR mutant we decided to compare the proteomes of the wild type and the mutant grown in the iron-poor casamino acids medium (CAA), in CAA plus H(2)O(2), and in CAA plus the strong iron chelator ethylenediamine-N,N-bis(2-hydroxyphenylacetic acid) (EDDHA). Especially in the presence of hydrogen peroxide the oxyR cells increase the production of stress proteins (Dps and IbpA). The superoxide dismutase SodM is produced in higher amounts in the oxyR mutant grown in CAA plus H(2)O(2). The PchB protein, a isochorismate-pyruvate lyase involved in the siderophore pyochelin biosynthesis is not detectable in the extracts from the oxyR mutant grown in the presence of hydrogen peroxide. When cells were grown in the presence of EDDHA, we observed a reduction of the ferric uptake regulator (Fur), and an increase in the two subunits of the succinyl-CoA synthetase and the fumarase FumC1.
Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery.
Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.
We present a detailed analysis of the genome architecture, structural proteome and infection-related properties of three Pseudomonas phages, designated LUZ7, LIT1 and PEV2. These podoviruses encapsulate 72.5 to 74.9 kb genomes and lyse their host after 25 min aerobic infection. PEV2 can successfully infect under anaerobic conditions, but its latent period is tripled, the lysis proceeds far slower and the burst size decreases significantly. While the overall genome structure of these phages resembles the well-studied coliphage N4, these Pseudomonas phages encode a cluster of tail genes which displays significant similarity to a Pseudomonasaeruginosa (cryptic) prophage region. Using ESI-MS/MS, these tail proteins were shown to be part of the phage particle, as well as ten other proteins including a giant 370 kDa virion RNA polymerase. These phages are the first described representatives of a novel kind of obligatory lytic P. aeruginosa-infecting phages, belonging to the widespread "N4-like viruses" genus.
We have applied our recently designed proteomics apparoach to search for protein changes in the in vivo adipocyte-enriched proteome from 8 overweight/obese subjects who underwent an intervention of 5 weeks of a very low calorie diet followed by 3 weeks of a normal diet. On average, persons lost 9.5 kg body weight largely contributed by the loss of fat mass (7.1 kg). Various parameters of adiposity and lipid metabolism changed significantly. Proteomics analysis revealed 6 significantly changed proteins. Analysis indicates that fructose-bisphosphate aldolase C and tubulin beta 5 are potential biomarkers for the present intervention. Further, identified proteins indicate a reduced intracellular scaffolding of GLUT4 (ALDOC, TUBB5, ANXA2), an increased uptake of fatty acids (FABP4), an improved inflammatory profile of the adipose tissue (ApoA1, AOP1) and a change in fat droplet organization (vimentin). Correlation analysis between changes in protein spot intensities and parameters of adiposity or lipid metabolism points to a link between aldo-ketoreductase 1C2 and parameters of adiposity, between FABP4 and parameters of lipid metabolism, and between proteins for beta-oxidation (HADH, ACADS, ACAT1) and FFA levels. Altogether, our findings underscore the potential value of in vivo proteomics for human intervention studies.
The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes.
We examined the genetic diversity of lytic Pseudomonas aeruginosa bacteriophage PB1 and four closely related phages (LBL3, LMA2, 14-1 and SN) isolated throughout Europe. They all encapsulate linear, non-permuted genomes between 64 427 and 66 530 bp within a solid, acid-resistant isometric capsid (diameter: 74 nm) and carry non-flexible, contractile tails of approximately 140 nm. The genomes are organized into at least seven transcriptional blocks, alternating on both strands, and encode between 88 (LBL3) and 95 (LMA2) proteins. Their virion particles are composed of at least 22 different proteins, which were identified using mass spectrometry. Post-translational modifications were suggested for two proteins, and a frameshift hotspot was identified within ORF42, encoding a structural protein. Despite large temporal and spatial separations between phage isolations, very high sequence similarity and limited horizontal gene transfer were found between the individual viruses. These PB1-like viruses constitute a new genus of environmentally very widespread phages within the Myoviridae.
The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1muMCd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10muMCd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.
Giant bacteriophages phiKZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phiKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The phiKZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201phi2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.
A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.
We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida. Its 42.6 kb genome is related to the "epsilon15-like viruses" and the "BPP-1-like viruses", a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida-infecting phage ?15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation.
A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks.
Enlarged white adipose tissue (WAT) is a feature of obesity and leads to changes in its paracrine and endocrine function. Dysfunction of WAT cells is associated with obesity-associated disorders like type 2 diabetes and cardiovascular diseases. Resveratrol (RSV), a natural polyphenolic compound, mimics beneficial effects of calorie restriction. As such, RSV seems a promising therapeutic target for obesity-associated disorders. The effect of RSV on the human adipokine profile is still elusive. Therefore, a proteomic study together with bioinformatical analysis was performed to investigate the effect of RSV on the secretion profile of mature human SGBS adipocytes. RSV incubation resulted in elevated basal glycerol release and reduced intracellular TG content. This increased intracellular lipolysis was accompanied by profound changes in the adipocyte secretion profile. Extracellular matrix proteins were down-regulated while processing proteins were mostly up-regulated after RSV treatment. Interestingly, RSV induced secretion of proteins protective against cellular stress and proteins involved in the regulation of apoptosis. Furthermore, we found a RSV-induced up-regulation of adiponectin and ApoE accompanied by a down-regulation of PAI-1 and PEDF secretion which may improve anti-inflammatory processes and increased insulin sensitivity. These effects may contribute to alleviate obesity-induced metabolic complications. In addition, two novel RSV-regulated adipocyte-secreted proteins were identified.
The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage.
A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology.
The bacterium Dickeya solani, an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with Dickeya solani, the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.