Interspecies Comparison of the Bacterial Response to Allicin Reveals Species‐Specific Defense Strategies

Allicin, a broad‐spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity‐conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram‐negative species, protein synthesis of the majority of proteins is downregulated while the Gram‐positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy‐based assays further indicate that in B. subtilis cell wall integrity is impaired.     

Analysis of cotton (Gossypium hirsutum) root proteomes during a compatible interaction with the black root rot fungus Thielaviopsis basicola

A proteomic approach was used to uncover the inducible molecular defense mechanism of cotton root occurring during the compatible interaction with Thielaviopsis basicola. Microscopic observation of cotton root inoculated with a suspension of conidia showed that this necrotrophic hemibiotroph fungus interacts with the plant and completes its life cycle in our experimental system. 2‐DE analysis of root extracts taken after 1, 3, 5, and 7 days postinoculation and cluster analysis of the protein expression levels showed four major profiles (constant, upregulated, one slightly downregulated, and one dramatically downregulated). Spots significantly (p<0.05) upregulated were analyzed by LC‐MS/MS and identified using MASCOT MS/MS ion search software and associated databases. These proteins included defense and stress related proteins, such as pathogenesis‐related proteins and proteins likely to be involved in the oxidative burst, sugar, and nitrogen metabolism as well as amino acid and isoprenoid synthesis. While many of the identified proteins are common components of the defense response of most plants, a proteasome subunit and a protein reported to be induced only in cotton root following Meloidogyne incognita infection were also identified.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Regulation by ABA of osmotic-stress-induced changes in protein synthesis in tomato roots

Polypeptide synthesis and accumulation were examined in the roots of tomato seedlings exposed to a polyethylene glycol‐imposed water deficit stress. In these roots, the synthesis of a number of polypeptides was induced, while that of several others was enhanced or repressed. To examine the role played by abscisic acid (ABA) in co‐ordinating the accumulation of these proteins, water‐deficit‐stress‐responsive polypeptide synthesis was investigated in the roots of the ABA‐deficient mutant flacca. In the roots of this mutant, the ability to accumulate a complete set of water‐deficit‐stress‐responsive polypeptides was impaired, indicating that ABA is required for their synthesis. The role of ABA was further examined by exposing the roots of both genotypes to exogenous ABA, which, with one exception, elicited the accumulation of all water‐deficit‐stress‐responsive proteins. Polyethylene glycol‐induced polypeptide accumulation was accompanied by a 1·6‐fold increase in the level of endogenous ABA in the roots of wild‐type plants and a 5‐fold increase in the roots of flc. Thus, although the absolute level was lower than that of the wild‐type, flc has the capacity to accumulate ABA in its roots. When fluridone was used to prevent the biosynthesis of ABA, the accumulation of several water‐deficit‐stress‐responsive polypeptides was reduced further. The synthesis of polypeptides was also examined in the roots of salt‐treated seedlings. Salt altered the accumulation of several polypeptides, all of which were previously observed in water‐deficit‐stressed roots, indicating that their synthesis was the result of the osmotic component of the salt stress. However, the accumulation of these polypeptides was not impaired in flc roots, indicating that the role played by ABA in regulating their accumulation in salt‐and polyethylene glycol‐treated roots differs. As such, salt‐ and water‐deficit‐stress‐induced changes in gene expression may be effected by different mechanisms, at least at the level of polypeptide accumulation.     

Comparative Proteomic Profiling of Methicillin‐Susceptible and Resistant Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin‐susceptible and methicillin‐resistant S. aureus (MSSA; MRSA) recovered from non‐healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty‐two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.     

New changes in the plasma‐membrane‐associated proteome of rice roots under salt stress

To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma‐membrane‐associated proteins following treatment with 150 mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt‐sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two‐phase partitioning, and plasma‐membrane‐associated proteins were separated by IEF/SDS‐PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5‐fold in response to salt stress. Of these proteins, nine were up‐regulated and nine were down‐regulated. MS analysis indicated that most of these membrane‐associated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine‐rich‐repeat type receptor‐like protein kinase, OsRPK1, was identified as a salt‐responding protein. Immuno‐blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno‐histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.     

Factors that interact with the antibacterial action of thyme essential oil and its active constituents

The viable counts of Salmonella typhimurium on nutrient agar (NA) decreased upon the addition of either the essential oil of thyme or its constituent thymol, especially under anaerobic conditions. Antagonistic effects of thymol against Staphylococcus aureus were also greater under anaerobic conditions. In contrast to the phenolic constituents of the oil, thymol and carvacrol, the chemically related terpenes p -cymene and y -terpinene had no antagonistic effects against Salm. typhimurium.
The addition of Desferal to NA counteracted the antibacterial effects of both thyme oil and thymol. No support was obtained, however, for a possible role of iron in the oxygen-related antibacterial action of the thyme oil and thymol or for the observed effect of Desferal. In the presence of thymol, the viable counts of Salm. typhimurium obtained on a minimal medium (MM) were lower than those obtained on NA. Addition of bovine serum albumin (BSA) neutralized the antibacterial action of thymol. It is suggested that the effects of BSA or Desferal are due to their ability to bind phenolic compounds through their amino and hydroxylamine groups, respectively, thus preventing complexation reactions between the oil phenolic constituents and bacterial membrane proteins. This hypothesis is supported by the marked decrease in the viable counts of Salm. typhimurium caused by either thyme oil or thymol when the pH of the medium was changed from 6.5 to 5.5 or the concentration of Tween 80 in the medium was reduced.     

Localisation and interactions of the Vipp1 protein in cyanobacteria

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C‐terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live‐cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull‐downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high‐light exposure. These include not only photosynthetic and stress‐related proteins but also RNA‐processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress‐induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.     

Nep1‐like protein from Moniliophthora perniciosa induces a rapid proteome and metabolome reprogramming in cells of Nicotiana benthamiana

NEP1 (necrosis‐ and ethylene‐inducing peptide 1)‐like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP‐treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short‐term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2‐DE gels in each proteome, with one‐third showing more than twofold differences in the expression values. Fifty‐three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide‐binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi‐quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo‐inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction‐oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP‐mediated cell death signaling in plants.     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

Proteomic analysis of symbiosome membranes in Cnidaria–dinoflagellate endosymbiosis

Symbiosomes are specific intracellular membrane‐bound vacuoles containing microalgae in a mutualistic Cnidaria (host)–dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin‐XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X‐100 soluble and insoluble fractions, were subjected to 2‐D SDS‐PAGE and identified by MS using an LC‐nano‐ESI‐MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti‐apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.