Abstract: While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). The impact of the biofilm on SIP motility (swimming, swarming and twitching) and ability to adhere was also investigated, together with the amount of signal molecules (N-acyl homoserin lactones) in the planktonic chamber. Results showed that the presence of the biofilm did not modify the motilities of SIPs and their ability to adhere on PVC. Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C(4)-HSL (rhlR-rhlI-dependent QS) and 3-oxo-C(12)-HSL (lasR-lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C(12)-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.
Abstract: The in vitro colonization of three commercial heart valve leaflets by Staphylococcus aureus was investigated. The leaflets, made of pyrolytic carbon alloyed with or without silicon, displayed similar surface properties (wettability, roughness) and were readily colonized by S. aureus that formed patchy biofilms on the three supports. A proteomic approach was used to assess the physiological status of biofilm populations by comparing their protein maps to those of bacteria cultured as free cells in the presence or absence of biofilm substratum. Principal component analysis (PCA) revealed, for each tested leaflet, statistical relationships between the protein maps of the biofilm and free-floating microbial populations. A spot-by-spot comparison of protein levels on two-dimensional electropherograms showed that many proteins were accumulated or underproduced by microbial populations grown in the presence of a leaflet compared with protein levels in control free populations. The number of accumulated proteins was noticeably higher than that of underproduced polypeptides. This protein overproduction was emphasized in biofilm populations. Several proteins, some of which were identified, were differentially produced by both surface-associated planktonic and biofilm-grown cell populations compared with control free-cell ones cultured in the absence of leaflet, whatever the leaflet tested. The potential of this proteomic approach for fighting against microbial adhesion and biofilm formation is discussed.
Abstract: Acinetobacter baumannii and Pseudomonas aeruginosa are known for their intrinsic resistance to antibiotics. Between mechanisms involved in this resistance, diminished expression of outer membrane proteins and up-regulation of efflux pumps play an important role. The characterization of membrane proteins is consequently necessary because of their importance in the antibiotic resistance but also in virulence. This review presents proteomic investigations aiming to describe the protein content of the membranes of these two bacterial species.
Abstract: Abstract An antibacterial compound, S07-2, was purified to homogeneity by hydrophobic interaction, anion exchange, C18 reverse-phase and HS PEG HPLC. The molecular mass of S07-2 was 905.6 Da as determined by MS. The S07-2 compound was resistant to high temperatures (up to 100 degrees C) and could withstand a wide range of pH from 3 to 10. In addition, its antibacterial activity was preserved after treatment with proteases. Biochemical characterization revealed its cyclic peptide structure. This compound showed a bactericidal effect against important food-spoilage bacteria and food-borne pathogens including Listeria monocytogenes and Enterococcus faecalis with lethal concentration values of 62.5 mug mL(-1) and against Salmonella enteritidis at a concentration of 31.25 mug mL(-1). However, no cytotoxic effect against human erythrocytes was recorded. Furthermore, the S07-2 compound displayed a remarkable Fe(2+)-chelating activity (EC(50)=9.76 mug mL(-1)) and 1-diphenyl-2-picrylhydrazyl-scavenging capacity (IC(50)=65 mug mL(-1)). All these chemical and biological features make S07-2 a useful compound in the food industry as a natural preservative.
Abstract: Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells.
Abstract: The genome of Legionella pneumophila reveals the presence of a large number of genes coding for eukaryotic-like proteins. By using database searches and homology investigations, we identified three proteins in L. pneumophila whose sequences share similarities with that of eukaryotic polypeptides (lpg0211, lpg1974 and lpg1982). In eukaryotes, the corresponding proteins (PBR, peripheral benzodiazepine receptor; VDAC, voltage-dependant anion channel; and CypD, cyclophilin D) participate in the formation of the mammalian mitochondrial permeability transition pore (MPTP), a complex involved in cell apoptosis. Intriguingly, the presence of these proteins has never been reported in the same bacterium and constitutes, up to now, a unique feature of L. pneumophila. In Legionella, we hypothesize that these proteins are recruited in a multiprotein complex close to the MPTP that may regulate intracellular survival and/or proliferation.
Abstract: To investigate the role of rpoS in gene expression of Escherichia coli cells grown as biofilms, we compared the proteomes of a rpoS mutant and the wild-type strain. Experiments were performed on planktonic cells (in exponential or stationary growth phase) and biofilms developed on glass wool. Spot-by-spot comparison of gels obtained from biofilm and planktonic wild-type organisms showed that the intensity of between 22 and 30% of detected spots was affected by the growth mode, depending of the control used. Principal component analysis, used to interpret the variations in protein spot densities, discriminated exponential-phase cells (wild-type and mutant) from the other incubation conditions and secondarily 72-old cultures. The statistical analysis demonstrated that the rpoS mutation did not significantly modify the proteome of exponential-growth phase cells, the differences involving only 3% of the proteome. However, increasing the incubation time from 8 to 72 h noticeably increased the number of changed proteins. A cluster analysis showed that RpoS plays a role in the special nature of the gene expression of biofilm cells but lower than in stationary-phase bacteria. We identified 35 rpoS-regulated proteins that were already or not described as controlled by this sigma factor. For some of them, the mode of regulation by RpoS was obviously dependent on the culture condition (planktonic vs biofilm).
Abstract: Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.
Abstract: The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is known to counteract in vitro the deleterious effects of toxic agents on cerebellar granule cell survival and differentiation. The potent antiapoptotic action of PACAP is mediated through inhibition of caspase-3 activity; however, additional proteins are likely involved and remain to be identified. Two-dimensional gel electrophoresis analysis coupled with mass spectrometry characterization led to the identification of a protein, peroxiredoxin 2, which was induced after a 6-h treatment with PACAP. Western blot analysis confirmed the regulation of peroxiredoxin 2 by PACAP and revealed that this protein is induced by both cyclic AMP and protein kinase C stimulators. Inhibition of peroxiredoxin 2 expression, using two distinct small-interfering RNAs (siRNAs), reduced the effect of PACAP on caspase-3 activity and cerebellar granule cell survival. Peroxiredoxin 2 expression was also induced in vivo and in vitro by ethanol. Although ethanol and PACAP exert opposite effects on caspase-3 activity, inhibition of peroxiredoxin 2 expression, using siRNAs, only reduced the ability of PACAP to prevent ethanol-induced caspase-3 activity. Taken together, these data indicate that peroxiredoxin 2 is probably involved in the neurotrophic effect of PACAP and suggest that this protein may have a therapeutic potential for the treatment of some neurodegenerative diseases.
Abstract: The replacement of crude allergen extracts by selected allergens currently represents a major goal for the improvement of allergy diagnosis and immunotherapy. Indeed, the development of molecularly defined vaccines would facilitate both standardization and enhance batch-to-batch reproducibility as well as treatment specificity. In this study, we have investigated the potential of tobacco plant cells to produce biologically active forms of the two major allergens from the house dust mite. A detailed characterization of these plant-made allergens has shown similar proteolytic maturation and folding as well as comparable immunoreactivity to their natural counterparts. Altogether, our results exemplify that suspension-cultured BY-2 tobacco cells represent a low cost and environmentally safe expression system suitable to produce recombinant allergens from Dermatophagoides pteronyssinus under a form appropriate for diagnostic and therapeutic purposes.
Abstract: Bacteria undergo significant changes during adherence to surfaces and biofilm development. Cell-to-cell signalling molecules are known to be involved in these phenotypic adaptations to the sessile mode of life. We demonstrated previously that indole can act as an extracellular signal to regulate biofilm formation in E. coli. To identify proteins over- or under-expressed in response to E. coli biofilm formation and indole signalling, we compared the proteomes of the E. coli S17-1 wild-type and 3714 (S17-1 tnaA::Tn5) tryptophanase-negative mutant cells (which don't produce indole) grown as suspensions or biofilms in the presence or absence of exogenous indole. From computer-assisted image analysis, 407 spots were discriminated on two-dimensional electropherograms. Principal component analysis (PCA) of the electropherograms did not discriminate between the proteomes of the wild-type and mutant cells grown as suspensions indicating that indole has a limited impact onto protein expression of planktonic cells. The first principal component extracted by PCA, after standardization of the observations, opposed planktonic and biofilm cells confirming the existence of changes in protein expression during E. coli biofilm formation. Among proteins over- or under-expressed by both sessile wild-type and mutant cells, we identified metabolic enzymes, transporters, proteins involved in the translation and transcription machinery, stress response and regulation, and signalling proteins. The wild-type and mutant strains grown as biofilms in the presence of indole were discriminated by the second component. The role of some proteins whose expression was altered in biofilm bacteria compared to suspended counterparts is discussed.
Abstract: A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.
Abstract: The rat pheochromocytoma PC12 cell line has been widely used as a model to study neuronal differentiation. In particular, after serum depletion, PC12 cells stop to proliferate and undergo apoptosis. Under such conditions, treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) promotes cell survival and induces neurite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable information concerning the mechanisms controlling neuronal cell survival and differentiation. To this aim, PC12 cells cultured in serum-free medium were treated with PACAP (10(-7) M), proteins were extracted, separated by two-dimensional gel electrophoresis (2-DE), and identified by MALDI-ToF mass spectrometry. The comparison between 16 2-DE maps led to the characterization of 110 proteins regulated by PACAP among which 22 have been identified by automatic query of the Mascot, Aldente, and Profound servers with the ProGeR-CDD database. Seventy-six percent of these proteins, including the p17 subunit of caspase-3, the heat shock protein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin beta-5, and calmodulin were overexpressed. Investigation of the putative functions indicated that some of the proteins regulated by PACAP and identified in the present article could control cell survival or differentiation.
Abstract: Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.
Abstract: Acinetobacter baumannii causes severe infections in compromised patients. We combined SDS-PAGE, two-dimensional gel electrophoresis and mass spectrometry (LC-MS/MS and MALDI-TOF) to separate and characterize the proteins of the cell envelope of this bacterium. In total, 135 proteins (inner and outer membrane proteins) were identified. In this analysis, we described the expression by this bacterium of RND-type efflux systems and some potential virulence factors. We then compared the membrane subproteome of a clinical multidrug-resistant (MDR) isolate with that of a reference strain. We found that the MDR strain expressed lower levels of the penicillin-binding-protein 1b, produced a CarO protein having different primary and quaternary structures to that of the reference strain, and expressed OmpW isoforms. We also showed that the clinical strain has a high ability to form biofilms consistent with the accumulation of some outer membrane proteins (OMPs) such as NlpE or CsuD that have already been described as involved in bacterial adhesion. These features may partly explain the MDR emergence of the clinical isolate.
Abstract: The discharge of chemicals such as oil associated or not with derived products constitutes a real threat for the environment. We report here the differential expression of the blue mussel (Mytilus edulis) gill proteins corresponding to two contaminated environmental conditions: crude oil and offshore produced water. In order to evaluate and understand contaminants, effects and adaptive response of these organisms, we identified proteins using MS. The latter can be grouped into three main classes: proteins involved in the cellular structure, in metabolism, and in defence proteins.
Abstract: The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. A previous study led to the isolation of eight antimicrobial peptides, termed ascaphins, from norepinephrine-stimulated skin secretions. Peptidomic analysis (HPLC separation followed by MALDI mass spectrometry and Edman degradation) of these secretions has led to the identification and structural characterization of 13 additional peptides present in relatively high concentration. In addition to bradykinin (BK; RPPGFSPFR), a C-terminally extended bradykinin (peptide RD-11; RPPGFSPFRVD), a bradykinin-like peptide (peptide AR-10; APVPGLSPFR), and a C-terminally extended form of this peptide (peptide AV-12; APVPGLSPFRVV) were obtained in pure form. These peptides produced concentration-dependent relaxation of precontracted mouse tracheal rings with a rank order of potency of BK>RD-11>AR-10>AV-12 but only RD-11 caused the same maximal relaxation as bradykinin. Four small peptides were also isolated from the skin secretions that contain the Pro-Trp motif that is a characteristic of the tryptophyllin family of peptides previously identified in skins of frogs of the family Hylidae. The data show that the synthesis of dermal peptides that may play a role in defense against predators arose early in the evolution of anurans.
Abstract: We compared the outer membrane protein (OMP) pattern of 2-day-old immobilized Yersinia ruckericells (IC) with that of early (FC24) and late (FC48) stationary-phase planktonic counterparts. Fifty-five OMPs were identified. Principal component analysis discriminated between the protein maps of FC and IC. Some OMPs involved in bacterial adaptation were accumulated by both FC48 and IC but the expression of other proteins was controlled by the sessile mode of growth.
Abstract: The members of the Aquarana (or Rana catesbeiana species group) form a well-supported monophyletic clade but phylogenetic relationships between species within the group are incompletely understood. Peptides that differentially inhibited the growth of bacteria were purified from electrically stimulated skin secretions of the carpenter frog Rana virgatipes. Structural characterization identified members of the ranatuerin-2 (3 peptides) and temporin (3-peptides) families, previously found in the skins of R. catesbeiana, R. clamitans, R. grylio and R. septentrionalis. Ranalexin, a peptide previously found only in the Aquarana, was isolated together with a variant (FFGLHNLVPSMLCVVRKKC) that lacks the propensity to adopt an alpha-helical conformation and so was devoid of antimicrobial activity. Two C-terminally alpha-amidated peptides belonging to the brevinin-2 family were isolated from the skin secretions that, like an ortholog from R. septentrionalis, lacked the C-terminal cyclic heptapeptide domain associated with members of this family. Ranatuerin-1, previously isolated from R. catesbeiana, R. clamitans and R. grylio but absent from R. septentrionalis, was also not identified in R. virgatipes. Synthetic replicates of temporin-1Va (FLSSIGKILGNLL.NH2), temporin-IVb (FLSIIAKVLGSLF.NH2) and temporin-1Vc (FLPLVTMLLGKLF.NH2) potently inhibited growth of Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus). Temporin-1Va was also active against Gram-negative bacteria and the opportunistic yeast pathogen Candida albicans and had relatively weak hemolytic activity (LD50=120 microM) and may therefore represent a candidate for drug development. Our data support the placement of R. virgatipes in the Aquarana and indicate a closer phylogenetic relationship of R. virgatipes with R. septentrionalis than with R. catesbeiana, R. clamitans and R. grylio.
Abstract: The problems associated with biofilm infections in humans result from the distinct characteristics of biofilms, in particular their high level of resistance to antibiotics. One of the hypotheses that have been advanced to explain this resistance to antimicrobials is the phenotypic differentiation of biofilm cells. Although many studies on biofilms have highlighted physiological alterations following the attachment of bacteria to a surface, no studies have explicitly demonstrated a "biofilm" physiology. To contribute to this topical debate, we used principal component analysis to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis of crude protein extracts from planktonic and sessile Pseudomonas aeruginosa cells. These analyses showed that the proteome of attached P. aeruginosa cells differs from that of their planktonic counterparts. Furthermore, we found that the proteome of sessile P. aeruginosa is strongly dependent on the nature of the biofilm substratum.
Abstract: We have compared the protein maps of agar-entrapped Pseudomonas aeruginosa cells to those of free counterparts grown in the presence or absence of the immobilized-cell gel support. Principal component analyses (PCAs) were used to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis. PCA of the data matrix (923 rows x 6 columns) in which spot density values were standardized horizontally extracted three principal components (PCs) with eigenvalues higher than 1, accounting together for 71.6% of the variability in the data. Principal component 1 (PC1) opposed free (F) and agar-entrapped (AE) cultures, with a low contribution of agar-released, free (ARF) cultures to PC1. Inversely, the contribution of ARF cultures to PC2 was high, opposing those of AE and F cultures. Component 3 was related to the duration of incubation. Only 10% of total proteins were upregulated in AE cells during the first 18 h of incubation, the number of underexpressed peptides balancing that of overexpressed ones. Downregulation clearly became the dominant tendency when the incubation time was extended to 48 h. These results demonstrate that AE and ARF bacteria are physiologically different from F organisms.
Abstract: The protein maps of Pseudomonas aeruginosa cells from two natural (attached) and one artificial (gel-entrapped) immobilized-cell (IC) systems, together with their free (suspended) counterparts, were compared after incubation for 18 or 48 h in a minimal salt medium. Principal component analysis (PCA) was used to interpret the variations in protein spot densities that were observed on electropherogram obtained by two-dimensional electrophoresis (2-DE). PCA of the 2-DE data, a matrix of 933 rows (observations, i.e., spot density values) and 12 columns (variables, i.e., incubation conditions), in which observations were standardized horizontally, extracted four principal components (PCs) accounting for 78.75% of the variability in the protein expression profiles. PC1 opposed the two modes of growth (planktonic and immobilized) while PC2 discriminated between the incubation times of free cell cultures. The incubation conditions of ICs, including the immobilization procedure (entrapment vs attachment) and the nature of the biofilm substratum, were fairly separated in PC3xPC4. The dependence of the protein patterns on the cell immobilization process was further illustrated by the identification of a number of peptides whose amount remained unchanged or was altered in ICs compared to free bacteria. These results reinforce the topical assertion that bacteria in the immobilized state display a specific physiological behavior but also question the existence of a unique IC phenotype.
Abstract: The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C(10)) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C(10)). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C(10)-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (alpha or beta), 18C(10) did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies.
Abstract: Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.
Abstract: Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and L-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both sigma(B) and sigma(H), could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.
Abstract: A novel neuropeptide of the RFamide peptide family was isolated in pure form from a frog (Rana esculenta) brain extract by using reversed-phase high performance liquid chromatography in combination with a radioimmunoassay for mammalian neuropeptide FF (NPFF). The primary structure of the peptide was established as Ser-Leu-Lys- Pro-Ala-Ala-Asn-Leu-Pro-Leu- Arg-Phe-NH(2). The sequence of this neuropeptide, designated Rana RFamide (R-RFa), exhibits substantial similarities with those of avian LPLRFamide, gonadotropin-inhibitory hormone, and human RFRP-1. The distribution of R-RFa was investigated in the frog central nervous system by using an antiserum directed against bovine NPFF. In the brain, immunoreactive cell bodies were primarily located in the hypothalamus, i.e., the anterior preoptic area, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. The most abundant population of R-RFa-containing neurons was found in the periependymal region of the suprachiasmatic nucleus. R-RFa- containing fibers were widely distributed throughout the brain from the olfactory bulb to the brainstem, and were particularly abundant in the external layer of the median eminence. In the spinal cord, scattered immunoreactive neurons were found in the gray matter. R-RFa-positive processes were found in all regions of the spinal cord, but they were more abundant in the dorsal horn. This study provides the first characterization of a member of the RFamide peptide family in amphibians. The occurrence of this novel neuropeptide in the hypothalamus and median eminence and in the dorsal region of the spinal cord suggests that, in frog, R-RFa may exert neuroendocrine activities and/or may be involved in the transmission of nociceptive stimuli.
Abstract: The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.
Abstract: Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium. Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms. These observations highlighted the existence of high phosphate deprivation within biofilm-like structures. In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration. From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.
Abstract: Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic alpha-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113-135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependent channel formation but this was not absolutely required since the pore-forming ability of the 113-135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an alpha-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region.
Abstract: In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.
Abstract: Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.
Abstract: A new toxin (SGTx1) was purified from the venom of the spider Scodra griseipes by a combination of gel filtration and reverse-phase chromatography. The complete amino acid sequence of SGTx1, TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF, was established by direct automated Edman degradation, and is in perfect agreement with the molecular mass of 3775 Da found by mass spectrometry. The primary structure of SGTx1 exhibited sequence identity with other spider toxins such as hanatoxin (76%), TxP5 toxin (32%) and huwentoxin (26%). The six cysteines in the sequence suggested three disulfide bridges, the presence of which was demonstrated by mass spectrometry after dithiothreitol reduction. Analysis of secondary structure using circular dichroism spectrometry yielded more than 50% beta-sheet and about 15-20% beta-turn. The extent of the beta-content and the presence of disulfide bridges suggest a structure of interconnected beta-strands. In addition, a study of membrane/toxin interactions was carried out by reconstitution in planar lipid bilayers and by antibacterial assays. SGTx1 displays moderate pore-forming ability (conductance of about 100 pS in 1 M NaCl), but antibacterial activity was not observed against Gram-positive or Gram-negative strains. As a preliminary assay, the activity of SGTx1 was investigating using electrophysiological measurements. At 0.15 microM, SGTx1 reversibly inhibits more than 40% of outward potassium currents in rat cerebellum granular cells. This result is reminiscent with the effect described for hanatoxin extracted from the venom of Grammostola spatulata.
Abstract: The influences of peptide length, absence of a Glx (Gln/Glu) residue and the C-terminal amino alcohol on liposome permeabilization and ion-channel characteristics in planar lipid bilayers were examined with two 18-residue peptaibols, PA V and PA IX. As compared to the 20-residue alamethicin, both peptides belonging to the newly isolated trichorzin family, lack a proline in the N-terminal part and one of the two Gln/Glu residues in the C-terminal part of the sequence. The two analogues studied here differ among themselves in their C-terminal amino alcohol (tryptophanol for PA V and phenylalaninol for PA IX). These alpha-helical peptaibols modify to a similar extent the permeability of liposomes, as measured by leakage of a previously entrapped fluorescent probe. Monitoring tryptophanol fluorescence, a greater embedment of the peptide PA V is observed in cholesterol-free bilayers. Macroscopic conductance studies for PA V and PA IX display alamethicin-like current-voltage curves, with a similar voltage dependence, but a smaller mean number of monomers per conducting aggregate is estimated for the tryptophanol analogue, PA V. Single-channel recordings indicate faster current fluctuations for PA IX, while amplitude histograms show lower conductance levels for PA V. Apart from underlining the role of the mismatch between helix length and bilayer hydrophobic thickness, these results stress that the C-terminal tryptophanol favours a stabilization of the conducting aggregates.
Abstract: The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.
Abstract: Four peptides mimicking the four P-regions of the electric eel sodium channel were chemically synthesized to characterize their secondary structure and their contribution to the channel selectivity. Circular dichroism spectra of these peptides in trifluoroethanol demonstrate an important beta-sheet conformational component. This beta-sheet content is much enhanced upon interaction with phosphatidylcholine small unilamellar vesicles. As expected (and except for P of domain III), no significant voltage-dependence is revealed in either macroscopic or single-channel conductance experiments. The concentrations-dependences of macroscopic conductances suggest that tetramers are the membrane conducting aggregates. In asymmetric ionic conditions, these channels made up of P-peptides were mostly specific for sodium over chloride whilst caesium was largely excluded. Single-channel conductance analysis discloses a moderate selectivity for sodium over potassium for PI and PII. This selectivity is larger with PIII but inverted for PIV. Finally, a control random peptide of the same length and with a comparable mean hydrophibicity was also tested. Its conformation in TFE is mainly unordered and no activity was detected in planar lipid bilayers. The data suggest that the presumed selectivity filter may not assume a circular symmetry and that molecular recognition between the different P-regions has to be taken into account.
Abstract: Conformational studies of synthetic peptides corresponding to the pore-forming regions of voltage-gated sodium channels show a high tendency for beta-sheet conformation when interacting with lipid vesicles, as revealed by circular dichroism and infrared spectroscopy. These observations have guided our choice of possible molecular models for the P-region peptide of domain II of voltage-gated sodium channels: three alternative beta-hairpins, with differing turn assignments, or an alpha-helical hairpin. After generation of models by distance geometry-based methods, molecular dynamics (MD) simulations were run. in the absence of explicit solvent molecules but employing three different dielectric constants, to explore possible conformational preferences. The simulations in the different dielectric environments suggest that a 4-residue turn with the sequence LCGE yields more stable beta-hairpins. The MD results suggest that the SS1 part of the peptide may be more stable as an alpha-helix, whereas the SS2 part tends to adopt a beta-conformation.
