Biomedical subjects

N Orange

Publications and source records attributed to N Orange.

At least 19 recordsLinked to original sources

PubMed2002

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

Bacterial Adhesion↗

PubMed2002

Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase.

Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.

Bacterial Adhesion↗

PubMed2001

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora.

The purified major outer membrane protein (37275 Da) from the psychrotrophic phytopathogen Erwinia carotovora MFCL0 was structurally characterised by MALDI-TOF mass spectrometry, N-terminal microsequencing and DNA sequence determinations, and secondary structure prediction analyses. The deduced amino acid sequence showed 76% and 72% of similarities with the Serratia marcescens and Escherichia coli OmpA proteins respectively. Dendrogram analysis allowed to point out that E. carotovora is close to the genus Serratia. After reconstitution into planar lipid bilayers, this major protein induced ion channels with a major conductance level of 630 pS in 1 M NaCl and a weak cationic selectivity. These functional and structural features allowed to identify this major outer membrane component of E. carotovora as an OmpA-like protein, i.e., a channel-forming protein which could be involved in the infection process of this phytopathogen agent.

Amino Acid Sequence↗

PubMed2001

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells.

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.

Animals↗

PubMed2000

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties.

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.

Autoradiography↗

PubMed2000

Isolation and characterization of novel glycoproteins from fish epidermal mucus: correlation between their pore-forming properties and their antibacterial activities.

In fish, a layer of mucus covers the external body surface contributing therefore, among other important biological functions, to the defense system of fish. The prevention of colonization by aquatic parasites, bacteria and fungi is mediated both by immune system compounds (IgM, lysozyme, etc.) and by antibacterial peptides and polypeptides. We have recently shown that only the hydrophobic components of crude epidermal mucus of fresh water and sea water fish exhibit strong pore-forming properties, which were well correlated with antibacterial activity [N. Ebran, S. Julien, N. Orange, P. Saglio, C. Lemaitre, G. Molle, Comp. Biochem. Physiol. 122 (1999)]. Here, we have isolated novel glycosylated proteins from the hydrophobic supernatant of tench (Tinca tinca), eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss) mucus. The study of their secondary structure was performed by circular dichroism and revealed structures in random coil and alpha-helix in the same proportions. When reconstituted in planar lipid bilayer, they induced the formation of ion channels. This pore-forming activity was well correlated with a strong antibacterial activity (minimal inhibitory concentration < 1 microM for the three proteins) against both gram-negative and gram-positive bacteria. Our results suggest that fish secrete antibacterial glycoproteins able to kill bacteria by forming large pores (several hundreds to thousands of pS) in the target membrane.

Anguilla↗

PubMed2000

Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium.

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.

Bacterial Proteins↗

PubMed2000

Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF.

Lipopolysaccharide (LPS) was found to be associated with the major outer membrane protein OprF of the psychrotrophic bacterium Pseudomonas fluorescens MF0, using two OprF purification procedures. OprF, purified under mild conditions, presented two types of association with LPS: tight (tLPS) and slight (sLPS), both of type R. LPS protected OprF from heat modification and trypsin degradation and facilitated the reincorporation of purified OprF into an artificial lipid bilayer without affecting its pore-forming activity. The size of the OprF channel depended on cell growth temperature, as did the extent of LPS phosphorylation: we suggest that LPS may be involved in modifications of OprF pore formation.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Molecular Weight↗

PubMed2000

Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures.

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.

Cellulase↗

PubMed1999

Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish.

Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16-0.18 microM) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.

Animals↗

PubMed1996

Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio).

A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/Pk ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018-0.18 microM) against different strains of gram-negative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

Amino Acid Sequence↗

PubMed1995

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3.

The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).

Amino Acid Sequence↗

PubMed1994

Growth temperature regulates the induction of beta-lactamase in Pseudomonas fluorescens through modulation of the outer membrane permeation of a beta-lactam-inducing antibiotic.

The psychrotrophic bacterium, Pseudomonas fluorescens strain MFO, is more sensitive to the beta-lactam mezlocillin at a low growth temperature (i.e. 8 degrees C) than at a higher growth temperature (28 degrees C). An early effect of this antibiotic at all temperatures is bacterial filamentation, but this occurs later at 8 degrees C than at 28 degrees C, which suggests a lower permeability of the cell envelopes to mezlocillin at low growth temperature. beta-Lactamase production is later induced by mezlocillin, but the level of this induction also depends on the growth temperature, the overall induction being much less efficient at 8 degrees C. It is hypothesized that the periplasmic concentration of the drug might be too low at 8 degrees C to allow efficient beta-lactamase induction; this hypothesis was confirmed by the demonstration that beta-lactamase production is drastically enhanced in cells cultivated at 8 degrees C permeabilized for 10 min by Na-EDTA. In addition, induction kinetic curves display a marked dependence upon growth temperature. A rapid saturation was evident when mezlocillin concentrations were increased at 8 degrees C; this was not seen at 28 degrees C at up to 1000 micrograms mezlocillin ml-1. The results are discussed in terms of two different routes of drug permeation, depending on the growth temperature.

Cell Membrane Permeability↗

PubMed1993

Ionophore properties of OmpA of Escherichia coli.

Both porins OmpA1, from wild-strain K12 Escherichia coli, and OmpA2, from a K12 derivative deficient in both OmpF and OmpC, are able to form ion channels in virtually solvent-free membranes. The conductance has been shown to vary in a discrete fashion with different single increment values especially with OmpA2. This behaviour seems to indicate, beside monomers, the presence of aggregates of different sizes. The estimated small pore diameter (0.6-0.7 nm) for the monomeric would explain the weak permeability of this narrow channel toward different solutes. OmpA protein, from experiments of ion selectivity and zero-current potential, is determined weakly anion selective.

Escherichia coli↗

PubMed1993

Growth temperature controls the production of a single extracellular protease by Pseudomonas fluorescens MF0, in the presence of various inducers.

The psychotrophic strain Pseudomonas fluorescens MFO is known to express several enzymatic activities in milk, including extracellular proteolytic activity, optimally when cells are grown at 17.5 degrees C. In order to study the nature of the mechanisms controlling the production of the extracellular protease, we devised a defined medium in which this enzymatic activity was induced by an amino acid and small peptides. Regardless of the inducer, optimal proteolytic activity appeared at 17.5 degrees C. SDS-PAGE and isoelectrofocussing revealed a single protease produced by P. fluorescens MFO with all the inducers used and at all temperatures examined. The level of proteolytic activity correlated with the amount of enzyme in the supernatants.

Animals↗

PubMed1991

Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens.

In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MF0 is maximal at a growth temperature of 17.5 degrees C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MF0, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5 degrees C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.

Acid Phosphatase↗

PubMed1988

Identification of viral antigenic determinants by monoclonal antibodies directed against Chilo iridescent virus (iridovirus type 6). Brief report.

Monoclonal antibodies (McAbs) obtained against Iridovirus type 6 (CIV) were characterized by Western blotting and/or immunoprecipitation. Seven McAbs were found to be strongly reactive with viral polypeptides of molecular weights 16 K and 18 K by Western blotting. Two McAbs were directed against a complex composed of 30 K, 50 K, and 100 K polypeptides, but failed to react with either of these free polypeptides. This finding could explain the faint reactivity of these McAbs in Western blotting and immunoprecipitation. The reactivity of the other McAbs with their antigenic determinants is also discussed.

Antibodies, Monoclonal↗