FR2859218A1 - Microvesicles made from the outer membrane of bacteria, useful e.g. as vectors for therapeutic agents, include a detectable signature, preferably fluorescent, to facilitate visualization - Google Patents

Abstract

Microvesicles (A) made of the external membrane of bacteria have diameter below 250 nm; are formed from a single membrane and include a detectable signature (DS). An independent claim is also included for a method for preparing (A).

Description

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EXTERNAL MEMBRANE MICROVESICLES MARKED,
PRODUCTION PROCESS AND USES THEREOF
The present invention relates to marked outer membrane microvesicles, their method of production and their uses.

 Membrane microvesicles are protrusions of the outer membrane of Gram-negative bacteria, which detach from the cell to escape into the medium. These microvesicles are spherical compartments. The invention is particularly interested in microvesicles smaller than about 250 nm.

 Some microorganisms naturally produce these microvesicles, without the need for any stimulation. These microvesicles have long gone unnoticed, because they are very small and the analysis techniques did not identify them. Even today, the analysis techniques to be used to identify these microvesicles, in particular electron microscopy using or not immunostaining techniques are cumbersome and expensive.

 The outer membrane microvesicles are known from the prior art, and cited in particular by Beveridge, Journal of Bacteriology, Aug. 1999, Vol. 181, No. 16 pp.

4725-4733, or by Bernadac et al. Journal of Bacteriology. Sep 1998 Vol. 180, N, 18 pp. 4872-4878.

 External membrane microvesicles are used in particular to develop vaccines. EP 1248647 discloses a vaccine from outer membrane microvesicles against diseases caused by Neisseria meningitidis

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 Serogroup B. WO97 / 05899 discloses a vaccine using outer membrane microvesicles as a vector of an antigen associated with the surface of the microvesicle. WO97 / 05899 also discloses a pharmaceutical composition comprising membrane vesicles of one or more microorganisms comprising enzymes and having bactericidal effects. The microvesicles used in this patent application are either natural microvesicles, sizes between 10-200 nm, or different and larger size vesicles, including the outer membrane, the cytoplasmic or plasma membrane and the cytoplasm .

 The use of these vesicles larger size is dictated by the difficulties of visualization and monitoring of natural microvesicles, but it has many disadvantages: first, it is difficult to ensure that these vesicles are homogeneous and all the same nature ; moreover, some vesicles contain cytoplasm, and thus, statistically, some of them contain cytoplasmic DNA. The use of this type of vesicle therefore presents a risk of unwanted DNA transmission and difficult to control.

 The present invention overcomes the above drawbacks and proposes external membrane microvesicles which are labeled, which makes it possible to visualize them rapidly and at a lower cost, for example with the direct analysis of a bacterial culture by fluorescence microscopy, avoiding use much more expensive and expensive techniques of electron microscopy or purification, which can be produced massively.

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 The microvesicles according to the invention are formed of a single membrane, the outer membrane. To date, all the experiments carried out by the inventors on Escherichia coli show that no cytoplasmic material is found in the microvesicles according to the invention.

The microvesicles according to the invention therefore contain only intermembrane material, in particular periplasmic for gram-negative bacteria. The microvesicles of the invention thus have the advantage of being homogeneous with each other, and of ensuring the non-transmission of genetic material of cytoplasmic origin. Moreover, the microvesicles according to the invention contain a single compartment containing proteins in an oxidized state, and therefore more suitable for use.

 The object of the invention is therefore to propose detectable microvesicles, even though their size is very small.

 More particularly, the subject of the present invention is one or more outer membrane microvesicles, of diameter less than 250 nanometers, formed with a single membrane, and comprising a marker or a detectable signature, in particular by fluorescence, by RPE type techniques or by measurement of radioactivity, which is either at the intravesicular level or at the level of the membrane.

 According to a first embodiment of the invention, this signature is a detectable intravesicular protein, preferably a fluorescent protein. Many fluorescent proteins are suitable. By way of example, mention may be made of the Green Fluorescent Protein.

 According to another embodiment of the invention, this intravesicular signature is a protein which fixes

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 a detectable cofactor. Many cofactors are likely to be fixed by the appropriate proteins.

Among the suitable cofactors, mention is made in particular of cofactors detectable by EPR. By way of example, mention may be made, as a protein linked to a detectable cofactor, of the proteins of the oxidation-reduction system and for Escherichia coli, trimethylamine N-oxide reductase (TorA). Other periplasmic bacterial cofactor proteins such as nickel-iron core hydrogenases or recombinant proteins may also be contemplated. In the case of iron-receiving membrane proteins in iron form coupled to a siderophore, these proteins from various bacterial genus can be overproduced and found in purified microvesicles. Thus we can proceed to membrane labeling via the complex iron (ferric) -siderophore.

 This signature may also be derived from an in vivo or in vitro radiolabelling, using radioactive labeling techniques known to those skilled in the art; the signature may also consist of an in vitro fluorescent labeling, that is to say a marking by chemical coupling of commercial fluorescent probes on molecules present on the surface or in the microvesicles. As probes there may be mentioned Texas Red-X succinimidyl ester (Molecular Probes) or Oregon Green 488 carboxylic acid succinimidyl ester (Molecular Probes) which are fluorescent probes that can react and couple with the reactive functions of molecules such as the amine function present among others in proteins.

This signature can also be an antibody coupled to a fluorescent reagent. This antibody may recognize a protein or molecule exposed on the surface of the bacterium (see the use of an anti-Lamb antibody: Lloubes and

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 al. Research Microbiology Jul-Aug 2001 Vol. 152 N 6 pp.

523-529)
According to one particular embodiment of the invention, it is envisaged to combine several signatures or markings, in particular to provide for marking the microvesicles both at the intravesicular level and at the level of the membrane.

 The microvesicles according to the invention have many advantages, and can in particular be used as targeting tools. According to an advantageous embodiment of the invention, the microvesicles of the invention are associated with molecules of interest, intended for targeting or therapy, of the type comprising saccharides and in particular oligosaccharides, proteins and in particular enzymes, but also antibiotics.

 Among the molecules of interest, the invention is particularly interested in associating with labeled microvesicles addressing molecules, proteins that may be targets of cellular receptors, in particular toxins, specific receptors notably of the type allowing entry. DNA described by Lambert et al. Molecular Microbiology Nov 1998 Vol 30 N 4 pp.761-765, or antibodies of particular scFv type exposed on the surface of the outer membrane. These proteins of interest would be present on the bacterial cell used for the process according to the invention. The preparation of such a bacterium is feasible according to the methods described in Francisco et al. (Production and fluorescenceactivated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.) Proc Natl Acad Sci U S A. 1993 Nov 15; 90 (22): 10444-10448.

Francisco and Georgiou (The expression of recombinant

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 Escherichia coli.) Biotechnological applications. Ann N Y Acad Sci. 1994 Nov. 30; 745: 372-382. Earhart (Use of an Lpp-OmpA fusion vehicle for bacterial surface display.) Methods Enzymol.

2000; 326: 506-516.

 According to another embodiment, which can be combined with the above, the microvesicles of the invention are combined with at least one determinant exposed on the surface of the microvesicle, for example the lipopolysaccharide (LPS) present on the surface of the bacteria and also microvesicles, which plays a role in the inflammatory response (Chen et al., J Cell Biochem, Aug. 2003 Vol 89, N 6 pp 1206-1214); enterotoxin from pathogenic Escherichia coli bacteria, which is also found on the surface of microvesicles (Horstman and Khuehn J Biol Chem Sep 2002 Vol 277 N 36 pp. 32538-32545); the Helicobacter pylori cytotoxin VacA, which is also found active in microvesicles formed by this organism (Keenan and Allardyce, Dec 2000, Eur J Gastroenterol Hepatol, Vol 12 N 12 pp. 1267-1273).

 According to an advantageous variant of the invention, the microvesicle, which may or may not be labeled, contains a nucleic acid fragment, which has been introduced in vitro, and may thus serve as a transport for a genetic information intended for a particular target. In this case, microvesicles containing the membrane protein FhuA (overproduced from a plasmid) are purified. This protein will allow the entry of phage T5 DNA (Lambert et al.

Molecular Microbiology Nov 1998 Vol 30 N 4 pp.761-765). Thus genes of interest can be cloned into the DNA of this phage. This variant is particularly intended

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 in situations where the microvesicles according to the invention are used in gene therapy.

 The subject of the invention is also a process for the production of labeled microvesicles, characterized in that: (a) a detectable signature is introduced or is produced or labeled in a cell of bacterial origin which is wild or mutated, producing microvesicles, (b) said bacterium is cultured in a solid medium or in a liquid medium.

 According to a particular embodiment of the invention, the cell is a bacterial strain selected from tol, pal or lpp mutants or from bacterial genera that produce microvesicles.

 Suitable for carrying out the process according to the invention are pathogenic bacteria having these characteristics, of the type, but not exclusively, Escherichia coli, Shigella flexneri, Salmonella typhimurium, Helicobacter pylori ...

 In the case where the signature is protein or associated with a protein, the cell and in particular the bacterial strain used in the process according to the invention must have an export system or have the natural ability to export the signature present in the cytoplasm, to the outer membrane or periplasm. According to a preferred embodiment of the invention, the bacterium used for the production of microvesicles according to the invention has a non-functional Tol-Pal system; advantageously, the bacterium is selected

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 among the mutants tol, pal or 1pp or among the bacterial genera producing microvesicles.

 In the case where this signature is a folded protein, in particular a fluorescent protein or linked to a detectable cofactor, the cell and in particular the bacterium must have a system for exporting folded proteins or naturally allow such an export. Advantageously, the bacterium used in the process according to the invention possesses the export TAT (Tween Arginine Translocation) system.

 According to a first embodiment of the invention, a plasmid which enables the production of a detectable signature is introduced into a cell of bacterial origin. Advantageously, this plasmid comprises a gene coding for a labeled protein, for example a fluorescent protein.

 According to another embodiment of the invention, it is noted, in particular by using in vitro radiolabelling or labeling with fluorescent probes, or is detected without prior marking, the natural components of the outer membrane or content intravesicular, without the need to introduce a plasmid. This natural component can be a cofactor fixed by a protein.

 According to a preferred embodiment of the invention, a step intended to induce a more massive production of microvesicles is carried out prior to cell culture. This step consists, for example, in destabilizing the integrity of the outer membrane, in particular by the periplasmic production of destabilizing protein sequences. These sequences may be chimeric proteins, of the autofluorescent proteinprotein protein destabilizing the integrity of the membrane

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 external or proteins co-produced with the detectable protein in the same bacterium. Such destabilizing sequences of membrane integrity allowing the production of microvesicles have been described by Bouveret et al. Biochemistry. May-Jun 2002 Vol. 84 N 5-6 pp.413-421.

 According to another particular embodiment of the invention, the method comprises a purification step which occurs after the cell culture step. This purification step consists first of all in eliminating all the bacteria, so as to recover the microvesicles. In the second place, a step makes it possible in particular to concentrate the microvesicles but also to suspend them in a buffer of choice. According to the invention, it is also capable of providing a uniform production of microvesicles having a diameter of about 40 nm.

 According to a particular embodiment of the invention, the method further comprises a step of quantitative or qualitative measurement of the microvesicles marking, and in particular when the signature is a fluorescent protein, a step of measuring the fluorescence of the marked microvesicles. This measurement makes it possible to verify the production, and to quantify it.

 Another object of the invention is to use microvesicles by following and quantifying their production in vivo. This monitoring makes it possible to study the fate of the vesicles in vivo, in particular their fate in contact with other bacteria or cells. For this application, microvesicles carrying natural or artificial determinants exposed will be of great importance for targeting. The microvesicles according to the invention are useful as a tool for targeting molecules of therapeutic interest or of diagnosis, in particular for measuring the impact of an active principle or an active molecule.

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 The subject of the invention is also the use of labeled microvesicles according to the invention, in particular microvesicles containing a nucleic acid fragment, as a vectorization tool.

 Another use according to the invention comprises the use of labeled microvesicles for the identification of an active agent, active principle or active molecule on a bacterium, characterized in that the production of microvesicles of said bacterium is stimulated, in particular by a stimulating agent, and said agent is identified by visualization of the labeled microvesicles. The simplicity of the detection and the efficiency of the technique developed by the present invention allows the analysis by high throughput screening techniques where on multiwell plates. Thus, bacterial cultures would be treated by each of the compounds from chemical libraries. The supernatants obtained after centrifugation of the cultures would be analyzed by automated fluorescence microscopy. The presence of fluorescent dots and the amount of these dots being related to the activity of the test compound.

 The invention will be more explicit in the light of the following examples, which illustrate the invention without limitation.

Unique example
Two culture conditions were used:
Production conditions in liquid medium. The tolA cells (Bernadac et al., 1998, J Bacteriol 180, 4872-4878) transformed by the plasmid RR-GFP (Santini et al., 2001, Journal of Biological Chemistry, 276, 8159-8164) are cultured in medium rich (Luria-Bertani or LB) in

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 anaerobiosis and in the presence of glucose (0.4%). After 4 hours of incubation at 37 ° C., the culture is centrifuged and the supernatant is removed. The cells are then washed in a minimum medium and then taken up in the same minimum medium (medium M9 containing sucrose as carbon source, supplemented with 20 amino acids) containing 0.2% of L-arabinose to allow the induction of the expression of GFP during a 2 hour incubation at 30 C.

 Production conditions in solid medium. The tolA cells transformed with the RR-GFP plasmid were cultured on a solid support in LB-agar medium in the presence of 0.4% glucose (GFP repression conditions) and then assayed on LB-agar medium containing arabinose ( 0.1%, GFP induction conditions) and incubated overnight (about 16 hours) at 30 ° C.

The cells harvested from the solid medium (scraping colonies on Petri dishes and suspension in TN buffer: Tris-HCl pH8.0, 100 mM NaCl) or liquid medium are centrifuged at low speed to sediment the bacteria. The supernatant containing the outer membrane vesicles is again centrifuged under the same conditions and then filtered twice on a 0.2 mm filter to avoid any risk of contamination by bacteria or membrane debris. Two vesicle purification conditions are used:
Either the vesicles contained in the supernatants are directly sedimented by ultracentrifugation (45 min at 250,000 g average). After rapid washing of the pellet in TN buffer (and new ultracentrifugation) it is resuspended in TN buffer (VesC).

 The supernatant is deposited on the surface of a gradient formed of three layers of density: 1.08,3 ml (15

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 %), 1.16, 2 ml (30%) and 1.24, 1 ml (45%) (% Optiprep ™). After ultracentrifugation for one hour at 30,000 rpm (SW41Ti) the 2 ml fractions of the gradient are recovered (VesG).

 We chose to measure the amount of GFP by spectrofluorimetric assay and to follow the blactamase activity by enzymatic assay (using a chromogenic cephalosporin substrate, CENTATM).

 All the results were expressed in arbitrary units using for reference (100) the measurements made on the periplasmic content of the producing cells (obtained after osmotic shock). The intravesicular content analysis is performed on the resuspended vesicle pellet (VesC) as well as on the microvesicles obtained in the 1.24 (VesG) density fraction.

 In order to measure the β-lactamase activity within membrane microvesicles and to avoid following the diffusion of the substrate through the membrane, we sought the minimum concentration of detergent necessary to solubilize the outer membrane of the microvesicles (tests carried out by light microscopy with fluorescent vesicles). Thus the Triton X100 detergent was added at 0.02% in all enzyme assay experiments.

 On the other hand we measured the effect of optiprepTM concentrations on the activity of blactamase and took into account a weak inhibition in the expression of our results when it was present: these measurements are reported in the Table I below, in which the values are reduced to the value

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100 which corresponds to the determination of the periplasmic contents of the bacteria. VesC and VesG correspond respectively to purified microvesicles by ultracentrifugation and by sedimentation gradient respectively.

<Tb>
<tb> b-lactamase <SEP> GFP
<tb> Medium <SEP> Liquid
<tb> VesC <SEP> 0.2 <SEP> 0.5
<tb> VesG <SEP> 0.8 <SEP> 2.8 <SEP>
<tb> Medium <SEP> Solid
<tb> VesC <SEP> 3.1 <SEP> 5.4
<tb> VesG <SEP> 1.5 <SEP> 2,3 <SEP>
<Tb>

Table I. assay of the β-lactamase by enzymatic measurement and determination of GFP by fluorescence measurement.

 The fluorescent marking according to the invention is therefore particularly effective. The inventors have even found that, unexpectedly, the labeling obtained by implementing the method of the invention with a fluorescent protein is stronger than that of a natural periplasmic protein. Thus the microvesicles are directly and easily detectable by fluorescence optical microscopy. Direct observation would be impossible in the absence of fluorescence due to the small size of microvesicles. In addition, this detection confirms the presence of sealed microvesicles that contain GFP in a concentrated state.

Claims (31)

1) An outer membrane microbe of bacteria characterized in that its diameter is less than 250 nanometers, that it is formed by a single membrane, and that it comprises a detectable signature.  2) microvesicle according to claim 1, characterized in that the bacteria are Gram-negative bacteria, 3) Microvesicle according to any one of claims 1 or 2, characterized in that the detectable signature is located either at the intravesicular level or at the level of the membrane.  4) Microvesicle according to any one of claims 1 to 3, characterized in that said signature is detactable by fluorescence, by RPE type techniques or by measurement of radioactivity.  5) Microvesicle according to any one of claims 1 to 4, characterized in that it contains only intermembrane material, including periplasmic.  6) Microvesicle according to any one of claims 1 to 4, characterized in that said signature is a detectable intravesicular protein, preferably fluorescent.  7) Microvesicle according to any one of claims 6, characterized in that said signature is a detectable protein, in particular by RPE, of the intravesicular protein type which fixes a cofactor or iron-binding membrane protein in iron form coupled to a siderophore.  8) Microvesicle according to any one of claims 1 to 6, characterized in that said signature is derived from an in vivo or in vitro radiolabeling. <Desc / Clms Page number 15>  9) Microvesicle according to any one of claims 1 to 6, characterized in that said marker is a fluorescent probe.  10) Microvesicle according to any one of claims 1 to 9, characterized in that said microvesicle is associated with at least one molecule of interest, of the type comprising oligosaccharides, proteins and especially enzymes, antibiotics.  11) Microvesicle according to any one of claims 1 to 10, characterized in that said microvesicle is combined with at least one determinant exposed to the surface of the microvesicle.  12) Microvesicle according to any one of claims 1 to 10, characterized in that said microvesicle is associated with at least one addressing determinant.  13) Microvesicle according to any one of claims 1 to 12, characterized in that it contains a nucleic acid fragment introduced in vitro.  14) A method for producing labeled microvesicles, characterized in that: (a) a detectable signature is introduced or is produced or labeled, in a microvesicles-producing cell of wild or mutated bacterial origin, (b) said bacterium is cultured in a solid medium or in a liquid medium.  15) A method according to claim 14, characterized in that when the signature is a detectable intravesicular protein or a protein which fixes a detectable cofactor, said cell has a folded protein export system. <Desc / Clms Page number 16>  16) Method according to any one of claims 14 or 15, characterized in that said bacterium is selected from mutants tol, pal or Ipp.  17) Method according to any one of claims 14 to 16, characterized in that is introduced into a cell of bacterial origin a plasmid that allows the production of a detectable signature.  18) Method according to claim 17, characterized in that said plasmid contains a gene coding for a fluorescent protein.  19) Method according to any one of claims 14 to 16, characterized in that is marked, in particular by radiolabelling, or is detected without prior marking the natural components of the outer membrane or intravesicular content, without that it is necessary to introduce a plasmid, especially using in vitro radiolabelling or labeling with fluorescent probes.  20) Method according to claim 19, characterized in that said natural component is a cofactor fixed by a protein.  21) Method according to any one of claims 14 to 20, characterized in that said method comprises a prior step of exporting protein sequences inducing the destabilization of the membrane integrity and the production of outer membrane microvesicles.  22) Method according to claim 21, characterized in that said destabilizing protein sequences may be chimeric proteins, autofluorescent protein-destabilizing protein type of the integrity of the outer membrane or proteins co-produced with the detectable protein in the same bacterium. <Desc / Clms Page number 17>  23) Method according to any one of claims 14 to 22, characterized in that said method further comprises a microvesicles purification step.  24) A method according to any one of claims 14 to 23, characterized in that one purifies microvesicles containing the membrane protein FhuA and then is introduced in vitro in the microvesicle, labeled or not, a nucleic acid fragment .  25) Method according to any one of claims 14 to 24, characterized in that it further comprises a step of quantitative and / or qualitative measurement of microvesicles marking.  26) The method of claim 25, characterized in that said signature is a fluorescent protein, and said method comprises a step of measuring the fluorescence of the labeled microvesicles.  27) Use of labeled microvesicles according to any one of claims 1 to 10, in the preparation of a composition for the in vivo detection of said vesicles, and in particular for monitoring their fate in contact with other bacteria or cells.  28) Use of the labeled microvesicles according to any one of claims 1 to 13, in particular microvesicles containing a nucleic acid fragment, in the preparation of a composition for vectorization.  29) Use of the labeled microvesicles according to any one of claims 1 to 13, in the preparation of a composition for the targeting of molecules of therapeutic interest or diagnosis.  30) Use of the labeled microvesicles according to any one of claims 1 to 13 for measuring the impact of an active ingredient or a <Desc / Clms Page number 18>  active molecule, in particular of the enzyme or detergent type, on the integrity of the membrane of the microvesicle.  31) Use of the labeled microvesicles according to any one of claims 1 to 13 for the identification of an active agent on a bacterium, characterized in that the production of microvesicles of said bacterium is stimulated, in particular by an agent of stimulation, and said agent is identified by visualization of labeled microvesicles.