FR2867388A1 - Reducing concentration of infectious prions, useful for treating animal tissue and fodder to prevent spread of spongiform encephalopathy, by incubation with bacteria that secrete hydrolytic enzymes active on prion protein - Google Patents

Abstract

Reducing the concentration of infectious prion protein (I) in organic material (A), as liquid, particles or fibers, that is (suspected of being) contaminated by (I), comprises: (a) treating (A) with at least one bacterium that secretes one or more hydrolytic (including proteolytic) enzymes (E) active against (I) and (b) incubating for long enough to produce the required reduction in (I) content. An independent claim is also included for a cleaning composition that reduces the concentration of (I) comprising at least one of the specified bacteria and a support.

Description

Field of the invention

  The present invention relates to compositions and methods for the destruction of infectious prion proteins associated with spongiform encephalitis affecting animal species, in particular bovine and ovine species. More specifically, the invention relates to the use of bacteria for the destruction of infectious prion proteins contained in animal tissues.

  PRIOR ART Prion proteins are abnormally conformed proteins that are associated with infectious neuro-degenerative diseases. Prion-related diseases in non-human mammalian species include scrapie in sheep, some encephalopathies such as bovine spongiform encephalopathy (BSE), feline spongiform encephalopathy, simian spongiform encephalopathy.

  The pathogenic prion protein catalyzes the transformation of the normal, physiological protein into an insoluble amyloid form. Prion proteins of mammals include a high degree of homology (85 to 97%). These proteins are therefore capable, after introduction into another organism, to cause the transformation of endogenous normal prion proteins (Prusiner 1998). The infection of cattle has thus been associated with the presence in their food of animal meat and bone meal, hereinafter referred to generically as animal meal, from animals infected with a pathogenic prion protein.

  The existence of animal meal from animals contaminated with BSE, due to the accumulation of the insoluble amyloid form of the prion protein, presents many economic, technical and environmental problems.

  Indeed, the insoluble amyloid form of the prion protein (PrPsc), which is the infectious and pathogenic form of the protein, is very stable. This protein is rich in p-sheets, resistant to heat and the most active proteolytic enzymes, such as proteinase K (Prusiner, 1998). Therefore, in many countries today, animal products that could be used as a nutritional supplement for animals, such as animal meal, are incinerated. Then, the ash residues are destroyed so as to prevent transmission of the infectious prion protein to other animals.

  In Europe, the use of animal meal and their by-products as food has been banned. In the United States, several cases of bovine spongiform encephalitis have been reported, and industrialists have been forced into stricter regulations to prevent the development of prion-related diseases.

  A wide variety of tests to determine the presence of infectious prion proteins in animal tissues has been developed, including Western blot assays, sandwich-type immunodetection assays, ELISA assays, fluoro-immunological assays, assay tests, and assays. capillary electrophoresis, and plasminogen binding assays. However, few industrially applicable methods for destroying the infectious prion protein have been developed.

  Infectious prion proteins are resistant to conventional methods of destruction that can denature proteins, including, for example, autoclaving, freezing, and exposure to highly deleterious reagents for bacteria, such as formaldehyde, carboxylic acid, and the like. chloroform. By way of illustration, the infectious prion protein is resistant to temperatures of the order of 200 ° C. At present only incineration or treatment with bleaching agents are used in practice to destroy the pathogenic isoform of the prion protein.

  Another method of destroying the prion protein is described in patent application WO 02/083082. This method involves the use of enzymes, including keratinases from a bacterium Bacilus licheniformis to reduce the amount of infectious prion protein. Nevertheless, this method has technical and economic disadvantages.

  Indeed, the implementation of this method involves many steps such as the culture of bacteria, the extraction of proteolytic enzymes or the storage of the latter, which can be expensive on an industrial level.

  There is thus in the state of the art a need for novel compositions and methods for destroying the infectious prion protein that are applicable to the treatment of biological materials, such as animal tissues, comprising an infectious prion protein.

  This need is all the more important as more than 180,000 cases of BSE and 100 cases of Creutzfeldt-Jakob disease have been reported in Europe since 1992, and cases in the human species are expected to increase significantly. The expansion of these diseases is difficult to contain since such diseases do not benefit today from any therapeutic treatment. In particular, the pathogenic prion protein is not immunogenic. Significant efforts have been made to study BSE, as well as the process of contamination of human food by the prion protein.

  In the human species, neurodegenerative diseases associated with prion proteins include Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal insomnia, kuru, and variants of Creutzfeldt-Jakob disease. These human pathologies are associated with the consumption of meat food (beef infected with BSE, causative agent of the new variant of Creutzfeldt Jakob disease), with the use of human growth hormone contaminated with infectious prions ( causing Creutzfeldt-Jakob disease) and ritual cannibalism (which causes Kuru's disease).

  The search for new compositions and new methods for destroying the infectious prion protein is therefore of importance, both in the agronomic domain and in the field of human health.

  Advantageously, these new techniques should preferably be less energy consuming, technically simpler, and more economical than known techniques. In addition, these new techniques should not result in the total destruction of the material to be decontaminated as may be the case with incineration.

Summary of the invention

  The invention relates to a method for reducing the concentration of infectious prion protein of a material in liquid form, in particle form or in fiber form, contaminated or suspected of being contaminated with the infectious prion protein comprising: - a step (a) contacting and mixing said contaminated material with at least one bacterium which secretes one or more hydrolytic enzymes, including proteolytic enzymes, with respect to said infectious prion protein and a step (b) of incubating the bacteria or bacteria with said contaminated material for a period of time sufficient to obtain a desired percentage reduction in the concentration of infectious prion protein in said contaminated material.

  The invention also relates to processes as defined above, for which step (a) is preceded by a step of heating the contaminated material to a temperature and for a period of time sufficient to increase the sensitivity of the infectious prion protein to proteolysis.

  The invention also relates to a cleaning composition for reducing the concentration of infectious prion protein of a contaminated material or suspected to be contaminated with the infectious prion protein comprising at least one bacterium as defined in the above process.

Description of the fictures

  Figure 1 Zymograms of supernatants from thermophilic bacteria and S290 SDS-PAGE 10%, lane 1: supernatants of strain AT1243 (incubation at 80C), lane 2: supernatants of strain AT1222 (incubation at 80C). lane 3: supernatants of strain S290.

  Figure 2 Hydrolysis of the recombinant prion protein by supernatants of thermophilic bacteria.

  (a) lanes 1 to 6: native prion protein, prion protein hydrolysed by the supernatants of strains S290 (60 C), AT1243 (80 C), AT1222 (80 C), AT1243 (60 C), and AT1222 (60 C) For five hours, respectively.

  (b) lanes 1 to 6: thermally denatured prion protein (PrPde) PrPden hydrolyzed by supernatants of strains S290 (60C), AT1243 (80C), AT1222 (80C), AT1243 (60C), and AT1222 ( 60 C) for five hours, respectively.

  (c) lanes 1 to 6: native prion protein in the presence of Cul + (PrPc "), PrPc" hydrolyzed by the supernatants of strains S290 (60C), AT1243 (80C), AT1222 (80C), AT1243 (60C) ), and AT1222 (60 C) for five hours, respectively.

  FIG. 3 Hydrolysis of animal meal by the supernatants of thermophilic bacteria and by proteinase K. (a) Lanes 1 to 16 represent respectively: wool / hair flours (1), wool / hair flours hydrolysed by the supernatants of strains AT1243 at 80 C (2), AT1222 at 80 C (3), and S290 at 60 C (4) meat flours (5), meat flours hydrolysed by the supernatants of strains AT1243 (6) and AT1222 ( 7), and S290 (8) blood flours (9), blood flours hydrolysed by the supernatants of strains AT1243 (10) and AT1222 (11), and S290 (12) mixed flours (13), flours mixed hydrolyzed by supernatants of strains AT1243 (14) and AT1222 (15) and S290 (16). The incubation times are 20 hours.

  (b) Lanes 1 to 8 represent respectively: wool / hair flours (1), wool / hair flours hydrolyzed by proteinase K (1 μM, 37 C) (2), meat flours (3) , protein-K 4 hydrolyzed meat-and-bone meal, blood meal (5), Proteinase K-hydrolyzed blood meal (6), mixed flours (7), protein-K-hydrolyzed mixed flours (8) ). The incubation times are 20 hours.

  Detailed description of the invention

  On the basis of the fact that the methods available in the state of the art for destroying the prion protein are rather complex and have technical or economic disadvantages, the inventors have endeavored to look for bacteria capable of multiplying on the substrates of the invention. decontaminate and simultaneously produce in situ proteolytic enzymes effective against the prion.

  In the first place, solving the problem of reducing the pathogenicity of animal meal with the aid of microorganisms has required, on the part of the inventors, the preliminary study of the activity of proteases secreted by such microorganisms on microorganisms. model substrates.

  Keratin has proved to be a good model substrate because it has many structural similarities with the infectious prion protein which gives these enzymes a high resistance to proteolysis. Mention may in particular be made of the insolubility of keratins due to the presence of numerous intra- and inter-molecular disulfide bridges, the important content in sheets (3, in the case of F3 keratins, and their important degree of aggregation (Arai et al., 1996, Goddard and Michaelis, 1934. Such proteins can be destroyed only by highly active proteases, such as subtilisins secreted by bacteria belonging to the genus Bacillus (Atalo and Gashe, 1993, Cheng et al., 1995; et al., 1992, Williams et al., 1990; Kristie et al., 2000) or by proteases active under denaturing conditions which destabilize the proteolysis-resistant substrates (Han and Damodaran, 1997). Substrate models insoluble keratin, forming fibrillar structures similar to PrPsc.

  Secondly, the resolution of the problem of reducing the pathogenicity of animal meal with the aid of microorganisms necessitated the selection of microorganisms producing proteolytic enzymes destroying the infectious prion protein which are able to multiply without nutritive supply. exogenously, on the substrate material to be decontaminated.

  To achieve this objective, the inventors have screened collections of microorganisms capable of growing on keratin or on animal meal and secreting proteases capable of degrading the PrPsc contained in animal meal. The inventors have discovered, according to the invention, thanks to this screening method, bacteria capable of degrading the PrPsc infectious prion protein and of growing on a medium composed of animal meal.

  The present invention is based on the use of thermophilic bacteria and bacteria belonging to the genus Streptomyces for the degradation of the infectious prion protein contained in tissues or animal meal.

  A first object of the present invention relates to a method for reducing the concentration of infectious prion protein of an organic material in liquid form, in particle form or in fiber form, contaminated or suspected to be contaminated with the infectious prion protein. comprising: - a step (a) of contacting and mixing said contaminated material with at least one bacterium which secretes one or more hydrolytic enzymes, including proteolytic enzymes, with respect to said infectious prion protein and, one step ( b) incubating the bacteria or bacteria with said contaminated material for a period of time sufficient to obtain a desired percentage reduction in the concentration of infectious prion protein in said contaminated material.

  The effectiveness of the method of the invention for degrading infectious prion protein was unexpected since exposure to elevated temperatures and enzymes such as proteinase K, known to degrade amyloid structures, is not sufficient to degrade the prion protein. It is therefore very surprising that the incubation of bacteria with the infectious prion protein succeeds in destroying the latter, at temperatures compatible with the survival of the bacteria.

  By hydrolytic enzyme is meant an enzyme usually referred to as hydrolase, belonging to class E.C.3 according to the International Classification of Enzymes.

  Preferably, the hydrolytic enzyme is a proteolytic enzyme or protease, belonging to the class E.C.3.4 according to the international classification of enzymes.

  By the terms contacting and mixing, the skilled person will understand that the bacteria are for example deposited on the contaminated material or likely to be contaminated by PrPsc, and then mixed with the contaminated material, so as to increase the surface of contact between the bacteria and the contaminated material.

  By the term incubation, it should be understood that the bacteria are placed in conditions that allow their survival, preferably conditions close to those which are usual in their natural environment. In particular, the temperature at which the bacteria are subjected during their incubation with the contaminated material will not be less than 20 C.

  The term organic matter in the form of particles includes organic matter in the form of powder and flour.

  The term contaminated organic material includes animal tissues, animal meals, their derivatives, and soils. The percentage reduction in the concentration of non-proteolyzed infectious prion protein means the value V, expressed in%, obtained using the following formula: V = [(XY) / X] * 100 In which: X is a value representative of the amount of unproteolysed prion protein in the contaminated material prior to treatment by the method according to the invention and Y is a value representative of the amount of unproteolysed prion protein in the contaminated material after treatment with the method according to invention.

  Preferably, the V value is at least 80%, preferably at least 90%, preferably at least 95%, most preferably at least 98%, and most preferably 100%.

  The value V of the percentage reduction in the concentration of non-proteolyzed infectious prion protein can be easily measured by those skilled in the art by techniques which are in themselves common.

  As an illustration, those skilled in the art use the electrophoresis technique advantageously comprising the following steps: (i) depositing a sample of organic material treated by the reduction method described above at the end of an electrophoresis gel (ii) migrating proteins present in the organic matter deposition on the electrophoresis gel, (iii) after protein migration, determining the amount Y of non-proteolyzed infectious prion protein, and (iv) calculating the value V at using the X value of amount of non-proteolyzed infectious prion protein in the organic material before treatment.

  The amount of non-proteolyzed infectious prion protein can be determined, for example, by contacting the surface of the electrophoresis gel with a solution containing labeled antibodies specific for this protein and then quantifying the intensity of the labeling in the gel region. electrophoresis in which proteins having a molecular weight identical or close to that of the unhydrolyzed infectious prion protein migrate. Antibodies specifically recognizing the infectious prion protein will be described hereinafter in more detail.

  The value X can be determined by applying steps (i) to (iii) of the above process to a sample of organic material before treatment by the method according to the invention.

  In order to calculate the percentage reduction in the concentration of infectious prion protein at the end of the process, the method according to the invention comprises an additional step (c) of measuring the concentration of infectious prion protein in the contaminated material. at the end of step (b). Advantageously, step (c) of the method according to the invention comprises carrying out a test selected from the group comprising: a Western Blot, a sandwich type immunoassay, a fluoroimmunological test, an immunoelectrophoresis, a test of plasminogen binding.

  In particular, those skilled in the art can perform immunoelectrophoresis to detect the infectious prion protein according to the method described in US Pat. No. 6,514,707. Those skilled in the art may also carry out the methods described above by using antibodies directed against the infectious prion protein derived from the hybridomas registered under the references ATCC HB-12403 and ATCC HB-12696 respectively described in patents US 6,165,784 and US Pat. No. 6,261,790. Those skilled in the art will also be able to measure the amount of infectious prion protein in the biological material before or after treatment with the method according to the invention by means of the kit for detecting the prion protein described in US Pat. Advantageously, the process of the invention is carried out in a general process for recycling contaminated materials, in which these materials are placed in a tank, before being traversed by a stream of water vapor. under pressure. In such a case, step (a) of the method of contacting and mixing the contaminated material with a bacterium, is done when the contaminated material is placed in the tank, and the incubation step (b) is performed during the establishment of the steam flow under pressure.

  Such an embodiment is particularly advantageous because it allows, through the use of water vapor, to use a wider variety of bacteria.

  Preferably, the bacteria used to carry out the process of the invention were screened during which the bacteria are selected according to their ability to grow on keratin, as illustrated in Example 3. In in addition, the bacteria may be subjected to a second screening step in which said bacteria are selected for their ability to degrade oligomers derived from the prion.

  This double screening makes it possible to have bacteria that are capable of growing on a medium consisting of animal meal, and which are also capable of degrading the prion protein, within these flours.

  In a particular embodiment of the invention, the bacteria used to carry out the process of the invention are chosen from anaerobic thermophilic bacteria and bacteria belonging to the genus Streptomyces.

  In a particular embodiment of the invention, said bacterium is a thermophilic anaerobe bacterium selected from the group comprising the AT1243 strain belonging to the Archaea Thermococcus species, deposited with the National Collection of Microorganism Cultures of the Institute. Pasteur (CNCM) under the registration number CNCM 1-3140, and the strain AT1222 belonging to the species Thermosipho, registered with the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) under the registration number CNCM 1-3139.

  Alternatively, the bacterium chosen is a bacterium of S290 strain belonging to the genus Thermoanaerobacter, deposited with the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) under the registration number CNCM 1-3177.

  The arrangement of several kinds and species of bacteria makes it possible to apply the process of the invention to substrates of variable porosity, grain size, and chemical composition. For example, depending on the compactness of the contaminated material to be treated and therefore the importance of gas exchange within the material to be treated, a thermophilic anaerobe bacterium or an aerobic bacterium belonging to the genus Streptomyces will be chosen to carry out the method. Those skilled in the art may also consider making mixtures between bacteria of different species to carry out the process of the invention.

  Nevertheless, the process according to the invention is not limited solely to the use of the bacterial strains described above but also comprises other bacteria having similar enzyme characteristics to those described in Examples 1 and 2 and in the table. 1 for the bacteria belonging to strains AT1243, AT1222 and S290. To compare the enzymatic characteristics of different bacteria, those skilled in the art will use conventional techniques such as zymograms or peptide degradation assays as illustrated in Examples 1 and 2 and in Table 1.

  A primary benefit of using living bacteria directly on the organic material to be treated is that the bacteria (i) produce the specific enzymes continuously and (ii) multiply in said organic material, which further amplifies the destructive effect vis-à-vis the infectious prion protein.

  A second advantage is that living bacteria simultaneously produce a variety of proteolytic enzymes of varying peptide bond cut-off specificity, further increasing the killing efficiency of the infectious prion protein.

  In a preferred embodiment of the invention, step (a) is carried out at a temperature between 20 C and 150 C.

  This temperature range makes it possible to reconcile at the same time the requirements necessary for the survival of bacteria, the activity of the proteolytic enzymes synthesized by them, and the sensitivity of the prion protein to proteolytic enzymes.

  As the temperature range described above is rather wide, the inventors have endeavored to find temperature optima for carrying out the method according to the invention, which are the subject of particular embodiments, described below: In a particular embodiment of the invention step (a) is carried out using strain AT1243 or AT1222 at a temperature of 80 ° C.

  Alternatively step (a) is carried out using strain S290 at a temperature of 60 C.

  Furthermore, the tests carried out by the inventors made it possible to measure the minimum duration of step (b) compatible with complete disappearance of the prion protein. In a particular embodiment of the invention, the incubation step (b) has a duration of 4 hours. The embodiments described above are based on Examples 3 and 4, showing that enzymatic extracts of strains AT1243, AT1222 and S290 are capable of growing on animal meal and completely degrading the native or denatured prion protein after 4 hours. at 5 hours of incubation at a temperature of 60 ° C. for strain S290 or 80 ° C. for strains AT1243 and AT1222.

  In a preferred embodiment of the invention, step (a) is preceded by a step of heating the contaminated material at a temperature and for a period of time sufficient to increase the sensitivity of the infectious prion protein to the proteolysis. Performing a step of the process prior to step (a) of contacting and mixing the bacteria with the contaminated material makes it possible to increase the sensitivity of the protein to proteolytic enzymes and consequently to lower the temperature. necessary for the correct operation of step (b). This alternative of the method according to the invention therefore has an advantage in the case where prolonged heating of the contaminated material during step (b) is not possible, for economic or technical reasons, for example.

  In a preferred embodiment of the invention, said step of heating the contaminated material is carried out at a temperature of 80 ° C. and in a particular embodiment of the process described above, step (a) is carried out at a temperature of 80 ° C. temperature between 20 C and 80 C.

  In another particular embodiment of the method described above, step (a) is carried out using a bacterium belonging to the genus Streptomyces, at a temperature of 20 C, for 20 to 40 hours.

  The embodiments described above are particularly advantageous since it is known to those skilled in the art that the use of proteinase K under the same conditions would have led to a partial degradation of the prion protein into large fragments of 14 kDa. which is not the case with the method of the invention.

  In order to ensure total safety of the organic material treated by the process of the invention, it is possible to provide an additional step of heating the contaminated material to a temperature, pressure and for a period of time sufficient to destroying bacteria introduced during step (a). For example this temperature can be 120 C, and this pressure can be 1.5 at. At the end of this heating step, the contaminated material is freed of the prion protein, and the bacteria introduced to carry out step (a) of the process are destroyed. In the case where the process is applied to animal meal these can then be reused as a food supplement or fertilizer for example, which is an economically attractive alternative to incineration.

  In a particular embodiment of the process of the invention, copper ions are added during step (a).

  The addition of copper ions to the contaminated material to be treated makes it possible to increase the efficiency of the degradation of the prion protein by the bacteria described above. As illustrated in Example 4, the addition of copper ions to strains AT1243 and AT1222 makes it possible to increase the efficiency of the degradation of the prion protein.

  Preferably, the contaminated material is selected from the group consisting of: animal tissues, animal meals, and derivatives thereof Cleaning compositions Another object of the invention is to provide cleaning compositions which reduce the concentration of infectious prion protein of a contaminated material or suspected to be contaminated with the infectious prion protein comprising at least one bacterium as defined in the above description, in combination with a suitable carrier.

  In this embodiment, the cleaning composition comprises the cells of the strain (s) of bacteria as defined above, preferably in the form of particles.

  The manufacture of a cleaning composition in the form of particles can be done for example by lyophilization of the bacteria that will be associated with a support. The support in which the bacteria are incorporated can be of various kinds. For example, said support may consist wholly or essentially of starch, including rice or maize starch which has been mixed with the bacterial cells before its transformation into particles, for example into granules.

  The support in which the bacterial cells are incorporated may also consist exclusively or consists essentially of 6-cyclodextrin, the 33-cyclodextrin then being mixed with the bacterial cells prior to a drying step, and then to manufacture particles, in particular granules.

  For example, for the manufacture of a cleaning composition according to the invention, one skilled in the art can mix the β-cyclodextrin with a suitable amount of Streptomyces aerobic bacterial cells, so as to obtain a composition comprising from 103 to 108 colony-forming units (cfu) of said bacterium (s) per gram of the final composition containing the β-cyclodextrin, and then subjecting the composition thus obtained to a drying step, before the production of particles, in particular granules, according to conventional methods.

  Preferably, the support may be composed of calcium carbonate, optionally in combination with lactose. The support used for the manufacture of a composition according to the invention may also consist exclusively or essentially of lactose, where appropriate in combination with sodium silico-aluminate, to which is added the culture medium of the strain or mixture of bacterial strains of interest, which has been previously subjected to a drying step.

  In another aspect, to manufacture a food prophylactic composition according to the invention, one skilled in the art can use commercial supports, such as porous glass, Porolon, carbon fibers, alginate or chitin.

  In a preferred embodiment, the cleaning composition according to the invention may comprise copper ions. Without wishing to be bound by theory, the inventors believe that the addition of copper ions at a concentration of 1 mM to the composition of the invention makes it possible to increase the effectiveness of the composition with respect to degradation. of the prion protein.

  Materials and Methods The following compounds, namely salts, media, bovine insulin B-chain, bovine gelatin, MOPS and all other chemical compounds were obtained from Sigma Chemical Company St. Louis MO. Purified preparations of recombinant ovine prion were obtained as described by Rezaei et al, 2000. Chicken keratin was obtained from Yu. Zuev, Kazan Center of Science, Russian Academy of Sciences, Kazan, Russia. Pig hairs were obtained from SIFDDA, Plouvara, France.

  Bacterial strains, media and growth conditions The anaerobic thermophilic bacterial strains were isolated from strains collected at the Rainbow site (36, 14 'North, 33, 54' West, 2300 meters deep). Strains AT1243 and AT1222 were isolated from a heat sink. Strain AT1222 was enriched at 60 ° C. in SP medium (30 g / l of marine salt and 2 g / l of porcine hair). The strain AT1243 was enriched at 80 ° C. in SPS medium (SP + 5 g / l of sulphide). The subcultures performed for the protease assays were carried out under the same conditions. PCR amplifications and sequencing indicate that strain AT1222 belongs to the species Thermosipho sp. (thermophilic bacterium) and that strain AT1243 is an archaic bacterium belonging to the thermophilic species Thermococcus genus.

  Strain S290 was isolated from the hot spot of Uzon caldera, Kamchatka and was identified as belonging to the genus Thermoanaerobacter by reactions with specific oligonucleotides namely oligonucleotides directed against 16S-rRNA. Strains S290 were cultured at 60 ° C for 5 days on Th mineral medium (0.33 g / l KH2PO4, 0.33 g / l NH4Cl, 0.33 g / l MgCl2, 0.33 g / l 1 g of CaCl 2, 0.33 g / l of KCl, 0.5 g / l of NaHCO 3, and 0.5 g / l of Na 2 S), supplemented with 2 g / l of pig wool or feathers.

  When the different species and strains of bacteria are cultured on animal meal, the keratin of all media has been replaced by 2 g / l of meat, bone, blood or flours meal comprising a mixture of these three components .

  Determination of proteases After culture, the bacteria and insoluble elements of the culture medium were precipitated by centrifugation for 15 minutes at 7000 rpm. Proteolytic and peptidase activities were measured in the resulting supernatants.

  The presence of proteases in the supernatants was determined by zymograms. This method makes it possible to determine the presence of proteolytic enzymes at low concentration and consists of identifying proteases after separation of all the protein components of the medium by electrophoresis. Proteases were separated by SDS-PAGE including hydrolyzed gelatin substrates. The low molecular weight products resulting from the hydrolysis of gelatin diffuse gel and, after staining, the presence of proteases has been identified by the presence of white bands in a colored gel. The polyacrylamide gel was polymerized in the presence of 0.01% bovine gelatin. 20 μl of lysis buffer was added to 40 μl of supernatant and 10, 12 or 15% SDS-PAGE gels were made (Laemmli, 1970). After electrophoresis, the gels were rinsed with 2.5% Triton X-100 solution to remove SDS, and incubated in 20 mM MOPS buffer, pH 7.2, 100 mM NaCl, 2 mM CaCl 2 different temperatures for 3 to 10 hours. The proteins on the gels were stained with silver nitrate (Nesterenko et al., 1994).

  The activity of proteases from supernatants of bacteria grown in hydrolysed keratin, in animal meal or with recombinant prion was determined by electrophoresis. 50 μl of supernatants were added to 50 μl of feather keratin at 1 mg / ml, or 2 mg / ml of pig hair keratin, or 2 mg / ml of animal meal or 1 mg / ml of recombinant prion in 20 mM MOPS buffer, pH 7.2 and incubated at different temperatures for 20-40 hours. Hydrolysis of the substrate with proteinase K [0.02 mg / ml (0.6 μM)] was carried out at 37 ° C. The lysis buffer was added to the aliquots from different proteolytic reactions. The aliquots were boiled for 10 minutes and then a 15% SDS-PAGE gel was made. The proteins on the gels were stained with Coomassie Brilliant R-450 blue.

  Specificity of proteases The peptidase activity towards amino acids or N-substituted peptides by p-nitroanilides was determined using a spectrophotometer. 900 μl of 0.1 mM substrate was added to 100 μl of supernatants in 20 mM MOPS buffer, pH 7.2 DMSO 10%. The reaction mixture is incubated at different temperatures for 3 and 20 hours, after which the absorption at 410 nm was measured (8410 p-nitroanilides = 8800 M-1 cm -1).

  The specificity of proteases from bacteria supernatants was determined by measuring the fragmentation of the oxidized B chain of bovine insulin. 100 μl of supernatant was incubated with 100 μl of insulin B-chain at 2 mg / ml in 20 mM MOPS buffer, pH 7.2 at different temperatures for 10 hours. The products of the hydrolysis were separated by reverse phase HPLC on a Nucleosil 300-5 C18 column with a gradient of 0 to 60% CH3CN, 0.1% TFA. The fractions collected were subjected to amino acid analyzes according to Bidlingmeyer et al. (1984). The amino acid content from the separated peptides was calculated by comparing the chromatograms obtained with standard chromatograms from PITC derivatives. The amino acid sequence of the peptides was established through knowledge of the primary structure of the B chain of insulin.

  Results Example 1 Zymograms of proteases from the microorganisms of interest In order to characterize the proteases secreted by the cultures of bacteria of interest, zymograms were performed on the supernatants from such cultures. The main characteristic of the proteases of the bacteria of interest is the stability of their structure to denaturation. Indeed, as shown in Figure 1, lanes 1 and 2, proteases subjected to electrophoresis retain their proteolytic activity. Zymograms made from the supernatants of strain AT1243 show that this strain has a set of proteases having an apparent molecular mass of the order of 45 to 100 kDa. Zymograms made from supernatants of AT1222 strains show a single band of proteolytic activity, corresponding to an approximate molecular mass of 100 kDa.

  Finally, zymograms made from supernatants of strain S290 show bands of proteolytic activity, corresponding to approximate molecular weights of 66, 100, 200 kDa and more (Figure 1, lane 3).

  EXAMPLE 2 Specificity of Proteases The ability of proteases secreted by thermophilic bacteria AT1243, AT1222 and S290 to hydrolyze substrates of low molecular weight, ie the N-benzoylarginine p-nitroanilides, the N-succinyl derivatives of phenylalanine, the Ala-Ala-Pro-Phe peptide and the Ala-Ala-Pro-Lys peptide was studied. After three hours of incubation, the supernatant activity of the S290 strains against the Suc-Ala-Ala-Pro-Phe-pNA peptide was observed (Table 1).

  The hydrolysis of the oxidized insulin B chain, a standard substrate for testing the activity of proteolytic enzymes, was studied. The insulin B chain is composed of a large number of hydrophobic amino acids, and also includes a large number of charged branched residues. The primary structure of the insulin B-chain is as follows: Phe'-Val-Asn-Gln-His-Leu-Cys * -Gly-Sers-His-Leu-Val-Glu-AlaLeu15-Tyr16-Leu-Val * -Cys-Gly-Glu-Arg-Gly-Phe-Tyr-PHE25-Thr-Pro-Lys-Ala.

  (* means that the cysteyl residue is oxidized).

  The inventors have shown using HPLC that the insulin B chain hydrolysis products accumulate in the reaction media after three hours of incubation with the supernatants of strain AT1243. Analysis of the amino acids from the hydrolysed peptides makes it possible to propose that the proteases of strain AT1243 cleave the following peptide bonds: Ser9-His10, Tyr16-Leu17 and Phe25-Tyr26. An additional cleavage site (Leu15-Tyr16) was observed in the case of strain AT1222. The proteases of strain S290 hydrolyze the B chain of insulin at the Leu15-Tyr16 and Tyr16-Leu17 peptide bonds. Thus, the majority of the proteases produced by the thermophilic microorganisms studied have a chymotrypsin specificity. Some of the bacteria also show an unexpected ability to hydrolyze peptide bonds after a seryl residue. Surprisingly, the proteases secreted by the thermophilic microorganisms do not attack the other hydrophobic sites located in the molecule of the B chain of the insulin. Analysis of the microenvironment around the cleavable bonds (the residues in the P2, P3, P1 'positions) does not reveal a strict regularity in the activity of the proteases towards these sites. Nevertheless, it can be observed that a hydrophobic residue is frequently located in the position P2 'of the cleavable bond, and that a flexible residue of glycine or alanine is in position P2 or in position P3. The proteases of the strains selected for the hydrolysis of keratins therefore recognize particular structural units in the substrate molecule. The prion protein contains many hydrophobic residues: Tyr (10), Phe (3), Trp (8), and IIe (4) and many Ser residues (8). Therefore proteases are able to degrade the prion until relatively short peptides are obtained. In addition, access to the cleavage sites will be increased during heating to the optimum temperature of protease hydrolysis, because under such conditions the protein structure will be partially or completely denatured.

  Example 3 Growth of microorganisms studied on media containing keratin or animal meal.

  The ability of thermophilic bacteria, strains S290, strains AT1243 and AT1222 to grow on media containing keratin pig hair or feathers was tested. The selected bacteria are able to use a keratin from pork or feather hair as the main source of nutrients.

  The growth on wool keratin was greatest for strains S290 and AT1222, strain AT1243 providing lower growth characteristics on this medium. The culture time of most bacteria was three days.

  The strains of bacteria studied also proved that they were able to grow on animal meal. In addition, these bacteria grow on animal meal faster than on keratin. The lowest growth was obtained when the substrate used was blood, probably because of the toxicity of some components present.

  EXAMPLE 4 Activity of Proteases Produced by Thermophilic Bacteria to the Recombinant Prion Protein

  It has been demonstrated that the ovine recombinant prion protein is denatured at 80 ° C. and forms amyloid structures. This protein has therefore been used as a model of cellular amyloid structure. The ability of the proteases from the thermophilic bacteria of the invention to hydrolyze the native ovine prion or thermally denatured has been studied.

  Figures 2a and 2b show that the proteases produced by strain S290 hydrolyze the native prion and completely denatured at 60 ° C in 4 hours. The proteases produced by strains AT1243 and AT1222 hydrolyze the native prion protein or thermally denatured at 80 C, which corresponds to their optimum growth temperature. However, at 60 ° C., the prion protein is not completely hydrolysed by these strains. In this case, as seen with proteinase K, a large hydrolytic fragment with a molecular weight of about 14 kDa accumulates. As already demonstrated, the resistance of this fragment to proteolysis is caused by its ability to aggregate. The formation of these aggregates is dependent on the concentration and activity of the proteases. If the concentration and activity of the proteases are lower than a critical value, the products of this limited proteolysis aggregate and become completely resistant to future hydrolysis. At 80 C, the aggregates are destabilized and / or denatured and the activity of the proteases increases considerably, therefore the accumulation of the 14 kDa fragment does not appear. The inventors have observed that the introduction of copper ions can facilitate the transformation of the prion molecules into their amyloid form. Using this model of amyloid structure, it has been shown that the proteases of strains S290 (at 60 ° C.), AT1243 and AT1222 (at 80 ° C.) completely hydrolyze the prion protein in 4 hours (FIG. 2c). During the study of the proteolytic activity of AT1243 and AT1222 strain proteases at 80 ° C., the accumulation of large hydrolytic prion fragments was not observed in the case of the hydrolysis of the native and denatured prion, unlike the activity of these strains at 60 C (Figure 2c). So amyloid structures are destabilized at 80 C facilitating their destruction by thermophilic proteases.

  Example 5: Proteolytic Activity of Bacterial Culture Supernatants

  After 24 hours of incubation with wool / hair, meat, blood and mixtures with the supernatants of strains AT1243, AT1222, and S290 a partial dissolution of the substrate was observed. Figure 3a shows that proteases of strains AT1243 and AT1222 effectively hydrolyze meat flours, wool / hair flours, blood flours and mixed flours. The enzymes of strains S290 show a lower but satisfactory activity. This could result from the low incubation temperatures of 80 C in the case of strains AT1243 and AT1222 and 60 C in the case of strain S290.

  In comparison, the data obtained for Proteinase K activity towards flour proteins are also shown in Figure 3b. Proteinase K effectively hydrolyzes wool / hair flours and with lower efficiency, meat flours and mixed flours. Proteinase K is virtually inactive against blood meal.

  TABLE 1 Activity of proteases (percentage of substrate hydrolysis) of supernatants of thermophilic bacteria to chromogenic substrates of low molecular weight. The reaction conditions are given in the material and method part. The substrates were incubated for three hours at 80 ° C (AT1243 and AT1222) and at 60 ° C (S290). The strains are represented in the left column, the substrates are represented on the first line.

  Bzl-Arg-pNA Suc-Phe-Suc-Ala-Ala-Suc-Ala-Ala-pNA Pro-Phe-pNA Pro-Lys-pNA AT1243 0.8 0.6 0 0 AT1222 1.0 0.6 1, S290 0.1 0.4 0.3 References Arai, K., Naito, S., Dang, VB, Nagasawa, N., Hirano, M. (1996). Cross-linking structure of keratin. VI. Number, type, and location of disulfide cross linkages in Iow-sulfur protein fiber and their relationship to permanent set. J. Appt. Polym. Sci 60, 169-179.

  Atalo, K., Gashe, B.A. (1993). Protease production by a thermophilic Bacillus species (P-001A), which degrades various kinds of fibrous proteins. Biotechnol. Lett. 15, 1151-1156 Bidlingmeyer, B.A., Cohen, S.A., and Tarvin, T.L., 1984. Rapid analysis of amino acids using precolumn derivatization. J. Chromatogr. 336, 93-104.

  Cheng, S.W., Hu, H.M., Shen, S.W., Takagi, H., Asano, A., Tsai, Y.C. (1995). Production and characterization of keratinase of a featherdegrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem. 59. 2239-2243 Goddard, D.R., Michaelis, L.k (1934). A study on keratin. J. Biol. Chem. 106, 604-614.

  Han, X.Q., Damodaran, S. (1997). Stability of protease against autolysis and sodium dodecyl sulphate and urea solutions. Biochem. Biophys. Res. Common. 240, 839-843.

  Kristie, L., Evans, J.C., Crowder, J., Miller, E.S. (2000). Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers. Can. J. Microbiol. 46, 1004-1011.

  Laemmli U.K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 225, 650-30657 Lin, X., Lee, C.G., Gasale, E.S., Shih J.C.H. (1992). Purification and characterization of a keratinase from a feather degrading Bacillus licheniformis strain. Apt. About. Microbiol. 58, 3271-3275.

  Nesterenko, M.V., Tilley, M., Upton, S.J. (1994). A simple modification of Blum 's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. J. Biochem. Biophys. Methods 28, 239-242.

  Prusiner, S.B. (1998). Let us pray. Proc. Nat /. Acad. Sci. USA 95, 13363-13383.

  Rezaei, H., Mark, D., Choiset, Y., Takahashi, M., Hui Bon Hoa, G., Haertle, T., Grosclaude, J., Debey, P. (2000). High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep protein prion protein linked to scrapie susceptibility. Eur. J. Biochem. 267, 2833-2839.

  Williams, C.M., Richter C.S., Mackenzie, Jr., J.M. Shih, J.C.H. (1990). Isolation, identification and characterization of a featherdegrading bacterium. Apt. About. Microbiol. 56, 1509-1515.

Claims (16)

  A method for reducing the concentration of infectious prion protein of an organic material in liquid form, in particle form or in fiber form, contaminated or suspected of being contaminated with the infectious prion protein comprising: - a step (a) comprising contacting and mixing said contaminated material with at least one bacterium which secretes one or more hydrolytic enzymes, including proteolytic enzymes, with respect to said infectious prion protein and - a step (b) of incubation of the or bacteria with said contaminated material for a period of time sufficient to obtain a desired percentage reduction in the concentration of infectious prion protein in said contaminated material.   2 Process according to claim 1, characterized in that said bacterium is a thermophilic anaerobe bacterium chosen from the group comprising the AT1243 strain belonging to the archaea species Thermococcus, deposited with the National Collection of Microorganism Cultures of the Institute Pasteur (CNCM) under the registration number CNCM 1-3140, and the strain AT1222 belonging to the species Thermosipho, registered with the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) under the registration number CNCM I-3139.   3 Process according to claim 1, characterized in that said bacterium is a bacterium of strain S290 belonging to the genus Thermoanaerobacter, deposited with the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) under the registration number CNCM 1 -3177.   4 Process according to any one of claims 1 to 3, characterized in that step (a) is carried out at a temperature between 20 C and 150 C.   Process according to Claim 2, characterized in that step (a) is carried out at a temperature of 80 ° C.   Process according to Claim 3, characterized in that step (a) is carried out at a temperature of 60 ° C.   Process according to any one of claims 1 to 6, characterized in that step (b) has a duration of four hours.   Process according to any one of claims 1 to 7, characterized in that step (a) is preceded by a step of heating the contaminated material at a temperature and for a period of time sufficient to increase the sensitivity. from infectious prion protein to proteolysis.   9 Process according to claim 8, characterized in that said step of heating the contaminated material is carried out at a temperature of 80 C.   Process according to Claim 8 or 9, characterized in that step (a) is carried out at a temperature of between 20 ° C and 80 ° C.   Process according to any one of claims 1 to 10, characterized in that it comprises an additional step (c) of measuring the concentration of infectious prion protein in the contaminated material, at the end of step (b). ).   Process according to claim 11, characterized in that step (c) comprises carrying out a test chosen from the group comprising: a Western blot, a sandwich immunoassay, a fluoroimmunological test, an immunoelectrophoresis, a test plasminogen binding.   Process according to any one of claims 1 to 12, characterized in that it comprises an additional step of heating the contaminated material at a temperature and for a period of time, sufficient to destroy the bacteria introduced during the course of time. step (a).   Process according to one of Claims 1 to 13, characterized in that copper ions are added during step (a).   Process according to one of Claims 1 to 14, characterized in that the contaminated material is chosen from the group comprising: animal tissues, animal meals, and their derivatives.   Cleaning composition that reduces the concentration of infectious prion protein of a material contaminated or suspected of being contaminated with the infectious prion protein comprising at least one bacterium as defined in one of claims 1 to 3, in combination with an appropriate carrier .   Cleaning composition according to Claim 16, characterized in that it comprises copper ions.