Using bacteria of nocardioform actinomycetes group for preparing pharmaceutical composition and method for using this pharmaceutical composition

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions concerns using live bacteria of nocardioform actinomycetes group for preparing a pharmaceutical composition and a method of using this composition. What is characterised is using nocardioform actinomycetes having an ability to survive in animal's macrophages; the live bacteria are attenuated by inactivating a gene, which codes a protein involved in destructing methylhexahydroindanedione propionate for preparing the pharmaceutical composition. The presented method for animal treatment for the purpose of protection against a disorder caused by infection by the bacterium of the nocardioform actinomycetes group, involves administering the pharmaceutical composition containing an effective amount of these bacteria into the animal.

EFFECT: presented inventions can be used for preventing the infections caused by nocardioform actinomycetes.

12 cl, 4 dwg, 11 tbl

The present invention relates to a pharmaceutical composition for protecting an animal from disorders resulting from infection by a bacterium that belongs to the group nocardioform actinomycetes. The invention also relates to the use of live attenuated bacteria of this group for obtaining this composition and to a method of treatment of an animal of this composition.

Among bacteria of the class Actinobacteria there is a group of bacteria called Actinomycetales, commonly referred to as actinomycetoma. Bacteria that belong to that order, represent the flagellar gram-positive bacteria (however, some species have a complex structure of the cell wall, which makes the classical staining gram less suitable or even unsuitable, that is, for example, with many species that belong to the family of Actinomycetales Mycobacteriaceae) with a high content of G+C (guanine+cytosine). They are best known as soil dwelling organisms, although different strains inhabit plants and animals, including humans. They form resistant spores, which are often attached to the air mycelium or hyphae.

Actinomyces play a role in the destruction of organic material. Several species are used in industry and pharmaceutical research due to their typical properties.

The majority of antinomic the LLC are non-pathogenic to animals (the term "animals", used in connection with the present invention, includes people). However, within many suborders of actinomycetes (along with other Streptosporangineae, Micrococcineae, Streptomycineae and Frankineae) has a single suborder, namely Corynebacterineae, which includes along with a large number of non-pathogenic bacteria, a significant number of pathogens of animals. It seems that these pathogens belong to phylogenetic group, known as nocardioforms actinobacteria, which includes the family Mycobacteriaceae, Nocardiaceae and Corynebacteriaceae (see among other sources Chapter 11, entitled: Rhodococus equi: Pathogenesis and Replication in Macrophages, in "Opportunistic Intracellular Bacteria and Immunity", by Lois J. Paradise et al. (eds.), New York, 1999). Notably, against the larger part of the diseases associated with infection with these pathogens, hardly there is any adequate preventive treatment (i.e. treatment before or substantially simultaneously with causing the disease pathogen, which may be capable or prevent disease, or at least to mitigate the impact of the disease). In recent years, was confirmed by the recognition that the family Mycobacteriaceae, Nocardiaceae and Corynebacteriaceae filogenia group nocardioform actinomycetes are very closely related families within the suborder Corynebacterineae (see also source the University of California, San Diego, Outline of Senior Project, Marelle Yehuda L., June 2, 2005). It also became clear that, in particular, pathogenic bacteria in this group, at least those regarding infection which there is no adequate preventive treatment (such as, for example, Mycobacterium uberculosis, Nocardia seriolae and Rhodococcus equi), have important common property: the infection usually occurs through the skin or mucous membrane with subsequent dissemination of bacteria inside macrophages and replicate within these macrophages (see, along with other sources of Microbes and Infection 7, 2005, 1352-1363; Proceedings of the National Academy of Sciences, June 7, 2005, Vol. 102, No. 23, pp 8327 - 8332; Nature Medicine 13, 282-284, 2007; Transplantation Proceedings, Volume 36, Issue 5, June 2004, pp 1415-1418). Indeed, macrophages are on the front line of immune protection from microbial infections, but unlike bacteria, which are dependent on the avoidance of phagocytosis for survival in the host organism under currently pathogenic bacteria within this group focused on macrophages to survive and even replicate in the host organism. The present invention relates to these bacteria, which have the ability to survive inside macrophages animal and in connection with the present invention will be referred to as nocardioforms actinomycete surviving in macrophages.

Obviously, nocardioforms actinobacteria, surviving in macrophages, evolved DL the avoidance of the basic functions of animal protection from germs. In particular, Mycobacterium tuberculosis, the organism that causes tuberculosis, is a type that has been successfully exploited by macrophages as its primary niche in vivo, but other bacterial species that belong to the group nocardioform actinomycetes, including Mycobacteriaceae, Nocardiaceae and Corynebacteriaceae, adopted the same strategy. They represent, for example, Mycobacterium ulcerans, which causes Buruli ulcer, Mycobacterium avium paratuberculosis, which causes the disease of John in cattle and which is associated with Crohn's disease in humans, Mycobacterium bovis, which causes bovine tuberculosis, Mycobacterium avium, which is associated with opportunistic infection of individuals with impaired immunity, such as patients with AIDS, Nocardia seriolae and Nocardia farcinia that cause Nocardia fish, Nocardia asteroids, which causes infection in renal transplant recipients, Rhodococcus equi (formerly known as Corynebacterium), which causes pneumonia in foals and which is also associated with opportunistic infections in individuals with impaired immunity, Corynebacterium pseudotuberculosis, which causes abscesses along with other bodies in the lungs of sheep, goats, horses, and sometimes people, etc. All these bacterial species have the ability to survive inside macrophages, infect and replicate inside the specified type host cell.

This is a typical property with the riously prevents the treatment of disorders (in the present description, the term "disorder" is used as equivalent to "disease"), resulting from infection by a bacterium that belongs to the group of surviving in macrophages nocardioform actinomycetes. In many cases, when clinical signs are indeed present, is treated with antibiotics. However, this is cumbersome, because inside macrophages has a significant amount of bacteria and they are difficult to antibiotics. Therefore, antibiotic treatment often takes a lot of time and gives an ambiguous effect. About diseases such as tuberculosis in humans, Nocardia fish and pneumonia in foals, it would be preferable prophylactic treatment. This preventive treatment is usually based on the use of vaccines containing killed or live attenuated bacteria originating from bacteria of the wild type. It turned out that in terms of surviving in macrophages nocardioform actinomycetes killed (inactivated) vaccine (i.e., vaccines containing killed bacteria or one or more subunits as a therapeutic agent) is ineffective. Now in General it is considered that for successful preventive treatment against surviving in macrophages nocardioform actinomycetes necessary living bacteria, because only they are capable of achieving macrophages and mimic the wild-type bacteria to a degree sufficient to start the adequate immune response. Indeed, for the treatment of tuberculosis has a live vaccine (BCG, Bacillus Calmette-guérin (BCG), based on the form of Mycobacterium bovis, which is closely related to the species Mycobacterium tuberculosis. However, the protective effect was insignificant. In relation to the type Nocardiaceae, such as Rhodococcus equi and Nocardia seriolae, there is currently no commercially available vaccines. In relation to the species Corynebacterium pseudotuberculosis has been attempted to combat the use of autogenous vaccines, but it had an ambiguous effect (RUMA Guidelines, National Office of Animal health, Hertfordshire, United Kingdom, 2006).

There is an obvious need for adequate preventive treatment to protect the animal from disorders resulting from infection of surviving in macrophages nocardioforms actinomycetoma. The treatment in this sense means the stimulation of the immune system of the animal target to the extent sufficient at least to reduce the negative effects of infection with wild-type microorganisms. The aim is that this leads to protection from disorder, i.e., prevents the occurrence of disorder or at least reduces the level of infection or clinical signs of disease in the animal and, therefore, also decreases the severity of disease. The fact that surviving in macrophages nocardioforms actinobacteria adopted the same strategy of survival in the body is ozaena, leads to the idea of a common strategy for prophylactic treatment against infection with these bacteria.

In this regard, it is noted description in the literature of the crucial role of cholesterol metabolism in the survival nocardioform actinomycetes in macrophages, which may be an important virulence factor (Proceedings of the National Academy of Science, February 6, 2007, vol. 104, no. 6, pp 1947-1952 consolidation). It was also assumed that the specified metabolism provides a logical target for new therapeutic agents to combat causing disease strains, i.e., drugs for treatment after infection. Indeed, in a retrospective analysis, we can find other supporting evidence established fact that all surviving in macrophages nocardioform actinomycetes cholesterol metabolism plays a role in the survival and persistence of bacteria in macrophages of the host. For example, from Chapter 11 (entitled: "Rhodococus equi: Pathogenesis and Replication in Macrophages (Pathogenesis and replication in macrophages) ") in the monograph "Opportunistic Intracellular Bacteria and Immunity (Opportunistic intracellular bacteria and immunity)", edited by Lois J. Paradise et al., New York, 1999) it is known that there are large similarities in the clinical symptomatology between infections caused by multiple nocardioforms actinomycete, and it was determined that x is lasterrorcode is the enzymatic component or virulence factor. In "Veterinary Microbiology (Veterinary Microbiology), vol 56, issue 3-4, June 1997, 269-276, it is shown that Corynebacterium pseudotuberculosis is involved in the process of cholesterolosis together with Rhodococcus equi.

So at first glance appears to be an attractive development of the pharmaceutical composition to protect an animal against a disorder arising from infection of surviving in macrophages nocardioforms actinomycetoma (such a composition may also be referred to as a vaccine) using a recognition that cholesterol metabolism plays a crucial role in the survival of these bacteria in macrophages. However, realizing that the suggestions made in the above in the present description the PNAS article (article, 2007), relative to the drug and thus is aimed at the complete destruction of bacteria by interfering in their metabolism of cholesterol, pursuing the same strategy, appear to be unsuitable for live vaccine: if an attempt is made to reduce bacteria by stopping its survival in significant replication site, the bacterium will not replicate and survive in an animal host. Indeed, for the treatment of medications this is the ideal situation. However, for live vaccines, if completely blocked the survival of bacteria, it is anticipated imitation vaccine, containing the she killed bacteria. It turned out that such vaccines are ineffective for treatment of infections of surviving in macrophages nocardioforms actinomycetoma. Yet attempts an evaluation of the use of live bacteria with defective cholesterol metabolism, in the pharmaceutical composition to protect an animal against infection with pathogenic nocardioforms actinomycete wild type. An example of such attempts is a live vaccine on the basis of mutant cholesterolosis (ChoE) strain 103+ Rhodococcus equi wild-type (Prescott in Veterinary Microbiology 118, 2006, pp 240-246). This attempt was unsuccessful. But not because she did not enjoy the protection that would be expected on the basis of technical provisions above in the present description by reference to the PNAS article (PNAS February 6, 2007, vol. 104, no. 6, pp 1947-1952 consolidation), but because the mutant strain was still too virulent. The mutant was still able to survive and multiply in macrophages at the level comparable with R. Equi wild type. It also seems that the antigenic load of this anomalous in relation to cholesterol mutant is comparable to the antigenic load of the body of the wild type. Therefore, the mutant was still able to cause disease. Indeed, while it was also found that living mutant Rhodococcus equi, which generally unable to capture cholesterol (mutant of permeate capture Sterol supAB, as not only is but in publications Van der Geize et al. at the 4^thHavemeyer Workshop on Rhodococcus equi, Edinburgh, 13-16 July, 2008; and Van der Geize et al.: "A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality" in the monograph Nucleic Acids Research 2008; doi: 10.1093/nar/gkn811, hereinafter cited as references as "Van der Geize et al., 2008), which means a full blockade of cholesterol metabolism (at least when cholesterol is used as the starting compound), still able to survive and continue to remain in macrophages (Van der Geize et al., 2008) and thus is still too virulent. Seems to weaken live Rhodococcus equi require additional mutation that effects beyond cholesterol metabolism (Prescott: Veterinary Microbiology 125, 2007, 100-110). Based on these results it was concluded that the metabolism of cholesterol is not an important virulence factor and cannot be used for sufficient attenuation data of bacteria. Obviously, bacteria with mutations in their metabolism of cholesterol, have the same or at least comparable antigenic load as the wild-type organism, and thus, although they are able to provide adequate protection (i.e., when discussing the animal survives the infection mutated bacteria), they are too virulent for use in pharmaceutical compositions. Thus it was believed that targeting farmace the political composition for prophylactic treatment, moreover, the composition comprising live bacteria, which are attenuated by inactivation of genes involved in the metabolism of cholesterol, is a dead end.

However, surprisingly, the applicant has found that in order to protect the animal from disorders resulting from infection of surviving in macrophage nocardioforms actinomycetes, you can apply a pharmaceutical composition comprising live bacteria of the species nocardioform actinomycetes (which is usually the same as the infectious bacterium, or, alternatively, which is very closely related species, thus having much in common epitopes for T-cells, as in the case of Mycobacterium tuberculosis in comparison with Mycobacterium bovis), and live bacteria attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate, and pharmaceutically acceptable carrier for the transfer of bacteria.

"Weak" in this sense means unable to invoke the full set of symptoms, which is usually associated with virulent (often wild type) pathogenic similar weakened bacteria.

"Inactivation" in the context of this invention means that the gene, for example, although it is part of the operon (i.e., the set of genes required for full expression of the protein on the functional level), or completely adalae the descendants of the genome, or replaced (any known or even be developed in the future technique; see, for example, the publication of Introduction to Biotechnology and Genetic Engineering, A. J. Nair, INFINITY SCIENCE PRESS LLC, 2008, Chapter 13 "Genetic Techniques", pp 476-496 and Chapter 15 "Recombinant DNA Technology", pp 563-612) so that it no longer encodes the corresponding wild-type protein, or was no longer available for full transcription, or any other change in the genome, so that the wild-type protein was not obtained weakened bacteria in vivo, at least at the level appropriate to maintain the normal catabolism of methylhexahydrophthalic propionate compared with the situation in which the gene (or operon, if applicable) are presented in a form suitable for maintaining normal metabolism.

"Encoding the protein" in the context of the present invention means that the gene (for example, although part of the operon) directly encodes the protein or subunit protein (multiple subunits that together form an enzymatically active protein), or encodes one or more intermediate compounds, which are converted either directly or through a number of stages in the protein or subunit (multiple subunits that together form an enzymatically active protein).

"Pharmaceutically acceptable carrier" may be a solvent, d is spersion Wednesday, floor, antibacterial and antifungal agent, isotonic or delaying the absorption agent and the like that are physiologically compatible with the animal target and acceptable to him, for example, along with other points made in sterile form. Some examples of such load-bearing media include water, saline, saline with phosphate buffer fluid for bacterial culture, dextrose, glycerol, ethanol and the like, and combinations thereof. They can provide a liquid, semi-solid and solid dosage forms depending on the intended method of administration. It is well known that the presence of the carrier medium is irrelevant to the effectiveness of the vaccine, but it can greatly simplify dosing and injection of antigen. In addition to the carrier and the antigen, a pharmaceutical composition may contain other substances such as adjuvants, stabilizers, viscosity modifiers or other components that are added depending on the intended application or the desired properties of the composition.

In the pharmaceutical compositions of the present invention contains live bacteria, and these bacteria mutate in order inaktivirovanie gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate. It is well known, climatesensitive propionate (also known as HIP or 3aα-H-4α(3'-propionic acid)-7αβ-methylhexane-1,5-indandione) and 5-hydroxy-methylhexahydrophthalic propionate (also known as HIL or 3aα-H-4α(3'-propionic acid)-5α-hydroxy-7aβ-methylhexane-1-indanone-δ-lactone) are formed during the destruction of cholesterol by actinobacteria, including surviving in macrophages nocardioforms actinobacteria. Recently in bacterial species that belong to the suborder Corynebacterineae, was identified operon (called ipdAB: the destruction of the indanone propionate alpha+Beta), encoding the α and β subunit transferase, which is involved in the destruction of the HIP (see at the same time the international patent application PCT/EP2008/060844, filed August 19, 2008, based on the primary application for U.S. patent filed on August 21, 2007). Known mutant with knockout transferase incapable of destroying HIP and HIL (see Fig.3 patent applications listed above in the present description by reference) and he is not growing at the HIP, HIL or 4-androsten-3,17-dione. In any case, "involved in the destruction of the HIP" means the knock-out mutant unable to grow more on the HIP as the sole source of carbon and energy or, at least, are unable to grow at HIP level, which can be obtained nematanthus bacterium. At present it is unclear catabolized whether transferase destruction of the HIP itself, or the reaction is catalyzed, what triggers the destruction of the HIP. However, HIP fracture occurs at a relatively late stage of cholesterol metabolism and is a very specifications the economic stage in the path of destruction cholesterol. On the basis of public views about the mutations in the metabolism of cholesterol (above in the present description reference to operation Prescott) would be expected that this mutation would lead to the fact that the living bacteria, while providing a protective effect, would be too virulent. However, to the surprise of the applicant proves that such a mutant adequately weakened, which is associated with significantly reduced survival of mutant inside macrophages. The reason why the gene, which participates in the destruction of the HIP, plays such an important role in survival within macrophages is unclear, it even seems at odds with the results of prior art, which show that even a complete blockade of cholesterol metabolism has insufficient dampening effect, apparently because nocardioforms actinobacteria have no effect on the survival of macrophages. It was therefore rather surprising discovery that this mutation, which affects the secondary stage of catabolism of cholesterol, creates serious obstacles to the survival of pathogenic nocardioform actinomycetes in macrophages. In particular, it was found that mutant living bacteria are still able to enter macrophages and remain in them (thus providing an incentive protective immune response), but at very low is smoother, which seems to be considerably reduces their virulence, which in turn makes them acceptable for preventive treatment.

In one embodiment, inactivated many genes in the same operon. By inactivation of many genes decreases the probability of changing the bacteria in the phenotype, resembling wild type. In particular, genes are inactivated and accessories > ipdB. By inactivation of these genes can be effective and safe weakening. In a preferred embodiment, the inactivation is achieved by deletion of at least one gene deletion unmarked gene. The advantage of unmarked mutations is that it provides an opportunity to re-introduce mutations in the same strain. Foreign DNA (vector DNA) is removed in the process of introducing mutations. Therefore, the newly introduced vector DNA for introduction of a second mutation could not be integrated in the website of the previous mutations by homologous recombination between the vector DNA). Integration will definitely happen if the vector DNA is still present in the chromosome, and will cause the emergence of a large number of false-positive integrants. The system provides the possibility of using the same antibiotic gene for the introduction of an infinite number of mutations. Unmarked mutation also provides the possibility of easy use in industry due to the lack of heterogeneous DNA providing easy removal of fermentation broth. Inactivation of genes by deletion of genes provides the possibility of designing sustainable RewriteRule mutants. Especially small genes (<500 base pairs) easier inactivated by deletion of genes compared with the rupture of genes by integration of one recombination. Gene deletion mutagenesis can also be used for inactivation of a cluster of several genes of the genome. The strategy of gene deletion mutagenesis can also be used to replace genes (for example, changes in wild-type to mutant gene).

In one embodiment, the bacteria belong to the family Nocardiaceae or Mycobacteriaceae. Preferably bacteria belong to the genera Rhodococcus, Nocardia or Mycobacterium and in particular refer to any species of Rhodococcus equi, Nocardia seriolae, Mycobacterium tubercolosis, Mycobacterium ulcerans, Mycobacterium bovis or Mycobacterium avium paratubercolosis. In respect of these species to date the industry has not produced adequate vaccine. The present invention provides pharmaceutical compositions that can be used as vaccines to combat these bacteria and therefore soften for relevant diseases that they cause the animal.

In one embodiment, the pharmaceutical composition is presented in a form suitable the La oral administration. In addition, this is a very convenient way of introduction, in particular, it became clear that this route of administration is safe. Parenteral administration can cause the development of abscesses. Preferably, the live bacteria are present in a concentration of from 1×10⁴up to 1×10¹⁰CFU (colony forming units) per dose.

The present invention also relates to the bacteria Rhodococcus equi, originating from the strain deposited at the National Collection of Cultures of Microorganisms of the Institute Pasteur in Paris, France, under the number CNCM 1-4108 or no CNCM 1-4109, and bacteria, which belong to this strain.

The present invention also relates to the use of live bacteria, which belong to the group nocardioform actinomycetes are able to survive inside macrophages animal, and live bacteria are attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate, to obtain a pharmaceutical composition for protecting an animal from disorders resulting from infection by the corresponding wild-type bacterium.

The invention also relates to a method of treatment of the animal to protect it from disorders resulting from infection by the bacterium belonging to the group nocardioform actinomycetes, which are able to survive inside macrophages vividly the tion, includes introduction to the animal a pharmaceutical composition comprising live bacteria of the species nocardioform actinomycetes, and live bacteria attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate.

The invention will be further explained using the following examples describe certain embodiments of the present invention, and these embodiments of distributed into three parts:

Part a: Identification and construction of strains

Part B: Survival in macrophages as a model for attenuation in vivo

Part C: the Effectiveness of mutant bacteria in protection against infection with wild type

Part a: IDENTIFICATION AND CONSTRUCTION of STRAINS

A1 Culture medium and growth conditions

Strains of R. equi were grown at 30°C (200 rpm) in Luria-Bertani (LB) consisting of bacteriophora (BD), Yeast extract (BD) and 1% NaCl (Merck) or mineral medium with acetate. M. smegmatis mc²155 (Snapper et al., 1990, Mol. Environ. 4:1911-1919) were grown at 37°C (200 rpm) in trypticase soy broth BBL (TSB; BD) with the addition of 0.05% tween 80. Mineral medium with acetate (MM-Ac) contain K₂HPO₄(4,65 g/l), NaH₂PO₄-H₂O (1.5 g/l), Na-acetate (2 g/l), NH₄Cl (3 g/l), MgSO₄·7H₂O (1 g/l), thiamine (40 g/l, sterilized by filtration; Sigma) is the main solution Vishniac (1 ml/l). The main solution Vishniac was prepared as follows (modified method Vishniac and Santer, 1957, Rev. 21: 195-213): EDTA (ethylenediaminetetraacetic acid) (10 g/l) and ZnSO₄·7H₂O (4.4 g/l) was dissolved in distilled water (pH 8 using 2 M KOH). Then added CaCl₂·2 H₂O (1.47 g/l), MnCl₂·7H₂O (1 g/l), FeSO₄·7H₂O (1 g/l), (NH₄)₆Mo₇O₂₄·4 H₂O (0,22 g/l), CuSO₄·5 H₂O (0,315 g/l) and CoCl₂·6 H₂O (0.32 g/l) in that order at pH 6, and finally kept at pH 4.

For growth on solid media was added baktagir (15 g/l; BD). The main solution of 5-fortitudine (Sigma - Aldrich) (10 mg/ml) was obtained in distilled water, was dissolved by heating to 50°C, sterilized by filtration and added to autoclaved medium.

Strain INS436 Nocardia seriola usual were grown at 26°C (200 rpm) in Eugon broth (BD) with the addition of tween 80 (0.05 per cent). For growth on solid media was added baktagir (15 g/l; BD). To agar medium was added sucrose (2%) for sacB-dependent sucrose selection.

A2 Identification accessories>, ipdB and fadE30 in strain 103+ R. equi and ipdAB in strain INS436 N. seriolae

As described above in the present description, discovered that genes and accessories > ipdB Rhodococcus are involved in the destruction of methylhexahydrophthalic propionate (HIP; 3aα-H-4α(3'-propionic acid)-7aβ-methylhexane-1,5-indandion) and 5-hydroxy-methylhexaneamine the ion propionate (HIL; 3aα-H-4α(3'- propionic acid)-5α-hydroxy-7aβ-methylhexane-1-indanone-δ-lactone). Bioinformatics analysis of protein sequences and accessories > IpdB strain R. erythropolis SQ1 in genomic databases revealed that genes encoding and accessories > IpdB, and their visible operanda organization is preserved in the genome of R. equi 103+ (wild-type strain, obtained from J. F. Prescott, Ontario, Canada; as cited as references in the manual of Veterinary Microbiology 118 (2006) 240-246). Genome sequence of R. equi 103+ was determined by the group sequencing of R. equi in Sanger Institute, Hinxton, Cambridge, UK (genome, published as R. equi 103S"). The genome analysis also revealed that R. equi 103+ contains additional analogichnye genes and accessories > ipdB, denoted respectively as ipdA2 and ipdB2. These genes are localized outside the cluster catabolic genes of cholesterol. Amino acid sequences Accessories>, IpdB, IpdA2 and IpdB2 depicted in the accompanying identities SEQ ID accordingly under No. 1, 2, 3 and 4. Identity of amino acid sequences of proteins and accessories > IpdB R. equi 103+ data analogichnymi and several other actinobacterial orthologues are shown in table 6. This table provides an overview of the genes identified in other genomes nocardioform actinomycetes encoding orthologues and accessories > IpdB Rhodococcus equi 103+. In connection with the present invention, data and other orthologues are called and accessories > IpdB. The IDA is lichnosti proteins indicates the percentage identity of amino acid sequences of full length and accessories > IpdB R. equi 103S. Actinobacterial genomic sequences were obtained from genome BLAST server for microbial genomes National Center for Biotechnology Information (NCBI). Sequence data in R. equi 103+ were obtained by the group sequencing R. Sanger Institute. The genome of strain 103+ (known as 103S) used for the identification purposes. Practical work with Rhodococcus equi, as illustrated below in the present description, was carried out with strain RE1 R. equi (highlighted in foal suffering granulomatoses pneumonia caused by infection with Rhodococcus equi).

The second gene involved in the destruction of methylhexahydrophthalic propionate, is a fadE30. Growth of the mutant ΔfadE30 significantly impaired on HIL and HIP essentially without signs of growth after 24 hours incubation. Gene fadE30 Rhodococcus equi was identified by the study of the similarity of protein sequences performed on the genome of R. equi 103+ available at the Sanger Institute (http://www.sanger.ac.uk). Annotated protein sequence FadE30 strain RHA1 Rhodococcus jostii (Ro4596, the access number in the Gene Bank ABG96382) was used as matrix protein sequence (McLeod et al., 2006, in Proc. Natl. Acad. Sci. U. S. A. 103:15582-15587; and Van der Geize et al., 2007, in Proc. Natl. Acad. Sci. U. S. A. 104:1947-1952 Consolidation). The study database similarity using Ro04596 revealed gene R. equi 103S, encoding a protein that showed identichnost the amino acid sequence of 73% with Ro04596. This protein was annotated as FadE30 R. equi 103S (SEQ ID No 43) and its corresponding gene was named fadE30. Ontologique genes encoding FadE30 other actinobacteria, can be identified similarly. Their selection are presented in table 8.

Genomic locus ipdAB N. seriola PCR amplified (PCR, polymerase chain reaction) in three parts using oligonucleotide primers designed to highly conserved nucleotide sequences actinobacterial locus ipdAB. Data conservative areas were identified by alignment of the nucleotide sequences of several known actinobacterial gene sequences ipdAB. Nucleotide genomic sequence region ipdAB Nocardia farcinica (nfa05080 - nfa05090) (DDBJ room access AP006618) was used as a primary matrix for the development of oligonucleotide PCR primers. Used oligonucleotide primernye sequences are listed in table 5. Chromosomal DNA of N. seriola INS436 used as template for PCR. Genes ipdAB N. seriola amplified using primers accessories > -actino-F and ipdB-actino-R (PCR 22), and above during transcription region ipdAB amplified using ipd-actin-F2 and accessories > -actin-R (PCR 23), and below during transcription region amplified using ipdB-actin-F and the id-actin-R (PCR 21). The PCR products were cloned into the cloning vector pGEM-T and determined the nucleotide sequence of the inserts. Were subsequently developed other pairs of primers on the DNA sequence and used to reklamiranje and resequencing locus ipd. This has led to the complete nucleotide sequence of the locus ipdAB N. Seriola covering 4139 base pairs. Sequenced DNA fragment contained the genes and accessories > ipdB N. Seriola and their neighboring genes. The deduced protein sequence and accessories > IpdB N. seriola INS436 shown, respectively, in SEQ ID NO 58 and SEQ ID NO 59.

A3 Cloning, PCR and isolation of genomic DNA

DH5α Escherichia coli used as a host for all cloning procedures. Restriction enzymes were obtained from company Fermentas GmbH. Chromosomal DNA of the cell cultures was isolated using a set of bacterial genomic DNA GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer's instructions.

PCR was performed in a reaction mixture (25 µl) consisting of Tris-HCl (10 mm, pH 8), 1x standard polymerase buffer, dNTP (deoxynucleotide triphosphate) (0.2 mm), DMSO (dimethyl sulfoxide) (2%), PCR primers (each at 10 ng/µl, table 5) and high-affinity DNA polymerase (Fermentas) or DNA polymerase Pwo (Roche Applied Science). For PCR colonies cellular material was mixed with 100 μl of chloroform and 100 μl of 10 mm Tris-HCl at pH 8,vigorously stirred vortex mixer and centrifuged (2 min, 14000×g). Sample upper aqueous phase (1 µl) was subsequently used as template for PCR. Standard PCR consisted of 5-minute stage of the DNA melting at 95°C followed by 30 cycles of 45 sec denaturation at 95°C, 45 sec annealing at 60°C and 1-3 min elongation at 72°C. the time of elongation depended on the length of the expected PCR amplicon, taking 1.5 minutes/1 thousand bases as a General rule.

A4 Electrotransformation R. equi, M. smegmatis and N. seriolae

Cells of strains of R. equi transformed by electrophoretogram essentially as described (Van der Geize et al., in accepted for publication in NAR article above as a reference; Navas et al., 2001, J. Bacteriol. 183: 4796-4805). Briefly, cell cultures were grown in 50 ml LB medium at 30°C until OD₆₀₀(optical density at wavelength 600 nm) was not reached 0.8 to 1.0. Cells were subjected to palletirovanie (20 min at 4500×g) and washed twice with 10% ice-cold glycerol. Subjected to palletirovanie cells resuspendable in 0.5-1 ml of ice-cold 10% glycerol and divided into aliquots of 200 µl.

Cells of M. smegmatis mc²155 transformed by electrophoretogram essentially as described in the literature (Jacobs et al., 1991, Methods Enzymol. 204:537-555). Briefly, cell cultures (250 ml) were grown at 37°C in an environment TSB+0.05% tween 80 up until OD₆₀₀was reached 0.8, put on ice for one hour and centrifuged (10 min the ri 5000×g) for pelletierine cells. Cell pellets washed twice with distilled water and resuspendable in a final volume of 1 ml 10% glycerol and divided into aliquots of 200 µl.

Bweremana MilliQ plasmid DNA (5-10 µl; kit GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added to 200 μl of cells in the cuvette with a gap of 2 mm Electroporative performed by a single pulse of 12.5 kV/cm, 1000 Ohms and 25 McGard. Subjected to electroporation cells are gently mixed with 1 ml of LB medium (R. equi) or 1 ml of TSB+0.05% tween 80 (M. smegmatis) and allowed to recover for 2 h (R. equi) or 5 h (M. smegmatis) at 37°C and 200 rpm Aliquots (200 µl) of recovered cells were sown on a selective agar medium. Transformants R. equi were selected on LB agar containing apramycin (50 µg/ml) and appeared after 2-3 days of incubation at 30°C. Transformants of M. smegmatis were selected on agar medium with TSB+0.05% tween 80 containing kanamycin (10 µl/ml), and they appeared after 4-5 days of incubation at 37°C.

For N. seriolae preculture (20 ml) strain INS436 were grown for 5 days at 26°C (200 rpm) in Eugon broth+0.05% tween 80 (OD_6oonm=6) and used for inoculation of 100 ml of fresh Eugon broth+0.05% environment tween 80 (1:100). The primary culture was grown overnight at 26°C for 20 h to OD_600Nm=1,3.

Cells were subjected to palletirovanie (20 min, 4000 g) at 4°C and washed twice with 50 ml ice-cold 10% glycerol. Subjected to palletirovanie tile and resuspendable in 500 ál of 10% glycerol, divided into aliquots of 200 ml and immediately used for electrotransformation. Subjected to elution MilliQ plasmid DNA (5-10 µl; kit GenElute Plasmid Miniprep Kit, Sigma-Aldrich) was added to 200 μl of cells in a cuvette with a gap of 2 mm, were mixed and left for 1 min on ice. Electroporative performed by a single pulse of 1.75 kV/cm, 200 Ω and 50 McGard (approximate pulse duration of 9.3 MS). Subjected to cavitation cells are gently mixed with 1 ml of broth medium Eugon with the addition of 0.05% tween 80 and allowed to recover for 3.5 h at 26°C and 220 rpm Aliquots of 50 and 100 µl of the recovered cells were sown on a selective Eugon agar with addition of 0.05% tween 80 and kanamycin (20 μg/ml). Transformants appeared after 7 days incubation at 26°C.

A5 unmarked Deletion of genes in strains of R. equi selection using 5-fortitudine (5-FC)

Mutants of R. equi was obtained by deletion of unmarked gene, essentially as described in the literature (Van der Geize et al., 2008). Transformants R. equi, the resulting electroporation cells wild-type or mutant cells, the strokes were sown on agar medium LB with the addition of apramycin to confirm resistance to apramycin (Apra^R). Four transformant Apra^Rper transformation were grown overnight (20-24 hours) at 30°C and 200 rpm in 25 ml of LB medium and seeding is whether the 10 ¹-10³-fold dilutions in medium MM-Ac to plates with agar MM-Ac with the addition of 5-FC (100 μg/ml) aliquot 100 ál. Resistant to 5-FC colonies appearing after 3 days incubation at 30°C, were sown strokes a reprint from Cup to Cup with LB agar and LB agar with the addition of apramycin (50 μg/ml) for selection of sensitive apramycin (Apra^sand resistant to 5-FC (5-FC^R) colonies. Apra^s/5-FC^Rcolonies were checked for the presence of desirable deletions of genes PCR colonies using primers amplificating locus deletions of genes (table 5). Genomic DNA was isolated from potential mutants with a deletion of the genes and used to confirm deletion of the gene using primers amplificating locus gene ipdAB or ipdAB2, as well as those above and below during transcription regions of these loci primers as shown in table 5.

A6 Construction of plasmids for deletions of genes ipdAB and ipdAB2 in R. equi

Plasmid pSelAct-ipd1 for the generation of unmarked deletions of the gene of the operon ipdAB in R. equi RE1 designed as follows. Above (1368 base pairs; primers ipdABequiUP-F and ipdABequiUP-R) and lower (1396 base pairs; primers ipdABequiDOWN-F and ipdABequiDOWN-R) during transcription flanking region of genes ipdAB amplified PCR (table 5, PCR1 and PCR2). The resulting amplicon ligated into EcoRV-digested pBluescript(ll)KS providing plasmids pEqui14 and pEqui16 respectively for those above and below during the transcription field. Spel/EcoRV (1,4, etc., O.), which fragment pEqui14, ligated in Spel/EcoRV, digested pEqui16, generating pEqui18. EcoRI/HindIII (2,9, etc., O.), which fragment pEqui18 containing a deletion of the gene ipdAB, and its flanking region was treated with the Klenow fragment and ligated into SmaI-digested, suicidal vector pSelAct (Van der Geize et al., 2008). The obtained plasmid was designated pSelAct-ipd1 to construct a mutant with a deletion of the gene ipdAB R. equi Δipd AB (also called RG1341). This mutant was deposited in the National Collection of Cultures of Microorganisms of the Institute Pasteur in Paris, France, under the number CNCM 1-4108.

Mutant of R. equi with double deletion of genes ΔipdABΔipdAB2 (also referred to as RG2837) was obtained by deletion of unmarked gene operon in the mutant strain of R. Equi ΔipdAB using plasmids pSelAct-ΔipdAB2. Plasmid pSelAct-ΔipdAb2 designed as follows. Above (1444 base pairs; primers ipdAB2equiUP-F and ipdAB2equiUP-R) and lower during transcription (1387 base pairs; ipdAB2equiDOWN-F, ipdAB2equiDOWN-R) region ipdAB2 PCR amplified using genomic DNA as template (table 5, PCR6 PCR7 and). The amplicon ligated into SmaI, digested pSelAct, resulting in the plasmids, respectively pSelAct-ipdAB2equiUP and pSelAct-ipdAB2equiDOWN. After digestion of both plasmids BgIII/SpeI fragment pSelAct-ipdAB2equiDOWN (1381 base pairs) ligated in pSelAct-ipdAB2equiUP, resulting in obtaining pSelAct-ΔipdAB2 used to construct the elechi gene ΔipdAB2. The resulting mutant of R. equi ΔipdABΔipdAB2 was deposited in the National Collection of Cultures of Microorganisms of the Institute Pasteur in Paris, France, under the number CNCM 1-4109.

A7 Construction of plasmid for deletion of the gene ipdAB in M. smegmatis mc²155

Plasmid pK18-ipdABsmeg used for unmarked deletions of the gene genes ipdAB in M. smegmatis mc²155, designed as follows.

Above (1502 base pairs; primers ipdABsmegUP-F and ipdABsmegUP-R) and lower (1431 base pairs; primers ipdABsmegDOWN-F and ipdABsmegDOWN-R) during transcription flanking region of genes ipdAB PCR amplified using genomic DNA of M. smegmatis mc²155 as a matrix (table 5, PCR12 and PCR13). The resulting amplicon ligated into SmaI-digested pK18mobsacB (Schafer et al., 1994, Gene 145:69-73), resulting in obtaining respectively pK18-ipdABsmegUP and pK18-ipdABsmegDOWN. The DNA fragment of 1.5 T. p. O. obtained from digested BamHI/SpeI pK18-ipdABsmegUP, subsequently ligated in pK18-ipdABsmegUP Linearisation SamHI/Spel, resulting in the construction of pK18-ipdABsmeg used for deletion of the gene ipdAB.

A8 Construction of plasmid for deletion of the gene fadE30 in R. equi

Plasmid pSelAct-fadE30 for the generation of unmarked deletions of the gene fadE30 in R. equi RE1 designed as follows. Above (1511 base pairs; primers fadE30equiUP-F and fadE30equiUP-R) and lower (1449 base pairs; primers fadE30equiDOWN-F is fadE30equiDOWN-R) during the transcription of flanking genomic region fadE30 amplified with standard PCR, using highly specific DNA polymerase (Fermentas GmbH) (table 5; PCR 15 and 16 PCR). The resulting amplicon ligated in clone pGEM-T vector (Promega Benelux), providing pGEMT-fadE30UP and pGEMT - fadE30DOWN. The DNA fragment BcuI/Bg/II (1,4, etc., O.) cut from pGEMT-fadE30DOWN and ligated in BcuI/Bg/II, linearized pGEMT-fadE30UP, resulting in obtaining pGEMT-fadE30. To construct pSelAct-fadE30, pGEMT-fadE30 Ncol digested and BcuI and treated with Klenow fragment. Blunt DNA fragment (2,9, etc., O.), carrying a deletion of the gene fadE30, ligated into SmaI-digested pSelAct (van der Geize et al., 2008). The obtained plasmid was designated pSelAct-fadE30 and was used to construct a mutant strain of R. Equi ΔfadE30.

A9 Construction of plasmid for deletion of the gene ipdAB in N. seriola INS436

Plasmid pK18ipdABNser used for unmarked deletions of the gene genes ipdAB in N. seriola INS436, designed in the following way. Above (1487 base pairs; primers ipdABNserUP-F and ipdABNserUP-R; PCR 24) and lower (1049 base pairs; primers ipdABNserDOWN-F and ipdABNserDOWN-R; PCR 25) during the transcription of the flanking region of genes ipdAB PCR amplified using genomic DNA of N. seriola INS436 as DNA matrix (table 5). The resulting amplicon ligated into SmaI-digested pK18mobsacB (Schafer ef al., 1994, in Gene 145: 69-73)), resulting in obtaining respectively pK18-ipdABNserUP and pK18-ipdABNserDOWN. The DNA fragment 1,07 T. p. O. obtained from SmaI-digested/ > PST pK18-ipdABNserDOWN, was later Legerova in pK18-ipdABNserUP, which was linearized SmaI/ > PST, resulting in the construction of plasmids pK18-ipdABNser that was used for the deletion of the gene ipdAB.

A10 Construction of mutant strains of R. equi ΔipdAB and R. equi ΔipdABΔipdAB2

Mutants of R. equi with unmarked deletion of genes ipdAB (RG1341) and ipdABipdAB2 (RG2837) were designed using the strategy of a two-step homologous recombination by contralesa using 5-fertilizin developed for R. equi (Van der Geize et al., 2008). To construct ΔipdAB-mutant strain RG1341 R. equi dereplication plasmid pSelAct-ipd1 was mobilized into strain RE1 R. equi by electrotransformation. 4 transformant Apra^Rresulting from homologous recombination between the plasmid pSelAct-ipd1 and genome RE1 subsequently subjected to selection using 5-FC to select the appearance of the second rare homologous recombination leading to deletion of the gene. 18 randomly selected colonies Apra^s/5FC^Rwere subjected to colony PCR, and 3 colonies FC^R/Apra^sgave an amplicon of the expected size (296 base pairs, table 5, PCR5). Genomic DNA was isolated from these three mutants ΔipdAB and subjected to PCR analysis locus ipdAB and its flanking regions above and below during transcription (table 5, PCR3 and PCR4). This analysis confirmed the presence of a genuine deletion of the gene ipdAB in two of the three cases did not reveal aberrant the x genomic rearrangements in the locus ipdAB. The presence of vapA as a marker plasmid virulence was confirmed by PCR (table 5, PCR11). Was selected as one ipdAB-mutant strain, designated R. equi RG1341, and was used for further work.

A mutant strain with a double deletion of genes was constructed from strain RG2837 using plasmids pSelAct-ΔipdAB2, essentially as described for the selection of one of the mutant ΔipdAB. 4 transformant Apra^Rresulting from electroporation cells of strain RG1341 using pSelAct-ΔipdAB2, were subjected to selection 5-FC to select colonies Apra^s/5-FC^R. Subsequent PCR analysis of 18 colonies Apra^s/5-FC^Rconfirmed that the 2 colonies contained a deletion of the gene ΔipdAB2, as evidenced by the resulting amplicon size 123 base pairs using the oligonucleotide obtained for the amplification of the operon ipdAB2 (table 5, PCR10). Further PCR analysis above and below the locus ipdAB2 during transcription regions confirmed the presence of the deletion of the gene ipdAB2 and showed no aberrant genomic rearrangements (table 5, PCR8 PCR9 and). The presence of virulence gene vapA was confirmed by PCR (table 5, PCR11). One mutant strain RG2837 with double deletion of the gene ΔipdABΔipdAB2 was selected for further work.

A11 Constructing mutant strain ΔipdAB M. smegmatis

Mutant M. smegmatis mc²155 with a deletion of unmarked gene is ipdAB designed using system controlactive sacB (Pelicic et al., 1996, MoI. Environ. 20:919-925; Van der Geize et al., 2001, FEMS Environ Lett. 205:197-202) as follows. Construction of mutant ΔipdAB strain M. smegmatis mc²155 dereplication plasmid pK18-ipdABsmeg mobilized in M. smegmatis by electrophoretogram. Received several transformants. One resistant to kanamycin, the transformant was grown for 2 days at 37°C selectivity in the environment TSB containing 0.05% tween 80, and subsequently were sown on plates with TSB agar containing 2% sucrose to select sensitive to kanamycin (Km^sand resistant to sucrose (Suc^Rdouble recombinants by contralesa sacB. Colonies appearing after 3 days of incubation, hatch, prints were sown on TSB agar and TSB agar with the addition of kanamycin (10 μg/ml) for selection of colonies Km^s/Suc^R. True colony Km^s/Suc^Rnext was checked by PCR colonies to detect the presence of the deletion of the gene ipdAB (table 5, PCR14). Genomic DNA was isolated from three potential mutants ipdAB. PCR analysis confirmed the presence of the deletion of the gene ipdAB, and one mutant strain ipdAB was selected for further work and designated as M. smegmatis ΔipdAB.

A12 Construction of mutant strains of R. equi ΔfadE30

The unmarked deletion of the gene fadE30 in R. equi RE1 generated using strategy a two-step homologous recombination by contralesa 5-fluorouracil, developed for R. equi (Van der Geizeet al., 2008). To construct a mutant strain ΔfadE30 dereplication plasmid pSelAct-fadE30 mobilized into strain RE1 R. equi by electrotransformation. Two transformant Apra^Rresulting from homologous recombination between the plasmid pSelAct-fadE30 and genome RE1, subsequently subjected to selection 5-FC for selection on the emergence of the phenomenon of the second rare homologous recombination results in deletion of the gene fadE30. 18 randomly drawn colonies Apra^s/5FC^Rwere subjected to PCR colonies using primers fadE30cont-F and fadE30cont-R (table 5; 19 PCR) and 13 colonies FC^R/Apra^sgave an amplicon of the expected size (428 base pairs). Genomic DNA was isolated from two potential mutants ΔfadE30 and subjected to PCR analysis. PCR analysis of the locus fadE30 using oligonucleotide primers fadE30contr-F and fadE30contr-R (table 5) confirmed the presence of the deletion of the gene fadE30 and the absence of the gene fadE30 wild type. PCR analysis of the flanking regions above and below during transcription showed the expected product having a length, respectively, 1,86, etc., O. and 1.76 T. p. O. Analysis confirmed the presence of the true deletion of the gene fadE30 in both cases and showed no aberrant genomic rearrangements in the locus fadE30. The presence of vapA as a marker of the virulence plasmids was confirmed by PCR. One mutant strain fadE30 was marked R. equi ΔfadE30 and used to further the work.

A13 Construction of mutant strains of N. seriola ΔipdAB

Mutant N. seriola INS436 with unmarked deletion of the gene ipdAB designed using system controlactive sacB (Pelicic et al., 1996, in MoI. Environ. 20:919-925; Van der Geize et al., 2001, in FEMS Environ Lett. 205:197-202) as follows. Dereplication plasmid pK18-ipdABNser mobilized in N. seriola INS436 electrotransformation. Then several resistant to kanamycin of transformants were grown selectivity for 7 days at 26°C in broth medium Eugon containing 0.05% tween 80. The selection is sensitive to kanamycin (Km^sand resistant to sucrose (Suc^Rdouble recombinants by contralesa sacB subsequently performed seeding on cups with Eugon agar containing 2% sucrose. Colonies that appeared after 7 days incubation at 26°C, dashed prints were sown on plates with Eugon agar and cups with Eugon agar with the addition of kanamycin (20 μg/ml) for selection of colonies Km^s/Suc^R. True colony Km^s/Suc^Rnext was checked by PCR colonies to identify the presence of the gene ipdAB using primers ipdABNser-F and ipdABNser-R (table 5). Genomic DNA was isolated from three potential mutants ipdAB. PCR analysis using a pair of primers ipdABNser-F and ipdABNser-R (PCR 23) led to the identification of PCR product size 222 base pairs and the absence of PCR product of wild-type size 1624 base pairs, confirming presets is by deletion of the gene ipdAB. A pair of primers IpdABNser-F2 and IpdABNserDOWN-Contr-R (26 PCR) was used to further confirm the deletion of the gene ipdAB. This pair of primers led to the expected product size 1148 base pairs for mutant ipdAB, whereas the PCR product size 2560 base pairs was obtained only for the wild-type strain. One of the mutant strain was denoted by N. seriola ΔipdAB and selected for further work.

PART B: SURVIVAL IN MACROPHAGES AS a MODEL FOR ATTENUATION IN VIVO

B1 strains Used

Virulent strains

Strain RE1: maternal wild-type strain. This strain grows on cholesterol.

Strain RE1ΔsupAB: deletion of the gene supAB. This strain is unable to grow on cholesterol.

Avirulent strain

Strain 103-: does not contain virulence plasmids ranging in size from 80 to 90, etc., of O., and, as you know, is autogeny in horses (Takai et al. 2000, Infect. Immun. 68: 6840-6847). This strain grows on cholesterol.

Strains in accordance with the invention

Strain RE1ΔipdAB (RG1341): gene ipdAB deleterule in the cluster catabolism of cholesterol. This strain does not grow on 4-androsten-3,17-dione (AD).

Strain RE1ΔipdAB-AD+: bacteria strain RE1ΔipdAB adapted for growth in AD (if the strain RE1ΔipdAB planted in high concentration (more than 10⁶CFU/ml) in the AD as the sole carbon source, then you may experience several colonies for Rostand AD).

Strain RE1ΔipdABipdAB2 (RG2837): the second set of genes ipdAB (not part of a cluster of catabolism of cholesterol) also deleterule. This strain does not grow on the AD.

Strain ΔfadE30 R. Equi. The growth of this strain is abruptly broken at the HIP/HIL and AD.

B2 Culture of Rhodococcus equi for infection of macrophages

Different strains of Rhodococcus equi to be tested in the analysis of survival in macrophages, were grown overnight (17 h) at 37°C and 100 rpm in a nutrient broth (Difco) to achieve a final concentration of 1-2×10⁸CFU/ml was Used only their culture. After inoculation of macrophages were used to define the number of live microorganisms (Cup counting) to confirm the infectivity titer.

B3 Culture of Rhodococcus for the control of infection foals

Strains RE1 Rhodococcus equi, RE1ΔipdAB and RE1ΔipdAB-AD+, were sown on blood agar and incubated for 24 hours at 37°C. Bacteria were collected 4 ml of sterile isotonic PBS per Cup. The bacterial suspension was diluted in sterile isotonic PBS to achieve a final concentration of 4×10⁴CFU/ml of Transportation was carried out at ambient temperature; diluted culture was used within 4 hours after receipt. After control of infection were used to define the number of live bacteria (Cup counting) to confirm the infectivity titer. Koli is esta living suspensions were respectively of 4.35×10 ⁴CFU/ml for RE1, 7,1×10⁴CFU/ml for REΔipdAB and 5.8×10⁴CFU/ml for RE1ΔipdAB-AD+.

B4 the test System

Cell line macrophages

Cell line U937 (human monocytes) were used to test the survival of strains of Rhodococcus equi. Monocytes were grown in medium RPMI 1640+NaHCO₃+NAPYR+glucose (medium RPMI 1640), buffered with 10 mm HEPES and adding 200 IU/ml penicillin and streptomycin and 10% fetal calf serum (FBS). Cells were grown in suspension at 37°C and 5% CO₂.

Foals

Used 8 foals: 7 foals aged 3 to 5 weeks and one foal at the age of 7 weeks (all mares). Foals were divided into 3 groups of 3, 3 and 2 colts, keeping a uniform age distribution in groups. Animals were kept in separate rooms. During the experiment, the foals were fed breast milk and mares were fed in accordance with standard procedures. Access to drinking water was unlimited.

At time T=0 all foals were subjected to control infection intratrahealno 100 ml of culture for infection controlstrain RE1, RE1ΔipdAB or RE1ΔipdAB-AD+ (see above in this description under the heading "Culture of Rhodococcus for the control of infection foals") using a syringe with gloy, the so-called transtracheal injection.

B5 Experimental procedures and parameters

Test of survival in macrophages

For analysis of survival in macrophages monocytes were grown in a few days, as described above in this application. The culture medium was replaced by fresh culture medium and cells activated during the night 60 ng/ml phorbol-12-myristate - 13-acetate (PMA) to induce their differentiation into macrophages.

Differentiated cells were besieged by centrifugation (5 min at 200×g) and the cell precipitate after centrifugation resuspendable in fresh not containing antibiotics medium RPMI 1640 with 10% FBS. For each subject test strain test tube containing 10 ml of cell suspension with approximately 10⁶cells/ml, were inoculable Rhodococcus equi at multiplicity of infection (MOI) of approximately 10 bacteria per macrophage.

Bacteria were incubated with macrophages for 1 hour at 37°C and 5% CO₂. The medium was replaced by 10 ml of medium RPMI 1640 with the addition of 10% FBS and 100 μg/ml gentamicin and again incubated for 1 hour to kill any extracellular bacteria. Macrophages (internalized R. equi) was besieged by centrifugation (5 min at 200×g) and the cell precipitate after centrifugation resuspendable in 40 ml of medium RPMI 1640, buffered with 10 mm HEPES, and with the addition of 10% FBS and 10 μg/ml gentami the ina. This suspension was divided by 4 culture vials (10 ml each) and incubated at 37°C and 5% CO₂. After 4, 28, 52 and 76 hours of incubation, the macrophages (one culture bottle on the strain) was besieged by centrifugation (5 min at 200×g) and the cell precipitate after centrifugation washed twice in 1 ml containing no antibiotic medium RPMI 1640. Finally, the cell precipitate was literally 1% Triton X-100 in 0.01 M saline phosphate buffer (PBS) with subsequent determination of the amount of live bacteria (Cup counting).

Control infectionfoals

1 - Rectal temperature was measured 1 day prior to infection control in day control of infection (directly in front of the control contamination) and then once a day after control of infection before opening.

2 - Clinical examination: within 3 weeks after controlling infection of horses examined daily to detect clinical signs.

3 - post-mortem examination and bacteriology: on day 21 after infection control foals were weighed and then were killed by anesthesia with xylazine (100 mg/100 kg) and ketamine (500 mg/100 kg) with subsequent bleeding to death. Lungs were weighed to calculate the ratio of the mass of lung to body weight. Full post-mortem examination was performed with particular attention paid to light the associated lymph nodes. In case of deviations from the norm pathologist, if considered necessary, samples were taken for histological study.

Tissue samples (1 cm³) was dissected from seven standard areas, representing the proportion of each half light (3 plots the half light + incremental share); for each section preferably selected diseased tissue, if it was present. Samples mirrored (two samples is equivalent to the share on each half) were combined to obtain samples for colt + sample incremental share. Each (combined) sample homogenized, serially diluted and inoculable the Cup with blood agar and then incubated at 37°C for 16-24 hours. Colony Rhodococcus were counted and expressed as CFU/ml of homogenate. Additional swabs were taken from all plots with pathological changes. Strokes strokes were sown on plates with blood agar and then incubated at 37°C for 16-24 hours. Rhodococcus equi originally identified by its typical non-haemolytic mucoides colony morphology. Further identification was performed by staining gram-API/Phoenix and/or PCR.

B6 Results

Survival in macrophages

The results of two separate experiments are shown in table 1 (in conjunction with Fig.1 and 2 in conjunction with Fig.2). The results show that Muta is t RE1ΔsupAB able to survive in macrophages in the same way, as the parent wild-type strain RE1, indicating that the metabolism of cholesterol is not essential for survival in macrophages. This is consistent with the recognition that the metabolism of cholesterol, per se, is not important for virulence. In contrast, survival in macrophages strain RE1ΔipdAB, strain RE1ΔipdABipdAB2, strain RE1ΔipdAB-AD+ and strain 103 were clearly reduced. However, it should be noted that bacteria are still able to survive in macrophages, but at a much reduced level (usually at a concentration of 100-1000 times lower than the level of the wild type). Strain 103 - does not contain plasmid virulence in size from 80 to 90, etc., of O., and is known to be avirulent in horses (Takai et al.). This strain 103 is not suitable as a vaccine strain, because it does not cause a protective immune response, probably due to the fact that it does not contain plasmid virulence. In table 9 (in conjunction with Fig.4) shows the results for a mutant strain of R. equi ΔfadE30, in comparison with the wild-type strain RE1 and strain RE1 ΔipdAB. It is clear that the mutant fadE30 has the characteristic of survival, comparable to the strain ΔipdAB, and less able to survive in macrophages.

The results with strains that have mutations in the operon, encoding a protein involved in the activity of the destruction of methylhexahydrophthalic propionate (i.e., strains RE1ΔipdAB, RE1ΔipdABipdAβ2, RE1ΔipdAB-AD+ and R. equiΔfadE30) show, is that these strains are less able to survive in macrophages. In particular, their ability to survive comparable with abilities to survive patogennogo strain 103-. This is already a good indicator of adequate attenuation. Strain RE1ΔipdAB-AD+ showed the same phenotype of macrophages, as strain RE1ΔipdAB, indicating that significant for survival in macrophages at the level of the wild type is more of an intact operon ipdAB, not intact cholesterol metabolism. Single deletion of the gene ipdAB (geneclustercatabolism of cholesterol) resulted in reduced survival in macrophages. Additional deletion in the copies of these genes (and accessories > ipdB unclusteredcatabolism of cholesterol, called ipdA2 and ipdB2) had no additional weakening effect in the test with macrophages.

Given these results, the strains RE1ΔipdAB and RE1ΔipdAB-AD+ (=strain RE1ΔipdAB, adapted for growth AD) was administered intratrahealno the colts (the usual procedure infection control) and compared with the parent wild-type strain RE1 for testing attenuation in vivo.

Rectal temperature

In table 3 (in conjunction with Fig.3) presents the results of the measurement of rectal temperature. Group 1 is a group, which received the strain RE1 wild type. Groups 2 and 3 received respectively RE1ΔipdAB and RE1ΔipdAB-AD+. Temperature from 3 to day 10 are not shown because they did not reveal what no significant changes from the normal rectal temperature. Anomalous temperature values are shown in bold. Two out of three foals (No. 18 and 19), subject to the control of infection with the parent strain RE1 wild type, showed no apparent increase in the values of rectal temperature with 14 days after control of infection and forth. The increase in the values of rectal temperature after 14 days coincided with the development of clinical signs (see below). The foal No. 21 (infected with a strain RE1ΔipdAB) showed a slight rise in temperature (39,1°C) at day 1 after control of infection, which is most likely not associated with Rhodococcus infection (incubation time in this model, the control of infection is > 7 days). In the subsequent foals subjected to control infection RE1ΔipdAB or RE1 ΔipdAB-AD+) were not found elevated temperature.

Clinical signs after infection control

Indeed, clinical scoring from 7 to 21 days showed that foals No. 18 and 19, subjected to the control of infection with the parent strain RE1 wild-type, developed signs of respiratory disease from the 13th day after the control of infection and forth. The stallion No. 17 (also from the control group contamination RE1) after control of infection showed only mild clinical signs. The foals, who were subjected to the control of infection with mutant shtam the om RE1ΔipdAB and RE1ΔipdAB-AD+, showed no clear signs of respiratory disease. Clinical effects of the latter two groups were mainly based on a slightly increased heart rate, which is also (partially) could be caused by stress associated with the treatment of animals, as it was present prior to infection control.

Post-mortem examination

Data postmortem studies of the lungs are shown in table 4. After control of infection by the parent strain RE1 wild type in foals developed signs of respiratory disease (in particular, the colts No. 18 and 19). Foals subjected to control infection by the mutant strains remained healthy. 21 days after infection control foals were slaughtered and had their showdown. At autopsy, all foals subjected to control infection by the wild-type strain, was typical pyogranulomatous pneumonia, from the hearth which was re-allocated R. equi in pure culture, and identity of the wild-type strain was confirmed by PCR. The foal No. 18, R. equi wild-type was also isolated from enlarged mediastinal lymph nodes.

In the lungs of foals subjected to control infection by the mutant strains were not revealed pneumonic area and Rhodococcus has not been allocated C is with the exception of a few enlarged bronchial lymph node foal No. 20 and healthy lung tissue foal No. 24. The identity of these isolates was confirmed as RE1ΔipdAB and RE1ΔipdAB-AD+ PCR, respectively, and growth on agar AD.

B7. Conclusion for PART B experiments

Strains RE1ΔipdAB and RE1ΔipdAB-AD+ clearly impaired survival in macrophages and are weak in foals. It seems that the knockout of the second copy of the gene ipdAB (leading to the RE1ΔipdABipdAB2) has no additional effect on the test of survival in macrophages and probably also in foals. Combined results for in vivo and in vitro, obtained with strains of RE1, RE1supAB, 103-, RE1ΔipdAB and RE1ΔipdAB-AD+ indicate a good correlation between the level of survival in macrophages and virulence in vivo for bacteria related to nocardioforms actinomycetes. In particular, it seems that, when mutant strain has a significantly reduced ability to survive in macrophages, usually about 2 to 3 logarithms relative to the virulent parent strain, the mutant strain is significantly weakened relative to the parent strain.

On the basis of the well-known fact that the route of infection and the virulence factors are divided among nocardioform actinomycetes, and therefore, the fact that Rhodococcus equi is usually used as models for investigating the Mycobacteriaceae, in particular, in respect of the virulence factors associated with survival in macrophages and persistence in them (the m along with other sources PNAS, February 6, 2007, vol. 104, no. 6, pp 1947-1952 consolidation), it is clear that the reduced survival of bacteria in the inactivation of a gene that encodes a protein involved in the destruction of methylhexahydrophthalic propionate is generic for weakening nocardioform actinomycetes.

PART C: the EFFECTIVENESS of MUTANT BACTERIA IN PROTECTION AGAINST INFECTION with WILD TYPE

C1. Introduction

It is believed that the pharmaceutical compositions containing the live mutant bacteria in accordance with the present invention, the inherent anti-infective efficacy. This can be understood by recognizing that the well-known views about the mutations of cholesterol metabolism clearly indicate that bacteria with mutations in the metabolism of cholesterol, all have the same antigenic load is comparable to that of wild-type organisms. In combination with presents in the present invention is recent evidence that mutant living bacteria are still able to enter macrophages and survive in them, leaves no doubt that remains stimulus immune response. However, the applicant conducted experiments to confirm this opinion. To do this, use a vaccine containing live Rhodococcus equi. There is currently no vaccine, as such, against this bacteria, which makes these experiments releva time. But more importantly, this bacterium was recognized as a good model for other nocardioform actinomycetes, in particular Mycobaterium tuberculosis. Other supporting experiments (for confirmation of results covering the full range nocardioform actinomycetes) can for example be carried out with Nocardia seriolae (see section C6). They are typical of fish pathogens, against which there is no adequate vaccines, for the same reasons that there is no adequate vaccines against other nocardioform actinomycetes, surviving in macrophages.

C2. The structure of the experiment

For this study used 16 foals between the ages of 2 to 4 weeks. Foals were divided into 4 groups of 4 colt and were vaccinated orally with 1 ml of vaccine containing different doses RE1ΔipdAB (prepared in sterile saline solution with phosphate buffer). Group 1 were vaccinated with 5×10⁹SOME, group 2 were vaccinated with 5×10⁸SOME; group 3 were vaccinated with 5×10⁷SOME and group 4 was left as unvaccinated controls. Vaccination was performed at time T=O and at time T=2 weeks. At time T=4 weeks all foals were subjected to the control of infection with 100 ml culture of a virulent strain of 85F Rhodococcus equi (containing 5×10⁶CFU per 100 ml).

During the 3-week period after the control of infection was conducted wedge the economic assessment of the condition of the horses. Foals were weighed on the first day of vaccination to day control of infection and on the day of opening. 3 weeks after control of infection (or earlier in case of severe clinical signs) foals were weighed and subjected to euthanasia and performed a full autopsy study with a focus on the lungs and respiratory lymph nodes. Lungs were weighed to calculate the ratio of the mass of lung to body weight. We studied tissue samples from all lung lobes.

C3. Materials and methods

Test product

A vaccine containing live Rhodococcus strain REIΔipdAB was in saline phosphate buffer (PBS). Culture for infection control was prepared as follows: the strain 85F Rhodococcus equi were sown on blood agar and incubated for 24 hours at 37°C. Bacteria were collected in 4 ml of sterile isotonic PBS per Cup. The bacterial suspension was diluted in sterile isotonic PBS to achieve a final concentration of 4×10⁴bacteria/L. Transportation was carried out at ambient temperature; diluted culture was used within 4 hours after receipt. After control of infection was determined by the number of live bacteria to confirm the infectivity titer. The titer was 5.3×10⁴CFU/ml

Experimental procedures and parameters</>

Re-isolation of bacteria was performed by taking rectal smears immediately before each vaccination and 0, 1, 2, 3, 6, 10, 14, 15, 16, 17, 20 and the 24th day (after vaccination). Swabs of the nasal cavity had foals at time T=O (immediately before the first vaccination). Obtained in the form of smear samples were serially diluted in physiological saline solution and were sown on blood agar and incubated at 37°C for 16-24 hours. Colony Rhodococcus initially identified the typical morphology of the non-haemolytic mucoides colonies were counted and expressed as CFU/ml In each day allocation randomly chose three re-isolate (from three different horses, if they were present) and used to verify identity: staining gram-API/Phoenix and PCR on genes ipdAB.

During the study, horses were daily observed by the biologist to identify any deviations from the norm in relation to General health and/or behavior. Starting one day before infection control, horses examined daily (before opening) to identify clinical signs. Weighing was carried out directly before the first vaccination, immediately before the first control infection and prior to necropsy. Thus it was possible to calculate the weight gain and the ratio of the light assy to body weight.

14-20 day after control of infection (or sooner in severe clinical signs) foals were killed by anesthesia with xylazine (100 mg/100 kg) and ketamine (500 mg/100 kg) and subsequent bleeding to death. Lungs were weighed to calculate the ratio of the mass of lung to body weight. Performed a full autopsy study. Tissue samples (1 cm³) was dissected from seven standard areas, representative of shares of each half light (3 plots the half light + incremental share); for each section preferably selected diseased tissue, if it was present. The samples, which is the mirror image (two samples of the equivalent share for each half), were combined to obtain 3 samples on colt + sample incremental share. Each (combined) sample homogenized, serially diluted and inoculable the Cup with blood agar and then incubated at 37°C for 16-24 hours. Colony Rhodococcus were counted and expressed as CFU/ml of homogenate. Scoring of lung with pneumonia for each animal was obtained by setting the percentage of consolidation for apical (left+right), caudal (left+right) and extra pulmonary sites. For each animal specified percentage folded to obtain figures scoring one lung. Still the way it turns out pulmonary numerical score from 0 to 500.

C4. Results

Results after control of infection are summarized in table 7. According to the summarized results it is clear that all four controls developed severe signs pyogranulomatous pneumonia caused by R. equi as the sole pathogen. The two vaccinated No. 4 and 5 had evidence comparable to the controls, but all other vaccination were much lighter signs or essentially no signs of pneumonia. The percentage weight of the lungs (objective quantitative measure of pneumonia) confirms partial protection most vaccinated.

Vaccinated colt No. 2 had pneumonia caused by specific bacteria. In fact, the foal can be considered as secure as Rhodococcus did not stand out, despite the massive infection control. In addition, three vaccinated foals with pneumonia were selected mixed infective pathogens. Data mixed infection, likely had an adverse impact on various security settings. Although the protective effect of the vaccine obvious effect based dose-response was not observed. In fact, it turned out that the lowest dose provided the best results. Based on this, combined with the fact that the colts have immature gastro-intestinal flora, with whom udaetsya, the optimal dose of from 1×10⁴up to 1×10¹⁰SOME.

C5. Conclusion under part C of experiments

It turned out that all three oral doses of the vaccine safe for young foals and caused significant protection against severe intratracheal infection control. In these experiments, genes and accessories > and ipdB operon ipdAB were removed from the genome of the bacteria in the vaccine. However, it is clear that other mutations involving the same operon, can be equally effective. The mutation, which, for example, affects only one of the genes accessories > or ipdB, by itself, can be equally effective. In any of the recent cases involved transferase cannot be obtained and thus achieves the same phenotype. Indeed, it can be also inferred from the data Rengarajan (PNAS, June 7, 2005, Vol. 102, No. 23, pp 8327-8332). In this reference presents relevant evidence for other nocardioforms actinobacteria, namely Mycobacterium tuberculosis: inactivation of any one of orthologic genes ipd (called respectively rv3551 and rv3552 in M. tuberculosis) leads to the same phenotype.

C6. Demonstration of efficacy as a vaccine mutant Nocardia seriolae

The above-described experiments for Rhodococcuse equi repeated for the other actinobacteria, a pathogen of fish Nocardia seriolae, which causes Nocardia fish. This is xperimenta used the wild-type strain INS436 and strain ΔipdAB, as described above in the present description in paragraph A13.

Groups of 20 gelthveau(Seriola quinqueradiata) was intraperitoneally injected with or strain wild-type or mutant strain (in various concentrations), and one group of 20 fish were left as control. Fishes observed within 2-3 weeks to detect mortality and other clinical reactions to assess the attenuation of the mutant strain. At the end of the specified follow-up period had a control contamination of surviving fish a fixed dose of the wild-type strain to assess the effectiveness of the mutant strain as a vaccine. Fish were observed for another 2 weeks.

For strain wild type inoculum was obtained from 2,25×10⁸CFU/ml For the mutant strain, it was a 7.85×10⁸CFU/ml (approximately 3 times higher). Of these similar inoculum was received twice, 20-fold, 200-fold and 2000-fold dilution for use in the study of abating. Material for intraperitoneal infection control consisted of 1.2×10⁶CFU/ml

The results of the experiment attenuation are presented in table 10 (which shows the mortality at the end of the observation period). From this it becomes clear that the mutant strain is attenuated relative to the wild-type strain. Although similarthe inoculum for the mutant strain was 3 times higher in CFU/ml, mortality was significantly lower in the group, to the which received the mutant strain. The results of experiments to determine the effectiveness presented in table 11. Although to get the full protection was impossible, it is clear that the mutant provides significant protection (taking into account the fact that in the control group, 60% of the fish died during the observation period). Even in the group of fish that had been diluted 2000 times, much less fish died in the control of infection with Nocardia seriolae wild-type.

It was shown that inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate, leading to a weakened and protective vaccine strain in the second nocardioforms actinomycete. This is an additional confirmation that this invention is applicable for a wide range nocardioform actinomycetes.

1. Pharmaceutical composition for protecting an animal from disorders resulting from infection by a bacterium that belongs to the group nocardioform actinomycetes, which are able to survive inside macrophages animal containing an effective amount of live bacteria species nocardioform actinomycetes, and live bacteria attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate, and a pharmaceutically acceptable carrier for the specified MS is s bacteria.

2. The pharmaceutical composition according to p. 1, wherein the inactivated multiple genes in the operon.

3. The pharmaceutical composition according to p. 2, wherein the inactivated genes and accessories > ipdB.

4. The pharmaceutical composition according to p. 1, where inactivation is a deletion of at least one gene by deletion of unmarked gene.

5. The pharmaceutical composition according to p. 1, wherein the bacteria belong to the family Nocardiaceae or Mycobacteriaceae.

6. The pharmaceutical composition under item 5, wherein the bacteria belong to the genera Rhodococcus, Nocardia or Mycobacterium.

7. The pharmaceutical composition according to p. 6, wherein the bacteria belong to the species Rhodococcus equi, Nocardia seriolae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium bovis or Mycobacterium avium paratuberculosis.

8. The pharmaceutical composition under item 1, characterized in that it is presented in a form suitable for oral administration.

9. The pharmaceutical composition under item 1, characterized in that the live bacteria are present in a concentration of from 1×10⁴up to 1×10¹⁰CFU per dose.

10. The pharmaceutical composition according to p. 7, characterized in that the said live bacteria are bacteria Rhodococcus equi strain deposited at the National Collection of Cultures of Microorganisms of the Institute Pasteur in Paris, France, under the number CNCM I-4108 or no CNCM 1-4109.

11. Use ivih bacteria, which belong to the group nocardioform actinomycetes, which are able to survive inside macrophages animal, and live bacteria attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate, to obtain a pharmaceutical composition for protecting an animal from disorders resulting from infection by the corresponding wild-type bacterium.

12. The method of processing animal to protect it from disorders resulting from infection by a bacterium that belongs to the group nocardioform actinomycetes, which are able to survive within macrophages of the animal, including the introduction of animal pharmaceutical compositions containing live bacteria nocardioform actinomycetes, and live bacteria attenuated by inactivation of the gene, which encodes a protein involved in the destruction of methylhexahydrophthalic propionate.