Recombinant strain escherichia coli zv1 as carrier of burkholderia pseudomallei chromosomal dna cloned sequence determining synthesis of protein 32 kda and resistance to pefloxacin and streptomycin

FIELD: microbiology, molecular biology, genetic engineering.

SUBSTANCE: invention relates to designing recombinant strains of E. coli carrying the cloned sequences of genome of meliodosis pathogen, B. pseudomallei, and determining different spectra of the medicinal resistance. Method involves transformation of E. coli JM107 competent cells with Kpn I-fragments of chromosomal DNA of the strain B. pseudomallei 56770 SMR2 ligated with Kpn I-restricts of vector pUC19 followed by selection of clones showing the combined resistance to antibacterial preparations of different classes. Then method involves carrying out the plasmid screening of the recombinant clones and hybridization analysis for detection of chromosomal DNA fragment by using B. pseudomallei chromosome sequences as a probe. Then method involves assay of the presence of the expression product of chromosomal DNA cloned sequence by immunoblotting method with immunoglobulins of specific meliodosis antsera. The prepared recombinant strain ZV1 is deposited in the State Collection of pathogenic microorganisms "Mikrob" at number KM167 and it shows resistance to pefloxacin and streptomycin. Use of the invention provides carrying out investigations of molecular-genetic bases of the multiple medicinal resistance of B. pseudomallei and to study the functional role of separate proteins in formation of the polyresistance in the meliodosis pathogen.

EFFECT: valuable properties of strain.

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The invention relates to Microbiology and molecular biology and concerns the construction of recombinant strains of E. coli carrying the cloned sequence of the genome of the pathogen melioidosis Century pseudomallei determining different spectra of drug resistance. The resulting strain E. coli ZV1 is a producer of recombinant plasmid DNA pJla1 with a cloned fragment of chromosomal DNA .pseudomallei determining the synthesis of the protein of 32 kDa and resistance to pefloxacin (PfR) and streptomycin (SmR). The strain is designed to investigate the molecular genetic basis of multidrug resistance Century. pseudomallei. The strain deposited in the Public Collections of Pathogenic Bacteria "Microbe" (GKPB "M") under number KM167.

Characteristic and important biological property of the pathogen melioidosis close to him and microorganisms is a high natural resistance to the effects of broad-spectrum antimicrobial compounds and protective factors of the immune system of the host [Vorachit M, Chongtrakool P., Arkomsean S., Boonsong S. Antimicrobial resistance in Burkholderia pseudomallei // Acta Trop. - 2000. - Vol.74. - P.139-144; Brett P., Woods, D. Pathogenesis of and immunity to melioidosis // Acta Trop. - 2000. - Vol.74. - P.201-210]. The presence of these properties determines the ability .pseudomallei to long and often asymptomatic, persistence in the infected host and creates significant is radnoti for the successful treatment of disease.

The study of the molecular basis for the implementation of various types of drug resistance .pseudomallei started relatively recently, however, and based on the obtained data it can be argued that the causative agent of melioidosis has an extremely vast genetic potential for the formation of either spectrum resistance. So, identified the chromosomal operon amrAB-oprA related to the multiple determinants of resistance of the transport type and defines high level of stability Century pseudomallei to aminoglycosides and macrolides [Moore, A., DeShazer D., Reckseidler, S., Weissman, A., Woods, D. E. Effluxediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. // Antimicrob. Agents Chemother. - 1999. - Vol.43. - P.465-470]. Identified and characterized chromosomal genes waaF, waaE, udg, gmhD, lytB, homologous components of the systems of LPS biosynthesis and exopolysaccharides from different gram-negative bacteria and are responsible for resistance Century. pseudomallei to the effects of cationic peptides and polymyxins [Burtnick .N., Woods, D.E. Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin resistance. // Antimicrob. Agents Chemoter. - 1999. - Vol.43. - P.2648-2656]. The presence in the genome of C. pseudomallei R-determinants of different classes of evidence and published the preliminary results of the project to sequence the genome of C. pseudomallei [Holden M, Titball R., Peacock, S., et. al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei // Proc. Nal. Acad. Sci. USA. - 2004. - Vol.101. P.14240-14245].

It should be noted that research aimed at deciphering the mechanisms of formation resistance .pseudomallei, can serve as a basis for understanding basic biochemical characteristics of an organism, determining its increased metabolic plasticity, adaptability to the effects of specific and nonspecific inhibitors and protective host factors, as well as to provide the necessary material to develop new approaches to the use of antibiotics in the treatment of melioidosis infection. The possibility of studying the mechanisms of drug resistance are determined by the presence of molecular-biological methods and model systems to identify and analyze sequences of the genome, determining the resistance. Necessary elements here are special strains carrying genetic determinants of altered sensitivity, methods of molecular analysis R-determinants of various types, as well as the cloning of the sequence determinants of resistance and analysis of their expression in heterologous systems.

Today there are a number of reports on the cloning and analysis of gene expression .pseudomallei determining resistance to antimicrobial compounds of different classes. Thus, in E. coli klonirovannyi blaA gene BPS.pseudomallei encoding β-lactamase, is able to hydrolyze penicillins and individual cephalosporins [Cheung So, But R., Woo P., et al. Cloning and expression of class A β-lactamase gene blaABPSin Burkholderia pseudomallei // Antimicrob. Agents Chemother. - 2002. - Vol.46. - P.1132-1135]. Based on the analysis of published sequences of the genome of the pathogen melioidosis ( the authors have designed specific primers containing sequences of the restriction sites EcoRI and XbaI, amplified chromosomal sequence blaABPSCentury pseudomallei and cloned in E. coli in the vector pBK-CMV. Recombinant plasmids pBKCMV01 and pBKCMV07 stably replicated in strains of Escherichia coli. Amino acid sequence of recombinant β-lactamase with an isoelectric point pI of 7.7 and a molecular weight 29.1 kDa had a high degree of homology with β-lactamases of class a, such as PenA Usersa, BlaI Yersinia enterocolitica, CTX-M-18 Klebsiella pneumoniae, SFO-1 Entero-bacter cloacae, FONTA-4 Serratia fonticola and OXY Klebsiella oxytoca.

In many respects, a similar approach was used for cloning and analysis of gene β-lactamase D-class .pseudomallei [Niumsup P., Wuthiekanun V. Cloning of the class D β-lactamase gene from Burkholderia pseudomallei and studies on its expression in ceftazidime-susceptible and - resistant strains // J. Antimicrob. Chemother. - 2002. - Vol.50. - P.445-455]. Strains of E. coli carrying the recombinant plasmid with the cloned PCR fragments of the genome Century pseudomallei, homologous g is us β -lactamases of class D, had a high level of resistance to ceftazidime and oxacillin. Analysis of cloned sequences allowed us to identify a gene, designated Okha-42 and encoding β-lactamase extended-spectrum homologous some oxacillin-gidrolizuut enzymes, such as OXY-22 Ralstonia pickettii, AsbB, AmpS and AmpH different species of Aeromonas, OXA-18 Pseudomonas aeruginosa and OKHA-9 Klebsiella pneumoniae.

Work on cloning sequences of the genome .pseudomallei determining multiple resistance to antimicrobial compounds of various classes, much less reflected in modern scientific periodicals. We should mention the study of structural and functional organization of plasmids .pseudomallei, which was carried out cloning and analysis of expression in E. coli fragments 12.95 TPN plasmids RRM [Zakharov IS, Viktorov D.V., Zamaraev VS Cloning and expression 7.55 TPN fragment of plasmid RRM Burkholderia pseudomallei in Escherichia coli. // Mol. Genet. microbiol. virusol. - 2002. No. 2. - P.19-22]. It is established that the recombinant clones carrying 7.55 TPN Cloned fragment RRM, vysokofrekvencny to individual aminoglycosides (streptomycin, kanamycin, gentamicin and fluoroquinolones (pefloxacin, ofloxacin). Two proteins of the outer membrane 23 and 27 kDa, missing the original strain of E. coli and capable of forming a multimeric complex 120 kDa,were detected by Western-blotting in the strain with the recombinant plasmid pSI 9, vector part of which, however, has undergone significant recombination of perestroika. According to the dot - and southern-hybridization analysis, was observed integration of the cloned sequence in the chromosome in E. coli clones carrying recombinant plasmids pSI 2 pSI and 4.

The aim of the invention is the construction of E. coli strain carrying a stably can replicate the recombinant plasmid with the cloned sequence of the chromosomal DNA Century pseudomallei determining resistance to antimicrobial compounds of different classes.

As the source of chromosomal DNA was used strain .pseudomallei 56770 SMR2 with a high level of resistance to cephalosporins, fluoroquinolones, aminoglycosides, β-lactams and different stable preservation of the phenotype of resistance with long-term cultivation in non-selective conditions. Chromosomal DNA was isolated by the method of neutral lysis [Maniatis T., Fritsch E., Sambrook D. Molecular cloning. - Lane. from English., M.: Mir, 1984. - 392 C.] of the 24-hour culture of the pathogen melioidosis grown on Nutrient-arape (Difco) with pefloxacin (60 μg/ml) at 37°C.

Chromosomal DNA Century pseudomallei 56770 SMR2 and the vector pUC19 was subjected to complete hydrolysis by endonuclease kpni restriction sites and after the resultant deposition rates ligated at a ratio of 25:1. In several series of experiments transformed ligase mix comp is competent cells E. coli JM107 received standard calcium method [Maniatis T., Fritsch E., Sambrook D. Molecular cloning. - Lane. from English., M.: Mir, 1984. - 392 S.], with subsequent plating on a dense nutrient medium Antibiotic medium 2 (Difco)containing Pfx 0.4 mg/ml

Transformants resistant to pefloxacin additionally investigated for resistance to aminoglycosides (kanamycin, streptomycin, gentamicin), β-lactams (ampicillin) and carbapenems (imipenem).

For plasmid screening used alkaline method for isolating plasmid DNA [Birnboim, H., Doly J. A rapid alkaline extraction method for screening recombinant plasmids //Nucleic Acids Res. -1979. - Vol.7. - P.1513] with subsequent detection of plasmid replicons by electrophoresis in 0.8% agarose gel.

For detection of the cloned fragment of chromosomal DNA .pseudomallei was used for hybridization analysis. Probe (sequence chromosome .pseudomallei 56770 SMR2) were labeled with bio-11-dUTP in PCR using forward and reverse primers pUC/M13 with the following parameters of reactions: denaturation 95°C - 30 s, annealing at 50°S - 40 s, elongation 72°C - 50 C. To visualize the results of hybridization used a set of "non-radioactive DNA identification" (NGO "Silex", Russia).

For detection of the products of expression of the cloned fragment of chromosomal DNA Century pseudomallei used the method of Western blotting [Towbin H., Staehelin T. Electrophoretic transfer of proteins from polyacrylamide gels to nirocellulose sheets: procedure and some applications // Proc. Natl. Acad. Sci. USA. - 1979. - N 76. - P.4350-4354] using antibodies polyclonal goat serum to cells .pseudomallei labeled with alkaline phosphatase.

The recombinant strain E. coli ZV1 (pJla1)obtained by transformation of the cells of E. coli JM107 Cloned fragments of chromosomal DNA Century pseudomallei 56770 SMR2, legirovannye with the vector pUC19, has resistance to pefloxacin (PfR) and streptomycin (SmR), loses a token resistance vector (ArSretains its basic cultural-morphological and biochemical properties of E. coli JM107. The strain E. coli ZV1 (pJla1) is the carrier vysokonapornogo plasmid replicon pJlal size 6.1 TPN output Level (synthesis) of plasmid DNA pJlal in strain is 0.25 mg/l in culture density OD/600=0.5. According to the restriction and hybridization analysis, plasmid pJlal shall be composed of ori replication of the vector pUC19, and the fragment cloned chromosomal DNA .pseudomallei approximately 5.5 TPN Product of expression of the cloned fragment is a protein with a molecular mass of 32 kDa interacting specifically to immunoglobulins melioidosis antisera in Western-blotting.

Example 1. The test recombinant strain E. coli ZV1 (pJla1) no expression of antibiotic resistance compared to the recipient strain E. coli JM107.

The resistant strains to antibiotics is determined by the serial dilution method on nutrient dense environments assessing the IPC product for 5×104M.K. Bacterial suspension prepared from 24 h agar cultures grown at 37°C. the results of the comparative analysis of the levels of resistance of the recombinant strain E. coli ZV1 (pJla1) and the source of the E. coli strain JM107 to antibiotics of different classes given in the table.

As follows from the obtained data, the recombinant strain E. coli ZV1 (pJla1) has resistance to individual antibiotics aminoglycoside (streptomycin) and fluoroquinolone (pefloxacin) rows.

Example 2. Analysis of the products of expression of the cloned sequences of chromosomal DNA .pseudomallei.

Bacterial cells grown for 24-48 h at 37°on nutrient agar, resuspended in lyse buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol), heated 5 min at 100°and remove nelsonbay material by centrifugation at 5000 g. 10-15 µl of the supernatant analyzed by electrophoresis in polyacrylamide gel. For electrophoresis using gels (11% polyacrylamide, 0.1% SDS)prepared according to the method of Laemmli [Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. - Nature. - 1970. - V.227. - P.680-685]. Electrophoresis is carried out at stabilizirovannom a current of 5 mA for 18 h in a vertical machine SE400 (HSI). To control the speed of fractionation in the way the s make 0.004% bromophenol blue. For staining gels use Kumasi G-250 (Serva).

Electrophoretic transfer of proteins to nitrocellulose membrane VA (LKB) with a pore size of 0.22 μm is conducted according to Towbin [Towbin H., Staehelin T. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications // Proc. Natl. Acad. Sci. USA. - 1979. - N 76. - P.4350-4354] in the vertical cell THE (HSI) for 18 h at stable voltage of 45 V and the temperature control with a 10°C. After electroblotting membrane was washed in TBST buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% tween-20) three times for 2 min, blocked in 2% BSA in TBST for 15 h at 4°repeat the washing step and incubated for 1.5 h at room temperature with specific IgG labeled with alkaline phosphatase, breeding 1:128 in TBST with 0.5% BSA. Then the membrane was washed five times for 2 min in TBST, activate the enzyme, incubare membrane 2 min in AR buffer (100 mM Tris-HCl pH 9.5,100 mM NaCl, 5 mM MgCl2) and treated with substrate solution (0.02% bromo-chloro-indolyl-phosphate, 0.02% nicrosini tetrazole buffer AR).

In Western blot turns (drawing) revealed a specific interaction melioidosis immunoglobulins with drug abdelmassih proteins E. coli E. coli ZV1 (protein a 32 kDa) and drugs used as positive controls (obselete proteins .pseudomallei 56770, B. thailandensis E264). The drug abdelmassih proteins of E. coli strain JM107 not contain components, specifically in aimogasta with immunoglobulins melioidosis antisera.

The strain Escherichia coli ZV1 KM167 (national collection of pathogenic bacteria "Microbe") is a carrier of recombinant plasmid DNA J1 1 with a cloned fragment of the chromosomal DNA of Burkholderia pseudomallei determining the synthesis of the protein of 32 kDa and resistance to pefloxacin (RfR) and streptomycin (SmR).


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