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Definitions

• a nucleotide sequence encoding a virulence associated ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Shigella sp . and enteroinvasive E. coli (EIEC) is disclosed. Further disclosed are hybridization probes comprising said nucleotide sequence, or a specific part thereof; a process for the specific detection of virulent Shigella sp . and EIEC comprising use of said hybridization probes; and a diagnostic kit for use in the above mentioned diagnosis .
• Dysentery caused by Shigella or related enteroinvasive E. coli could be fatal if it is not treated promptly.
• the incidence of mortality and morbidity is very high, especially amongst children in developing countries.
• Dysentery caused by Shigella is the manifestation of several intricate biochemical events in which both bacterial and host factors are involved.
• the invasion of colonic epithelial cells by the bacteria is an early essential step which is followed by intracellular multiplication and reinfection of adjacent cells (1) .
• Molecular genetic studies have identified a few regions on the chromosome as well as on the 220-230 kb megaplasmid of Shigella that code for bacterial proteins responsible for the virulence phenotype such as the Congo Red binding (reviewed in Ref.
• Nucleic acid (DNA and RNA) hybridization is now commonly used for the detection of causative agents of a variety of infectious diseases which include viral, bacterial, fungal and parasitic organisms (10) . Serodiagnosis at the early stage (acute phase) of Shigellosis is not possible while conventional microbiological and biochemical techniques are laborious and time consuming.
• Venkatesan et al . (14) used part of an unidentified gene sequence of the Shigella megaplasmid DNA for the specific identification of both Shigella and EIEC. In another approach, the gene for an identified virulence-specific antigen was used to identify the virulent organism. Thus, Venkatesan et al . (15) used portions of three genes, which are known to code for invasion positive antigens, for the detection of virulent Shigella species and EIEC.
• the present invention is based on the identification of an enzyme (ATP diphosphohydrolase or apyrase) which hydrolyses nucleoside triphosphates and nucleoside disphosphates to nucleoside monophosphates, and which is associated with the virulence of all species of Shigella (e.g. S. flexneri , S. dysenteriae, S. sonnei , S. boydi ) and the related EIEC.
• the gene coding for apyrase has been cloned and sequenced.
• the unique nucleotide sequence of the disclosed gene has its potential use in the detection of a virulence determinant in pathogenic bacteria as exemplified by virulent Shigella and enteroinvasive E. coli .
• a DNA, sequence, comprising the gene coding for the protein, is disclosed in the Sequence Listing (SEQ ID NO: 1) .
• the invention relates to (i) said DNA sequence, (ii) a DNA hybridization probe comprising said DNA sequence or a part thereof, (iii) a process for identification of Shigella sp . and EIEC using such a probe, and (iv) a diagnostic kit where the DNA sequence, or a part thereof, is used for the detection of the pathogen.
• the present invention relates to a DNA sequence which encodes for ATP diphosphohydrolase (apyrase) in Shigella and/or EIEC strains.
• the present invention relates to the DNA sequence shown in the Sequence Listing (SEQ ID NO: 1) .
• the invention relates to a DNA sequence comprising the nucleotide sequence shown in the Sequence Listing (SEQ ID NO: 1) from nucleotide position 310 up to and including position 978, or an analogue of said DNA sequence which (i) encodes a polypeptide, the amino acid sequence of which is at least 90% homologous with the amino acid sequence as indicated in the Sequence Listing (SEQ ID NO: 2), from position 1 up to and including position 223, or (ii) constitutes an effective subsequence of said DNA sequence.
• DNA sequence comprises in this context a single stranded DNA sequence as indicated in the Sequence Listing, as well as the complementary strand of the same DNA sequence and the corresponding double stranded sequence.
• the term "effective subsequence" as used above refers to a subsequence being a least partially functional with respect to the activities of apyrase in Shigella and/or EIEC strains .
• the subsequence may be the result of a truncation at either end of the DNA sequence, or of the removal of one or more nucleotides or nucleotide sequences within the DNA sequence.
• hybridization conditions is to be understood in its conventional meaning, i.e. that hybridization is carried out according to an ordinary laboratory manual such as Sambrook, J., Fritsch, E.F. and Maniatis, T.E.: Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor, NY, 1989)
• a further aspect of the invention is a hybridization probe comprising a DNA sequence as described above.
• the hybridization probe of the invention can thus suitably comprise a 738 nucleotide DNA fragment, starting from nucleotide 241 and ending with nucleotide 978 of the sequence disclosed in the Sequence Listing (SEQ ID NO: 1) .
• the hybridization probe of the invention is capable of specifically hybridizing to the megaplasmid DNA of virulent Shigella, by known methods. It is envisaged that also continuous segments of the DNA sequence of the invention, containing at least 30 nucleotides should have the same capability of hybridization. In other words, smaller as well as larger fragments of the DNA sequence described here can be used as hybridization probes.
• the hybridization probe can also be designed from the complementary strand to hybridize with the specific mRNA and thus increasing the concentration of hybridizable sequences. For this purpose it is desirable to synthesize probes from the ends of the sequence in order to hybridize with both DNA and intact mRNA sequences.
• the hybridization probe of the invention can be either a radioactive label or a non-radioactive reporter molecule, covalently attached to the probe.
• the probe can be labelled by e.g. incorporation of radioactive element such as 3 ⁇ P into the probe, either at many phosphodiester bonds or at the terminii of the probe DNA, ligation of an organic molecule which is either a chromophore or fluorophore or a molecule which can be detected by chemical or immunological methods (17) .
• a further aspect of the invention is a procedure for detecting the megaplasmid DNA of virulent Shigella and EIEC utilizing the hybridization of a hybridization probe as described above with the total DNA of Shigella and EIEC.
• oligonucleotides of desired lengths can be synthesized from the 738 nt DNA sequence of the apyrase gene.
• One such oligonucleotide which has been labelled with either a radioactive or a non- radioactive reporter molecule can be used to hybridize with the sample DNA.
• One or more additional oligonucleotides synthesized from different regions within the 738 nt DNA sequence can be used to coat a microtitre plate. This coated microtitre plate can then be used to capture the sample DNA, earlier hybridized with the labelled synthetic oligonucleotide, through a second hybridization on the microtitre plate.
• the capture hybrids can then be estimated using suitable protocols depending on the nature of the reporter molecule.
• the detection of hybrids on the plate will, in turn, indicate the presence of specific DNA in the sample.
• Fig. 1 shows a pictorial representation of such a protocol.
• the process for diagnosing Shigella and EIEC can suitably comprise the following steps :
• the bacteria can suitably be lysed in a solution containing 4 M guanidine hydrochloride, 12.5 mM EDTA, 0.5% sodium laurylsarcosine, 0.5% triton X-100.
• the extracted nucleic acid material can be amplified by PCR using standard procedures .
• step (c) Hybridization of the nucleic acids (DNA and/or RNA) obtained in step (b) with a hybridization probe as described above, which is labelled in a manner described above.
• step (d) Capturing of the hybrids as obtained in step (c) with a second hybridization probe as described above, synthesized from a different region of the sequence than the probe as in (c), said second hybridization probe suitably being coated on a solid support .
• a diagnostic kit for the detection of Shigella and EIEC can be designed by known techniques (10) .
• Such a kit is included in the scope of the invention, and can suitably comprise the following parts:
• a microorganism or cell culture transfected with DNA encoding the said polypeptide • A virulence associated apyrase protein from Shigella or EIEC strains in substantially pure form, to be used e.g. as a research tool.
• a process for obtaining the said apyrase protein in substantially pure form comprising (a) anionic exchange chromatography, and (b) preparative gel electrophoresis .
• a process for detection of virulent Shigella and/or EIEC strains comprising the detection of an apyrase protein, or its activity, in said strains by enzymatic and/or immunological methods.
• a method for protecting a host against Shigella and/or EIEC infection comprising interfering with the activity of the apyrase enzyme, as well as the use of an apyrase enzyme as a target for therapy of Shigella and/or EIEC infection.
• Bacteria were grown in LB medium overnight, harvested by low speed centrifugation (5000 x g, 10 min) and washed twice with 10 mM Hepes buffer, pH 7.5. The washed cells (3 x 10 9 ) were resuspended in 200 ⁇ l of 50 mM Tris-Cl, pH 7.5, 10 mM EDTA, 5 mM ATP (neutralised) and incubated at 37°C for 30 min. Released P j _ was estimated by Chen's method (16) , after removing the cells by centrifugation in Eppendorf centrifuge. Table 1 shows the ATPase activity of different strains of S. fl exneri grown under various growth conditions.
• the activity was seen only in virulent but not in the avirulent isolate or the plasmid cured strain. Like many other virulence associated properties of Shigella, the activity was greatly reduced when the virulent bacteria were grown at 30°C or at 42°C, the temperatures at which Shigella is avirulent (see ref. 2) .
• the table also shows that this enzyme activity was found in other virulent species of Shigella, viz. S. dysenteriae, S. sonnei and S. boydii and in related EIEC but not in noninvasive E. coli K-12.
• a simple two step purification scheme was developed to isolate the enzyme from the EDTA extract of the cell pellet which served as a convenient and a relatively enriched source (0.8 ⁇ mole/min/mg-protein) for the protein.
• a 50-fold enrichment was obtained with a yield of 60% using DEAE Sephadex A-50 chromatography.
• the enzyme eluted between 0.3 and 0.35 M NaCl as a single peak.
• This fraction after electrophoresis on preparative polyacrylamide gel containing Sarkosyl and electroelution, resulted in a highly enriched preparation as revealed by SDS-PAGE (Fig. 2) .
• the molecular mass of the protein was estimated to be 25 kDa.
• the purified enzyme preparation showed little specificity with respect to NTPs, the relative activities with respect to ATP being, 1.5, 1.0, and 0.8 for GTP, CTP and
• ATPase activity in the plasmid cured strain (BS176) of Shigella suggested that the enzyme was coded by the megaplasmid of virulent Shigella .
• a S. flexneri megaplasmid DNA library was constructed in the vector pUC8 at the HindiII site and transformed into host HB101. Recombinant clones were screened for the ATPase activity. From about 512 clones tested, one was found positive for ATPase activity and was named pARC 25. This pARC 25 clone was subjected to partial restriction map analysis. It had an insert of 2.1 kb (Fig. 4) .
• a 0.9 kb PvuII-Hindlll fragment (Fig. 4), cloned into M13 mpl8, was found to be the minimum size of the gene encoding for ATPase activity.
• the protein from both the 2.1 kb and 0.9 kb constructs had an apparent molecular mass of 25 kDa (Fig. 2) .
• the N-terminal first ten amino acids sequenced from the cloned enzyme matched with the enzyme isolated from virulent Shigella .
• Plasmid DNA from clone pARC 25 was used as template to amplify the apyrase gene sequence by Polymerase Chain Reaction (PCR) .
• the 27-base long forward primer having the sequence A A A C C A T G G A A A C C A A A A A C T T T C T T C
• bacteria were grown in Luria broth overnight, harvested by low speed centrifugation and washed with 10 mM Hepes buffer, pH 7.5. The washed bacteria (5 x 10°) were subjected to whole cell ATPase assay as described earlier.
• EIEC is further demonstrated in Table 2. A whole range of enteropathogens had been tested for the presence of the specific ATPase activity. None of these organisms showed any significant level of the enzyme activity as compared to virulent Shigella and EIEC.
• Sensitivity of the ATPase enzyme activity Constant numbers of non-Shi elIa organisms ( E. coli K-12) were mixed with various numbers of Shigella and assayed for ATPase as described.
• Sensitivity of the ATPase enzyme activity Shigella and E. coli K-12 were grown together overnight and 100 ⁇ l of the cells were assayed for ATPase.
• the sensitivity of the enzyme assay was 10 e organisms which represents 50 organisms or less as inoculum in the stool sample/mixed culture when grown overnight in a suitable medium.
• bacteria were grown overnight in Luria Broth. Culture (1.0 ml) was pelleted down and lysed in 100 ⁇ l of lysing solution (2% Triton X- 100 or 4 M guanidine HCl, 0.5% Na-lauryl sarcosine, 0.5% Triton X-100, 12.5 mM EDTA) . The lysed solutions were boiled for 10 min and following centrifugation, 5-10 ⁇ l of the supernatant was diluted with distilled water to 100 ⁇ l and then denatured with an equal volume of 0.5 N NaOH. The denatured DNA samples were spotted on to the nylon membranes which were pre-incubated in 0.5 M NaOH,
• the membranes were neutralized in 0.5 M Tris- Cl, pH 8.0, containing 1.5 M NaCl .
• Prehybridization was carried out in a sealed plastic bag for 2-3 hours at 55°C.
• the pre-hybridization buffer consisted of 6 x SSC, 1% SDS, 2 X Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA.
• Hybridization was carried out in the same bag after addition of the 32 P-labelled probe (0.5 Kb internal fragment of the apyrase gene) .
• the conditions of the PCR include incubations for 30 seconds at 94°C for denaturation, 30 seconds at 55°C for annealing and 1 minute at 72°C for extension. 10 ⁇ l of the PCR product was subsequently analysed on a 1% agarose gel. (Fig. 6, Panels A and B) .
• a normal stool sample which was suspended in saline was spiked with 10-fold dilutions of pure cultures of Shigella, lysed and the lysates were used for PCR analysis (Fig. 7, Panel A) .
• PCR analysis indicated that the apyrase gene was only present in different species of Shigella and EIEC.
• the sensitivity of detection by PCR was about 100 Shigella present in a mixed population. Further, the stool sample did not inhibit the PCR to any significant level.
• FIGURE 1 A first figure.
• FIGURE 3 shows the postion of the 25 kDa apyrase protein.
• reaction products of ATP hydrolysis Analysis of reaction products of ATP hydrolysis.
• the reaction products were analyzed by TLC in polyethyleneimine sheets using an isobutyric acid : ammonia : water (66:1:33) solvent system.
• the nucleoside phosphates were revealed with short wavelength UV.
• Partial restriction map of clone pARC25 containing apyrase gene The 2.1 kb DNA fragment obtained from the plasmid of clone pARC25 by Hindlll digestion was digested with various enzymes and analysed on 1% agarose gels. The fragments generated were subcloned into suitable vectors and tested for expression of ATPase activity. H, Hindlll; RV, EcoRV; P, Pstl; Rl, EcoRl; HI, Hpal.
• Panel A Row a: 1, S. flexneri 2a; 2, S. dysenteriae, 3, S. boydii ; 4, S. sonnei ; 5, EIEC; 6, S. flexneri 2a (plasmidless mutant) ; 7, S. flexneri 2a (avirulent) .
• Row b 1, EPEC; 2, ETEC; 3, S. typhimurium; 4, Aeromonas ; 5, Enterobacter; 6, Klebsiella; 7, S. typhi .
• Row c 1, Yersinia ; 2 , Pseudomonas ; 3, Vijbrio; 4, Normal stool flora; 5, Pleisomonas ; 6, C600 (E. coli ) ; 7, E. coli K-12.
• Panel B Row a: 1, S. flexneri 2a; 2, S. dysenteriae, 3, S. boydii ; 4, S. sonnei ; 5, EIEC; 6, S.
• FIGURE 6 Specificity of PCR
• Lane 1 S. flexneri 2a; 2, S . dysenteriae; 3, S. boydii ; 4, S. sonnei ; 5, EIEC; 6, Marker ( ⁇ DNA, EcoRI/Hindlll digest); 7, S. flexneri 2a (plasmidless mutant); 8, EPEC; 9, ETEC; 10, E. coli K-12; 11, S. typhi .
• Lane 1 S. typhimurium; 2 , Aeromonas ; 3, Enterobacter; 4, Klebsiella ; 5, Marker ( ⁇ DNA, EcoRl/Hindlll digest); 6, Yersinia ; 7, Pseudomonas; 8, Normal stool flora; 9, Vibrio; 10, Pleisomonas ; 11, M90T ( S. flexneri 2a virulent) .
• Lanes 1-7 and 9-11 Ten-fold serial dilutions of Shigella from 10 9 to 1, lane 8, Marker ( ⁇ DNA, EcoRI/HindiII digest) ; lane 12, Normal stool flora; lane 13, negative control .
• TELEPHONE +46-8-553 260 00
• TELEFAX +46-8-553 288 20
• TELEX 19237 astra s
• ORGANISM Shigella flexneri
• TATTTTTTGT TTTTCCATCA CTCTGTTCAA ATTTTTCCGC ATGACTTGTG TTTTTTGTAA 180 TACAGCTCGT TTTTTACAGC TGACCAAAAT CATCAATTAA TTATGCTAAG GAAATAAATT 240
• Lys Asp Glu Lys Met Ala lie Thr Gly Ser Tyr Pro Ser Gly His Ala 125 130 135
• Val lie Cys Gly Ala His Trp Gin Ser Asp Val Glu Ala Gly Arg Leu 170 175 180 185
• ORGANISM Shigella flexneri

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