AU660342B2 - Virulence-specific bacterial DNA sequence - Google Patents

Description

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66 0342 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1990 REGULATION 3.2 R 1131-1 AU r r Name of Applicant: Actual Inventor/s: Address for Service: AB ASTRA KRISHNAN SANKARAN; YERRAMILLI V.B.K. SUBRAHMANYAM; RAMAN K. ROY; and VASANTHI RAMACHANDRAN.

E.F. WELLINGTON CO., Patent and Trade Mark Attorneys, 312 St. Kilda Road, Melbourne, 3004, Victoria.

a Invention Title: "VIRULENCE-SPECIFIC BACTERIAL DNA SEQUENCE" Details of Associated Provisional Applications Nos: The following statement is a full description of this invention including the best method of performing it known to us.

1 R,1131-1 -1 A- TECHNICAL FIELD 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.

BACKGROUND ART Dysentery caused by Shigella or related enteroinvasive E.coli (EIEC) could be fatal if it is not treated promptly. The incidence of mortality and *.:.morbidity is very high, especially amongst children (WHO Report, 1986) 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 Molecular genetic studies have identified a few regions on the chromosome as well as on the 220-230 kb megaplasmid 3f Shigella that code for bacterial proteins responsible for the virulence phenotype such as the Congo Red binding (reviewed in Ref. invasion intracellular spreading "and thermoregulation Earlier work revealed that the levels of two invasion plasmid antigens (ipa), viz. 63 kDa (ipa b) and 43 kDa (ipa c), are regulated during invasion of epithelial cells by Shigella.

R, 1131-1 -2- The.biochemical basis of host killing by virulent Shigella is still largely unknown. Sansonetti and Mournier concluded, from studies with J774 macrophages, that interference by the invading bacteria with the energy metabolism of host cells, as reflected in the rapid and dramatic drop in ATP concentration, increase in pyruvate concentration and cessation of lactate production, could account for cell death.

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 Serodiagnosis at the early stage (acute phase) of Shigellosis is not possible while conventional microbiological and biochemical techniques are laborious and time consuming.

Two approaches have been taken in the development of diagnostic procedures for the detection of E!EC and Shigella. In one group of studies, a unique sequence of DNA of the organism was identified and used as a probe. Thus, Small and Falkow (11) reported a 2.5 kb Hindlll DNA fragment from the megaplasmid of EIEC which specifically 20 hybridized to the DNA isolated from four virulent Shigella species and EIEC. A 21-base synthetic oligonucleotide corresponding to a sequenced segment of the above 2.5 kb fragment was reported to hybridize to the DNA of all Shigella species and EIEC In another report a 4 4* 17-kb EcoR1 fragment of S. flexneri 5 was used to detect both Shigella and EIEC DNAs. 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. used portions of three genes, which are known to code for invasion positive antigens, for the detection of virulent Shigella species and EIEC.

R 1131-1 -3- BRIEF DESCRIPTION OF THE INVENTION The present invention is based on the identification of an enzyme (ATP diphosphohydrolase or apyrase) which hydrolyses nucleoside triphosphates and nucleoside dispnosphates to nucleoside monophosphates, and which is associated with the virulence of all species of Shigella 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: The invention relates to 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 Spathogen.

DETAILED DESCRIPTION OF THE INVENTION In one aspect the present invention relates to a DNA sequence which encodes for ATP diphosphohydrolase (apyrase) in Shigella and/or EIEC strains. In another aspect the present invention relates to the DNA sequence shown in the Sequence Listing (SEQ ID NO: 1).

*o In yet another aspect, 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 encodes a polypeptide, R 1131-1 -4the amino acid sequence of which is at least 90% homologous with the amino acid sequence as indicated in the Sequence Listing (SEQ ID NO: from position 1 up to and including position 223, or (ii) constitutes an effective subsequence of said DNA sequence.

The term "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.

ScttAlso included in the invention is a DNA sequence which hybridizes with said DNA sequences, or a specific part thereof, under stringent hybridization conditions. The term "stringent 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, Fritsch, E.F. and Maniatis, 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).

R 1131-1 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.

It is preferred to use the hybridization probe of the invention in labelled form. The label can be either a radioactive label or a non-radioactive reporter molecule, covalently attached to the probe. The probe can be s 32 into the S labelled by e.g. incorporation of radioactive element such as P into the probe, either at many phosphodiester bonds or at the terminii of the S 20 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.

To carry out this procedure, 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 R.1131-1 -6sample 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.

Accordingly, the process for diagnosing Shigella and EIEC can suitably comprise the following steps: a) Growing the bacteria from a clinical specimen to be tested for the presence of Shigella or EIEC in a suitable culture medium.

Extracting the nucleic acids from the culture. The bacteria can suitably be lysed in a solution containing 4 M guanidine hydrochloride, 12.5 mM EDTA, 0.5% sodium iaurylsarcosine, triton X-100. Optionally, the extracted nucleic acid material can be amplified by PCR using standard procedures.

20 Hybridization of the nucleic acids (DNA and/or RNA) obtained in step with a hybridization probe as described above, which is labelled in a manner described above.

Capturing of the hybrids as obtained in step with a second hybridization probe as described above, synthesized from a different region of the sequence than the probe as in said second hybridization probe suitably being coated on a solid support.

Detection of the presence or absence of labelled hybridized material by e.g. radioactive, colorimetric, fluorometric or enzymatic methods.

S

On the basis of this process, a diagnostic kit for the detection of Shigella and EIEC can be designed by known techniques Such a kit is R 1131-1 -7included in the scope of the invention, and can suitably comprise the following parts: 1. A lysing solution to lyse the bacteria and which also contains a hybridization probe which has been labelled in a known manner.

2. Microtitre plates coated with a second hybridization probe, .synthesized from a different region of the sequence than the probe as in to capture the bacterial DNA and/or RNA on to the plate.

3. Appropriate reagents to detect the labelled probe which has hybridized to bacterial DNA/RNA as in and then been captured on to the microtiter plates as in Yet further aspects of the invention are: S A recombinant polypeptide which is obtainable from the described DNA sequence, to be used e.g. as a research tool.

A process for the preparation of the said recombinant polypeptide comprising expression in a host organism of DNA encoding the protein.

A recombinant cloning vector containing DNA encoding the said polypeptide.

20 A microorganism or cell culture transfected with DNA encoding the said polypeptide.

S* A virulence associated apyrase protein from Shigella or ElEC strains in substantially pure form, to be used e.g. as a research tool.

A process for obtaining the said apyrase protein in substantially 25 pure form comprising 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.

30 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.

R 1131-1 -8- Use of a DNA sequence; (ii) a hybridization probe; (iii) a substantially pure apyrase protein; and/or (iv) a recombinant polypeptide, obtainable from the disclosed DNA sequence, for the detection of virulent Shigella and/or EIEC strains.

EXAMPLES

Demonstration of a virulence specific adenosine triphosphatase (ATPase) activity in Shiqella and EIEC 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 109) were resuspended in 200 pJ of 50 mM Tris-CI, pH 7.5, 10 mM EDTA, 5 mM ATP (neutralised) and incubated at 370C for 30 min. Released Pi was estimated by Chen's method after removing the cells by centrifugation in Eppendorf centrifuge.

iis sr 20 Table 1 shows the ATPase activity of different strains of S. flexneri grown a 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 0 C, the temperatures at which Shigella is avirulent (see ref. 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.

R 1131-1 -9- TABLE 1 ATPase activity of different species of Shigella and E. coli. Released Pi was estimated by Chen's method (16).

Organism Specific activity in periplasmic prep.

(pmole/min)/mg S. flexneri 0.80 S. dysenteriae 1.30 S. sonnei 1.05 S. boydii 0.55 EIEC 0.83 S. flexneri, 30 0 C 0.18 S. flexneri, 42°C 0.08 S. flexneri, avirulent 0.14 S. flexneri, avirulent 0.17 E. coli K-12 0.17

C

S S

S

*SC

(ii) Purification of a 25 kDa protein that has the ATPase activity and its N-terminal amino acid sequencing 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 pmole/min/mg-protein) for the protein. A 50-fold enrichment was obtained with a yield of using DEAE Sephadex A-50 chromatography. The enzyme eluted between 0.3 and 0.35 M NaCI 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. The molecular mass of the protein was estimated to be 25 kDa. Its elution as a single peak in Sephadex G-100 gel filtration chromatography, at the position of a 30 kDa globular protein, suggested R 1131-1 that the enzyme in its native state was a monomer. The partial N-terminal amino acid sequence of this protein was found to be: Leu Lys Ala Glu Gly Phe Leu Thr Gin Gin Thr Ser Pro Asp Ser Leu Ser lie Leu Pro (SEQ ID NO: 3 in the Sequence Listing).

(iii) Identification of the protein as ATP diphosphohydrolase 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 UTP respectively. It also hydrolysed ADP, though at a lower relative rate of 0.16. It did not, however, hydrolyse p-nitrophenyl phosphate. TLC analysis of the reaction products of ATP hydrolysis showed that ADP was an intermediate in the conversion of ATP to AMP (Fig. 3) suggesting a sequential release of Pi by the enzyme from ATP.

The protein, therefore, was identified as ATP diphosphohydrolase (EC otherwise called apyrase.

(iv) Demonstration that the enzyme is encoded by a 0.9 kb fragment of the megaplasmid DNA The absence of ATPase activity in the plasmid cured strain (BS176) of Shigella suggested that the enzyme was coded by the megaplasmid of virulent Shigella. In order to isolate the gene coding for the ATPase enzyme, a S. flexneri megaplasmid DNA library was constructed in the vector pUC8 at the Hindlll 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. From this 2.1 kb insert, a 0.9 kb Pvull-Hindill fragment (Fig. cloned into M13 mp18, was found to be the minimum size of the gene encoding for ATPase activity. The R.1131-1 -11protein from both the 2.1 kb and 0.9 kb constructs had an apparent molecular mass of 25 kDa (Fig. Further, the N-terminal first ten amino acids sequenced from the cloned enzyme matched with the enzyme isolated from virulent Shigella.

Determination of nucleotide and amino acid sequence The nucleotide sequence of 1134 bp region on the 2.1 kb Hindlll fragment was determined on both strands (SEQ ID NO: 1 in the Sequunce Listing). A single open reading frame of 738 bases was found starting 38 bases downstream of the unique Pvull site and ending at a TAA triplet at position 978. The sequence did not match with any of the published sequence of Shigella megaplasmid DNA. The 20 N-terminal amino acids (positions 1-20 of SEQ ID NO: 2 in the Sequence Listing) of the peptide (SEQ ID NO: 2 in the Sequence Listing) translated from the nucleotide sequence showed a perfect match with the N-terminal amino acids determined by protein sequencing (SEQ ID NO: 3 in the Sequence Listing). The derived amino acid sequence revealed the presence of 23 amino acid long leader peptide that was not present in 20 the mature protein and was apparently part of the signal sequence essential for its translocation.

(vi) Cloninq and overexpression of the apyrase gene 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

AAACCATGGAAACCAAAAACTTTCTTC

(SEQ ID NO: 4 in the Sequence Listing) with a Ncol site forced into it started at position 236 of the sequence while the 21-base long reverse primer with the sequence

GCCGGATCCAGGCTGTCCAGC

R 1131-1 -12- (SEQ ID. NO: 5 in the.Sequence Listing) and a BamHI site forced into it,.

started at position 1003. The PCR amplified product was cloned into vector pTrc 99c at the Ncol/BamHI site and transformed into host BL21 (DE3). Positive clones were first identified by PCR amplification of transformed colonies. Subsequently they were confirmed by assaying ATPase acitivity after induction of clones with. IPTG. There was about fold increase in ATPase activity of the recombinant clones compared to wild-type S. flexnei.

The presence of the above enzyme activity in Shigella sp. and EIEC seems biologically significant, since it could act as a general cytotoxin in cells invaded by the bacteria and interfere with the energy metabolism of the host cells.

(vii) Specificity and sensitivity of the ATPase enzyme activity For determination of enzyme activity, 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 108) were subjected to 20 whole cell ATPase assay as described earlier.

The specificity of the ATPase activity in Shigella and EIEC is further demonstrated in Table 2. A whole range of enteropathogens had been S" tested for the presence of the spacific ATPase activity. None of these 0* organisms showed any significant level of the enzyme activity as compared to virulent Shigella and EIEC.

For determination of sensitivity of the enzyme assay, 10-fold serial dilutions of overnight grown Shigella was assayed in the presence and absence of fixed number of E.coli K-12 (Table In a parallel experiment, different numbers of Shigella were grown overnight along with E.coli K-12 and assayed for ATPase activity (Table Normal stool R-1131-1 -13sample spiked with constant number of Shigella also gave positive enzyme activity to the same level as pure Shigel! dicating the noninterference of the stool sample per se in the detection of Shigella ATPase activity.

S

S. S.

S

*4 TABLE 2 Specificity of the ATPase enzyme activity Organism TEnzyme activity* S. flexnei2a+++

EIEC...

S. flexneri 2a (Plasmidless mutant) Enterobacter cloacae Aeromonas Pleisomonas Pseudo monas Yersinia Normal stool flora Vibdio Kiebsellia S. typhimurium S. typhi ETEC EPEC E.coli K-i 2 A8 2 Onm l.

2 A82Onm O 0 3 S.

S

*5 5555 S.

S

S

.SS

S. S S

S.

R l131-l -1 4- TABLE 3 Sensitivity of the ATPase enzyme activity. Constant numbers of non- Shigella organisms (Ecoli K-1 2) were mixed with various numbers of Shigella and assayed for AT~ase as described.

fNo. of Shigella No. of non-Shigella Enzyme activity I ~(E.coli K-1 2) 107 106 151 ++,A8 2 nm 1.

2 A820nm 0.3 9 9* R 1131-1 TABLE 4 Sensitivity of the ATPase grown together overnight ATPase.

enzyme activity. Shigella and E.coli K-12 were and 100 p1 of the cells were assayed for No. of Shigella No. of E.coli K-12 Enzyme activity x 10 6 107 x 10 5 107 x 10 10 5x10 3 107 5x10 2 107 x 10 107 107 A820n m 1.2; A820nm 0.8; A820nm 2 0.4; A820nm 0.3 9.

*r *99.

.999 I 9 99 @9 9* 9 Si 99 .9r 99 The sensitivity of the enzyme assay was 108 organisms which represents 50 organisms or less as inoculum in the stool sample/mixed culture when grown overnight in a suitable medium.

(viii) Determination of specificity and sensitivity by dot blot hybridization For determination of specificity, bacteria were grown overnight in Luria Broth. 1.0 ml culture was pelleted down and lysed in 100 p1l of lysing solution Triton X-100 or 4M guanidine HCI, 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 p1 of the supernatant was diluted with distilled water to 100 pl and then denatured with an equal volume of 0.5 N NaOH. The denatured DNA samples were spotted R 1131-1 -16on to the nylon membranes which were pre-incubated in 0.5 M NaOH, M NaCI. The membranes were neutralized in 0.5 M Tris-CI pH containing 1.5 M NaCI. Prehybridization was carried out in a sealed plastic bag for 2-3 hours at 550C. The pre-hybridization buffer consisted of 6 x SSC, 1% SDS, 2 X Denhardt's solution, 100 p.g/ml salmon sperm DNA. Hybridization was carried out in the same bag after addition of the 3 2 P-labelled probe (0.5 Kb internal fragment of the apyrase gene). After overnight hybridization at 55°C the membranes were washed twice for min in 2 x SSC, 2% SDS at 55°C, once for 15 minutes in 2 X SSC, 0.2% SDS at 550C and finally once in 0.2 X SSC at 55°C. Membranes were exposed to X-ray films for 24 hrs kept at -70°C (Fig. 5, Panel A).

For determining the sensitivity of the dot blot analysis, various numbers of Shigella ranging from 50 5 x 106 were grown overnight along with E.coli K-12 (107 organisms) at 37°C. 1.0 ml of these cultures were processed as described before for dot blot hybridization (Fig. 5, Panel B).

In a parallel experiment, the minimum number of bacteria detectable by dot blot hybridization was determined by lysing known number of Shigella and using the lysates for dot blot analysis (Fig. 5, Panel The above experiments indicated that the probe used in the hybridization experiments was specific only for different species of Shigella and EIEC.

The sensitivity of detection was 107 Shigella which represents organisms or less as inoculum in the stool sample/mixed culture when grown overnight in a suitable medium.

(ix) Determination of specificity and sensitivity by PCR

A

Cell lysates were prepared as described earlier for dot blot analysis from 1.0 ml overnight cultures of Shigella and other organisms. 10 pL of a 30 1:100 dilution of the lysates were used in PCR reaction to amplify a Kb internal fragment of the apyrase gene using a 25-mer forward primer starting at nucleotide position 243 and a 27-mer reverse primer starting R 1131-1 -17at nucleotide position 794 of the apyrase gene. The PCR was performed for 30 cycles in a reaction volume of 100 pl using 125 p.M of all dNTP's and 200 ng of the primers. The conditions of the PCR include incubations for 30 seconds at 940C for denaturation, 30 seconds at 550C for annealing and 1 minute at 72°C for extension. 10 p. of the PCR product was subsequently analysed on a 1% agarose gel. (Fig. 6, Panels A and

B).

For determining the sensitivity of the PCR analysis 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).

In a separate experiment, 50 5 x 10 6 Shigella were inoculated along with 107 organisms of E.coli K-12 into Luria Broth and grown overnight at 37°C. The cells were lysed and processed for PCR as described above.

(Fig. 7, Panel B).

The 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.

R 1131-1 -18-

REFERENCES

1. LaBrec, Schneider, Magnani, T.J. and Formal, S.B.

(1964): J. Bacteriol. 88, 1503-1518.

2. Sankaran, Ramachandran, Subrahmanyam, Y.V.B.K., Rajarathnam, Elango, S. and Roy, R.K. (1989): Infect. Immun.

57, 2364-2371.

3. Venkatesan, Buysse, J.M. and Kopecko, D. (1988): Proc.

Natl. Acad. Sci. U.S.A. 85, 9317-9321.

4. Boudry, Kactoreh, M. and Sansonetti, P.J. (1988): Microb.

Pathog. 4, 345-357.

Makiao, Sasakawa, Kamata, Kurata, T. and Yoshikawa, M. (1986): Cell 46, 551-555.

6. Pal, Newland, Tall, Formal, S.B. and Hale, T.L.

(1989): Infect. Immun. 57, 477-486.

7. Bernardini, Mournier, Hauteville, Coquis- Rondon, M.

Sansonetti, P.J. (1989): Proc. Natl. Acad. Sci. U.S.A. 86, 3867-3871.

8. Maurelli, A.T. and Sansonetti, P.J. (1988): Proc. Natl. Acad. Sci.

U.S.A. 79, 2820-2824.

9. Sansonetti, P.J. and Mournier, J. (1987): Microb. Pathog. 3, 53-61.

Patent application GB-A-2,242,904.

R 1131-1 -19- 11. Small, P.L.C. and Falkow, S. (1986): In: L. Leive, P.F. Bonventre, J.A. Morello, S.D. Silver, and H.C. Wu Microbiology 1986, p. 121-124. American Society for Microbiology, Washington, D.C.

12. Pauda, Riley, Kumari, Khanna, K.K. and Prakash, K. (1990): J. Clinical. Microbiol. 28, 2122-2124.

13. Taylor, Echeverria, Sethabutr, Pitarangsi, C., Leksomboon, Blacklow, Rowe, Gross, R. and Cross, J. (1988): J. Clinical. Microbiol. 26, 1362-1366.

14. Venkatesan, Buysse, J.M. and Kopecko, D. (1989): J. Clinical.

Microbiol. 27, 2687-2691.

15. Venkatesan, Buysse, Vandendries, E. and Kopecko, D.

(1988): J. Clinical. Microbiol. 26, 261-266.

16. Chen, Toribara, T.Y. and Warner, H. (1956): Anal. Chem. 23, 1756-1758.

17. Viscidi, R.P. and Yolden, R.G. (1987): Mol. Cellular Probes 1, 3-14.

S

S.

O

a a.

R 1131-1 BRIEF DESCRIPTION OF DRAWINGS FIGURE 1 A) Solution hybridization of bacterial nucleic acid sequences with a labelled oligonucleotide probe synthesized from the 738 nt DNA sequence of the apyrase gene.

B) Microtitre well coated with another oligonucleotide synthezied from the 738 nt DNA sequence of the apyrase gene.

C) Capture hybridization of the hybrids from with the oligonucleotide as in D) Capture hybrids after washing, ready for detection of the label by known methods.

FIGURE 2 12% SDS-PAGE analysis of different fractions during purification of apyrase from S. flexneri or from clone Lane 1: Pooled ATPase positive fractions from DEAE Sephadex column chromatography; Lane 2: Electroeluted ATPase positive fractions from native PAGE (S.

flexnen); Lane 3: Molecular weight markers; Lane 4: Electroeluted ATPase positive fractions from native PAGE S(clone pARC25), indicates the postion of the 25 kDa apyrase protein.

.d FIGURE 3 Analysis of reaction products of ATP hydrolysis. Purified enzyme (8 or mU) (U gmole/min) were incubated with ATP in the assay medium for 30 either 15 min or 30 min. The reaction products were analyzed by TLC in polyethyleneimine sheets using an isobutyric acid ammonia water *O .t R 1131-1 -21- (66:1:33) solvent system. The.nucleoside phosphates were revealed with short wavelength UV.

Lane 1: Lane 2: Lane 3: Lane 4: Lane 5: Standard nucleoside phosphates; 80 mU enzyme, 15 min incubation; 80.mU enzyme, 30 min incubation; 8 mU enzyme, 15 min incubation; 8 mU enzyme, 30 min incubation.

FIGURE 4 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 vith 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; RI, EcoR1; HI, Hpal.

FIGURE Determination of specificity and sensitivity of the dot blot hybridization.

Panel A Row a: 1, S.flexneri 2a; 2, S.dysenteriae, 3, S. boydii; 4, S.sonnei; EIEC; 6, S.flexneri 2a (plasmidless mutant); 7, S.flexneri 2a (avirulent).

Row b: 1, EPEC; 2, ETEC; 3, S.typhimurium; 4, Aeromonas; 25 5, Enterobacter 6, Klebsiella; 7, S.typhi.

Row c: 1, Yersinia; 2, Pseudomonas; 3, Vibrio; 4, Normal stool flora; Pleisomonas; 6, C600 (E.coli); 7, E.coli K-12.

*d 5 t* 30 S.

S

Panel B 1-6, Ten-fold serial dilutions of Shigella from 5 x 106 to 50 were grown overnight along with 107 E.coli K-12.

R, 1131 -1 -22- Panel C 1-6, Ten-fold serial dilutions of Shigella from 10~ to FIGURE 6 Specificity of POR Panel A Lane 1, S.flexneri 2a; 2, S.dysenteriae; 3, S. boydf; 4, S.sonnei; 5, EIEC; 6, Marker DNA, EcoRi/Hindll digest); 7, S.flexneri 2a (plasmidless mutant); 8, EPEC; 9, ETEC; 10, E.coli K-1 2; 11, S.typhi.

Panel 8 Lane 1, S.typhimurium; 2, Aeromonas; 3, Enterobacter 4, Klebsiella; Marker DNA, EcoRl/Hindlll digest); 6, Yersinia; 7, Pseudomonas; 8, Normal stool f lora; 9, Vibrio; 10, Pleisomonas; 11, M90T flexneri 2a virulent).

'FGURE 7 Sensitivity of FOR Panel A Lanes 1-7 and 9-11, Ten-fold serial dilutions of Shigella from 109 to 1, lane 8, Marker DNA, ECORlHindlll digest); lane 12, Normal stool flora; lane 13, negative control.

Panel B Lanes 1-6, Ten-fold serial dilutions from 5 x 10~ to 50 were grown overnight along with 107 E.coli K-12; lane 7, Marker DNA, EcoRl/Hindlll digest); lane 8, Ecoli K-1 2.

R.1131-1 -23- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: AB ASTRA STREET: Kvarnbergagatan 16 CITY: Sodertalje COUNTRY: Sweden POSTAL CODE (ZIP): S-151 TELEPHONE: +46-8-55 32 60 00 TELEFAX: +46-8-55 32 88 TELEX: 19237 astra s (ii) TITLE OF INVENTION: Virulence-Specific Bacterial DNA Sequence (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PateintIn Release Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: IN 874/MAS/91 FILING DATE: 26-NOV-1991 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1134 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (vi) ORIGINAL SOURCE: ORGANISM: Shigella flexneri (vii) IMMEDIATE SOURCE: CLONE: pARC (ix) FEATURE: NAME/KEY: CDS LOCATION: 241..981 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 310..978 OTHER INFORMATION: /EC_number= 3.6.1.5 /product= "Apyrase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCACAAATCA TCATAATCAA GAGACAAAAC GATACGAAAA AATAAGATAA AAAACATCGT S TCTTTTACCA CATTATTTTC CGTGAATATG AAAAATAATG TTATTACTTT AATATAAGAC 120 TATTTTTTGT TTTTCCATCA CTCTGTTCAA ATTTTTCCGC ATGACTTGTG TTTTTTGTAA 180 TACAGCTCGT TTTTTACAGC TGACCAAAAT CATCAATTAA TTATGCTAAG GAAATAAATT 240 as* R 1131-1 -24- ATG AAA ACC AAA Met Lys Thr Lys -23 -20 AAC TTT CTT CTT Asn Phe Leu Leu

TTT

Phe -15 TGT ATT GCT ACA Cys Ile Ala Thr AAT ATG ATT Asn Met Ile 288 TTT ATC CCC Phe Ile Pro TCA GCA AAT GCT CTG AAG GCA GAA Ser Ala Asn Ala Leu Lys Ala Glu TTT CTC ACT CAA Phe Leu Thr Gin

CAA

Gin ACT TCA CCA GAC Thr Ser Pro Asp TTG TCA ATA CTT Leu Ser ile Leu CCG CCT CCG GCA Pro Pro Pro Ala AAT TCA GTA GTA TTT CAG GCT GAC AAA Asn Ser Vai Val Phe Gin Ala Asp Lys CAT TAT GAA TTC His Tyr Glu Phe GGC CGC 432 Gly Arg TCG CTC CGG Ser Leu Arg TAC GAG AAT Tyr Giu Asn GCT AAT CGT GTA Ala Asn Arg Val CTC GCT AGC GAA Leu Ala Ser Glu GAT GCA TAC Asp Ala Tyr ATG GAT ATT Met Asp Ile TTT GGT CTT GCA Phe Gly Leu Ala TCA GAT GCT TAT Ser Asp Ala Tyr TCA AGG Ser Arg GAA AAT ACC CCA Glu Asn Thr Pro TTA TAT CAG TTG Leu Tyr Gin Leu ACA CAA GTA CTA Thr Gin Val Leu

CAG

Gin GAT AGC CAT GAT Asp Ser His Asp GCC GTG CGT AAC Ala Val Arg Asn AAA GAA TAT TAT Lys Glu Tyr Tyr AGA GTT CGT CCA TTC GTT ATT TAT AAA Arg Val Arg Pro Phe Val Ile Tyr Lys 110 GCA ACC TGT ACA Ala Thr Cys Thr CCT GAT Pro Asp 120 AAA GAT GAG Lys Asp Glu TCC TTT GGT Ser Phe Gly 140 ATG GCT ATC ACT Met Ala lie Thr

GGC

Gly 130 TCT TAT CCC TCT Ser Tyr Pro Ser GGT CAT GCA Gly His Ala 135 AAT CCT CAA Asn Pro Gin 720 768 TGG GCA GTA GCA Trp Ala Val Ala

CTG

Leu 145 ATA CTT GCG GAG lie Leu Ala Glu a a.i a. a CGT AAA Arg Lys 155 GCG GAA ATA CTT Ala Giu Ile Leu

CGA

Arg 160 CGT GGA TAT GAG Arg Gly Tyr Glu GGA GAA AGT CGG Gly Giu Ser Arg

GTC

Val 170 ATC TGC GGT GCG Ile Cys Gly Ala TGG CAA AGC GAT Trp Gin Ser Asp GAG GCT GOG CGT Glu Ala Giy Arg

TTA

Leu 185 ATG GGA GCA TCG Met Gly Ala Ser GTT GCA GTA CTT Val Ala Val Leu AAT ACA CCT GAA Asn Thr Pro Glu TTT ACC Phe Thr 200 AAA AGC CTT Lys Ser Leu GAA GCC AAA AAA Glu Ala Lys Lys TTT GAA GAA TTA Phe Giu Giu Leu AAT ACT CCT Asn Thr Pro 215 ACC AAT GAA CTG ACC CCA Thr Asn Glu Leu Thr Pro 220 TAAAGCTGGA CAGCCTGTAT CAGGCTATGG 1008 1068 AGGGCCCATA GACAAATCTA CCCTATATGA GCATAGGAGG AGTCTATGGG CACACCACGT R.1131-1 TTTACCCCTG AATTTAAGGG ATTACTGGAA AGGCTGGGAC ATATCCTCCG GCAGAAGCAG 1128 AAAAAG 1134 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 246 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Thr Lys Asn Phe Leu Leu Phe Cys Ile Ala Thr Asn Met Ile -23 -20 -15 Phe Ile Pro Ser Ala Asn Ala Leu Lys Ala Glu Gly Phe Leu Thr Gin 1 Gin Thr Ser Pro Asp Ser Leu Ser Ile Leu Pro Pro Pro Pro Ala Glu 15 20 Asn Ser Val Val Phe Gin Ala Asp Lys Ala His Tyr Glu Phe Gly Arg 35 Ser Leu Arg Asp Ala Asn Arg Val Ar- Leu Ala Ser Glu Asp Ala Tyr r Tyr Glu Asn Phe Gly Leu Ala Phe Ser Asp Ala Tyr Gly Met Asp Ile 65 Ser Arg Glu Asn Thr Pro Ile Leu Tyr Gln Leu Leu Thr Gin Val Leu 80 Gin Asp Ser His Asp Tyr Ala Val Arg Asn Ala Lys Glu Tyr Tyr Lys 95 100 105 Arg Val Arg Pro Phe Val Ile Tyr Lys Asp Ala Thr Cys Thr Pro Asp 110 115 120 Lys Glu Lys Met Ala Ile Thr Gly Ser Tyr Pro Ser Gly His Ala 125 130 135 Ser Phe Gly Trp Ala Val Ala Leu Ile Leu Ala Glu Ile Asn Pro Gin 140 145 150 Arg Lys Ala Glu Ile Leu Arg Arg Gly Tyr Glu Phe Gly Glu Ser Arg 155 160 165 Val Ile Cys Gly Ala His Trp Gin Ser Asp Val Glu Ala Gly Arg Leu 170 175 180 185 e Met Gly Ala Ser Val Val Ala Val Leu His Asn Thr Pro Glu Phe Thr to 190 195 200 Lys Ser Leu Ser Glu Ala Lys Lys Glu Phe Glu Glu Leu Asn Thr Pro 205 210 215 Thr Asn Glu Leu Thr Pro 220 *t* 4 .4 R 1131-1 -26- INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (vi) ORIGINAL SOURCE: ORGANISM: Shigella flexneri (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Leu Lys Ala Glu Gly Phe Leu Thr Gin Gln Thr Ser Pro Asp Ser Leu 1 5 10 Ser Ile Leu Pro INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AAACCATGGA AACCAAAAAC TTTCTTC 27 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCCGGATCCA GGCTGTCCAG C 21 The matter contained in each of the following claims is to be read as part of the general description of the present invention.

Claims (19)

1. A DNA sequence which encodes for ATP diphosphohydrolase (apyrase) in Shigella and/or EIEC strains.

2. A DNA sequence as indicated in the Sequence Listing (SEQ ID NO: 1).

3. A DNA sequence comprising the sequence as indicated in the Sequence Listing (SEQ ID NO: 1) from position 310 up to and including position 978, or an analogue of said DNA sequence which encodes a polypeptide, the amino acid sequence of which is at least 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.

4. A continuous fragment of the DNA sequence according to any one of claims 1-3, which is at least 30 bases or basepairs in length. 15

5. A DNA sequence which hybridizes with the DNA sequence according to any one of claims 1-4, or a specific part thereof, under stringent hybridization conditions.

6. A hybridization probe comprising a DNA sequence according to any one of claims 20

7. A hybridization probe according to claim 6 which is in single stranded form.

8. A hybridization probe according to claim 6 or 7, which is able to capture nucleic acids from virulent Shigella and/or EIEC strains. R 1131-1 -28-

9. A hybridization probe according to any one of claims 6-8 which has been labelled with a radioactive or non-radioactive label.

A process for the detection of virulent Shigella and/or EIEC strains utilizing a hybridization probe according to any one of claims 6-9.

11. A process for the detection of virulent Shigella and EIEC strains comprising the following steps: growing bacteria from a clinical sample in a suitable culture medium; extracting the nucleic acids from the bacteria and optionally amplifying the nucleic acids by Polymerase Chain Reaction; hybridization of nucleic acids obtained in step to a labelled hybridization probe according to claim 6; capturing of the hybrids as obtained in step with a second hybridization probe according to claim 6, synthesized from a different region of the sequence than the probe as in step said hybridization probe being coated on a solid support; 20 detection of the presence or absence of labelled hybridized material.

12. A diagnostic kit for the detection of virulent Shigella and/or EIEC strains, said detection comprising the process according to claim 10 or 11.

13. A recombinant polypeptide-which is obtainable from the DNA sequence according to claim 3.

14. A process for the preparation of a recombinant polypeptide according to claim 13, comprising expression in a host organism of DNA encoding ,L IV the protein.

R 1131-1 -29- A recombinant cloning vector containing DNA encoding a polypeptide according to claim 13.

16. A microorganism or cell culture transfected with DNA encoding a polypeptide according to claim 13.

17. Use of a DNA sequence according to any one of claims 1-5 for the detection of virulent Shigella and/or EIEC strains.

18. Use of a hybridization probe according.to any one of claims 6-9 for the detection of virulent Shigella and/or ElEC strains.

19. Use of a polypeptide according to claim 13 for the detection of virulent Shigella and/or EIEC strains. DATED this 13th day of April, 1995 AB ASTRA, By its Patent Attorneys, SF--WELLINGTON CO., S. Wellington) go *o ooo