WO1994012211A1

WO1994012211A1 - Virulence-specific bacterial dna sequence

Info

Publication number: WO1994012211A1
Application number: PCT/SE1993/000996
Authority: WO
Inventors: Krishnan Sankaran; Yerramili V. B. K. Subrahmanyam; Raman K. Roy; Vasanthi Ramachandran
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1992-11-23
Filing date: 1993-11-19
Publication date: 1994-06-09
Anticipated expiration: 1995-05-23
Also published as: AU5582194A; SE9203506D0; BR9307508A; EP0668780A1; JPH08503609A

Abstract

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.

Description

VIRULENCE-SPECIFIC BACTERIAL DNA SEQUENCE

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 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. 2), invasion (3, 4), intracellular spreading (5,6,7), and

thermoregulation (8). 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 .

The biochemical basis of host killing by virulent

Shigella is still largely unknown. Sansonetti and

Mournier (9) 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 (10). 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 EIEC 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 HindIII DNA fragment from the megaplasmid of EIEC which specifically 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 (12). In another report (13), a 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. (15) used portions of three genes, which are known to code for invasion positive antigens, for the detection of virulent Shigella species and EIEC.

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 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.

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).

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 (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. 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.

Also 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, 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. 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 labelled by e.g. incorporation of radioactive element such as ³²P 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.

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 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.

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.

(b) 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 laurylsarcosine, 0.5% triton

X-100. Optionally, the extracted nucleic acid material can be amplified by PCR using standard procedures.

(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. (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.

(e) Detection of the presence or absence of labelled hybridized material by e.g. radioactive,

colorimetric, fluorometric or enzymatic methods.

On the basis of this process, 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:

(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 according to (1), 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 according to (1) and then been captured on to the microtiter plates according to (2).

Yet further aspects of the invention are:

• 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.

• 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.

• Use of (i) 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

(i) Demonstration of a virulence specific adenosine triphosphatase (ATPase) activity in Shigella 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 μl 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_i 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. flexneri 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.

TABLE 1

ATPase activity of different species of Shigella and E. coli . Released P_i was estimated by Chen's method (16).

(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 μmole/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. Its elution as a single peak in Sephadex G-100 gel filtration

chromatography, at the position of a 30 kDa globular protein, suggested 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 P_i by the enzyme from ATP. The protein, therefore, was identified as ATP diphosphohydrolase (EC 3.6.1.5), 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. 4). From this 2.1 kb insert, a 0.9 kb PvuII-Hindlll fragment (Fig. 4), cloned into M13 mp18, 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). Further, the

N-terminal first ten amino acids sequenced from the cloned enzyme matched with the enzyme isolated from virulent Shigella .

(v) Determination of nucleotide and amino acid sequence

The nucleotide sequence of 1134 bp region on the 2.1 kbHindlll fragment was determined on both strands (SEQ ID

NO: 1 in the Sequence 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 20 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 the mature protein and was apparently part of the signal sequence essential for its translocation.

(vi) Cloning 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

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

(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 G C C G G A T C C A G G C T G T C C A G C

(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/BamRl 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 15-fold increase in ATPase activity of the recombinant clones compared to wild-type S. flexneri.

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 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 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.

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 3). In a parallel experiment,

different numbers of Shigella were grown overnight along with E. coli K-12 and assayed for ATPase activity (Table 4). Normal stool sample spiked with constant number of Shigella also gave positive enzyme activity to the same level as pure Shigella indicating the non-interference of the stool sample per se in the detection of Shigella ATPase activity.

TABLE 2

Specificity of the ATPase enzyme activity

* + + +, A_820nm ≥ 1.2

-, A_820nm ≤ 0.3 TABLE 3

Sensitivity of the ATPase enzyme activity. Constant numbers of non-ShigelIa organisms ( E. coli K-12) were mixed with various numbers of Shigella and assayed for ATPase as described.

* + + +, A_820nm ≥ 1.2

-, A_820nm ≤ 0.3

TABLE 4

Sensitivity of the ATPase enzyme activity. Shigella and E. coli K-12 were grown together overnight and 100 μl of the cells were assayed for ATPase.

* + + +, A_820nm ≥ 1.2

+ + , A_820nm ≥ 0.8

+, A_820nm ≥ 0.4

-, A_820nm ≤ 0.3

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. Culture (1.0 ml) was pelleted down and lysed in 100 μl 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 μl of the supernatant was diluted with distilled water to 100 μl 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,

1.5 M NaCl. 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 μg/ml salmon sperm DNA. Hybridization was carried out in the same bag after addition of the ³²P-labelled probe (0.5 Kb internal fragment of the apyrase gene). After overnight

hybridization at 55°C the membranes were washed twice for 15 min in 2 x SSC, 2% SDS at 55°C, once for 15 minutes in 2 X SSC, 0.2% SDS at.55°C 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 C). 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 10 Shigella which represents 50

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 Cell lysates were prepared as described earlier for dot blot analysis from 1.0 ml overnight cultures of Shigella and other organisms. 10 μl of a 1:100 dilution of the lysates were used in PCR reaction to amplify a 0.5 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 at nucleotide position 794 of the apyrase gene. The PCR was performed for 30 cycles in a reaction volume of 100 μl using 125 μM of all dNTP's and 200 ng of the primers. 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 μl 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, 5 - 50 x 10⁶ Shigella were inoculated along with 10⁷ organisms of E. coli K-12 mto 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. REFERENCES

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J. Clinical. Microbiol. 27, 2687-2691. 15. Venkatesan, M., Buysse, J.M., Vandendries, E. and Kopecko, D. (1988): J. Clinical. Microbiol. 26, 261-266.

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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 (A) with the oligonucleotide as in (B).

(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 pARC25. Lane 1: Pooled ATPase positive fractions from DEAE

Sephadex A-50 column chromatography;

Lane 2: Electroeluted ATPase positive fractions from native PAGE ( S. flexneri );

Lane 3: Molecular weight markers;

Lane 4: Electroeluted ATPase positive fractions from native PAGE (clone pARC25), indicates the postion of the 25 kDa apyrase protein. FIGURE 3

Analysis of reaction products of ATP hydrolysis. Purified enzyme (8 or 80 mU) (U = μmole/min) were incubated with ATP in the assay medium for either 15 min or 30 min. 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. Lane 1 Standard nucleoside phosphates;

Lane 2 8 mU enzyme, 15 min incubation;

Lane 3 8 mU enzyme, 30 min incubation;

Lane 4 80 mU enzyme, 15 min incubation;

Lane 5 80 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 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, Pst1; Rl, EcoR1; HI, Hpa1. FIGURE 5

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 ; 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

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

1-6, Ten-fold serial dilutions of Shigella from 10⁹ to 10⁴.

FIGURE 6

Specificity of PCR

Panel A

Lane 1, S. flexneri 2a; 2, S . dysenteriae; 3, S. boydii ; 4, S. sonnei ; 5, EIEC; 6, Marker (λ DNA, EcoRI/Hindlll digest); 7, S. flexneri 2a (plasmidless mutant); 8, EPEC; 9, ETEC; 10, E. coli K-12; 11, S. typhi .

Panel B

Lane 1, S. typhimurium; 2 , Aeromonas ; 3, Enterobacter; 4, Klebsiella ; 5, Marker (λ DNA, EcoRl/Hindlll digest); 6, Yersinia ; 7, Pseudomonas; 8, Normal stool flora; 9, Vibrio; 10, Pleisomonas ; 11, M90T ( S. flexneri 2a

virulent). FIGURE 7

Sensitivity of PCR

Panel A

Lanes 1-7 and 9-11, Ten-fold serial dilutions of Shigella from 10⁹ to 1, lane 8, Marker (λ DNA, EcoRI/Hindlll digest); lane 12, Normal stool flora; lane 13, negative control.

Panel B

Lanes 1-6, Ten-fold serial dilutions from 5 x 10⁶ to 50 were grown overnight along with 10 E. coli K-12; lane 7, Marker (λ DNA, EcoRI/Hindlll digest); lane 8, E. coli K- 12. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: AB ASTRA

(B) STREET: Kvarnbergagatan 16

(C) CITY: Sodertalje

(E) COUNTRY: Sweden

(F) POSTAL CODE (ZIP) : S-151 85

(G) TELEPHONE: +46-8-553 260 00

(H) TELEFAX: +46-8-553 288 20

(I) TELEX: 19237 astra s

(ii) TITLE OF INVENTION: Virulence-Specific Bacterial DNA Sequence

(iii) NUMBER OF SEQUENCES: 5

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IN 874/MAS/91

(B) FILING DATE: 26-NOV-1991

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: SE 9203506-2

(B) FILING DATE: 23-NOV-1992

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1134 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : both

(D) TOPOLOGY: linear

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Shigella flexneri

(vii) IMMEDIATE SOURCE:

(B) CLONE: pARC 25

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 241..981

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 310..978

(D) OTHER INFORMATION: /EC_number= 3.6.1.5

/product= "Apyrase"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TCACAAATCA TCATAATCAA GAGACAAAAC GATACGAAAA AATAAGATAA AAAACATCGT 60

TCTTTTACCA CATTATTTTC CGTGAATATG AAAAATAATG TTATTACTTT AATATAAGAC 120

TATTTTTTGT TTTTCCATCA CTCTGTTCAA ATTTTTCCGC ATGACTTGTG TTTTTTGTAA 180 TACAGCTCGT TTTTTACAGC TGACCAAAAT CATCAATTAA TTATGCTAAG GAAATAAATT 240

ATG AAA ACC AAA AAC TTT CTT CTT TTT TGT ATT GCT ACA AAT ATG ATT 288 Met Lys Thr Lys Asn Phe Leu Leu Phe Cys lle Ala Thr Asn Met lle

-23 -20 -15 -10

TTT ATC CCC TCA GCA AAT GCT CTG AAG GCA GAA GGT TTT CTC ACT CAA 336 Phe lle Pro Ser Ala Asn Ala Leu Lys Ala Glu Gly Phe Leu Thr Gln

-5 1 5

CAA ACT TCA CCA GAC AGT TTG TCA ATA CTT CCG CCG CCT CCG GCA GAG 384 Gln Thr Ser Pro Asp Ser Leu Ser lle Leu Pro Pro Pro Pro Ala Glu

10 15 20 25

AAT TCA GTA GTA TTT CAG GCT GAC AAA GCT CAT TAT GAA TTC GGC CGC 432 Asn Ser Val Val Phe Gln Ala Asp Lys Ala His Tyr Glu Phe Gly Arg

30 35 40

TCG CTC CGG GAT GCT AAT CGT GTA CGT CTC GCT AGC GAA GAT GCA TAC 480 Ser Leu Arg Asp Ala Asn Arg Val Arg Leu Ala Ser Glu Asp Ala Tyr

45 50 55

TAC GAG AAT TTT GGT CTT GCA TTT TCA GAT GCT TAT GGC ATG GAT ATT 528 Tyr Glu Asn Phe Gly Leu Ala Phe Ser Asp Ala Tyr Gly Met Asp lle

60 65 70

TCA AGG GAA AAT ACC CCA ATC TTA TAT CAG TTG TTA ACA CAA GTA CTA 576 Ser Arg Glu Asn Thr Pro lle Leu Tyr Gln Leu Leu Thr Gln Val Leu

75 80 85

CAG GAT AGC CAT GAT TAC GCC GTG CGT AAC GCC AAA GAA TAT TAT AAA 624 Gln Asp Ser His Asp Tyr Ala Val Arg Asn Ala Lys Glu Tyr Tyr Lys

90 95 100 105

AGA GTT CGT CCA TTC GTT ATT TAT AAA GAC GCA ACC TGT ACA CCT GAT 672 Arg Val Arg Pro Phe Val lle Tyr Lys Asp Ala Thr Cys Thr Pro Asp

110 115 120

AAA GAT GAG AAA ATG GCT ATC ACT GGC TCT TAT CCC TCT GGT CAT GCA 720 Lys Asp Glu Lys Met Ala lle Thr Gly Ser Tyr Pro Ser Gly His Ala

125 130 135

TCC TTT GGT TGG GCA GTA GCA CTG ATA CTT GCG GAG ATT AAT CCT CAA 768 Ser Phe Gly Trp Ala Val Ala Leu lle Leu Ala Glu lle Asn Pro Gln

140 145 150

CGT AAA GCG GAA ATA CTT CGA CGT GGA TAT GAG TTT GGA GAA AGT CGG 816 Arg Lys Ala Glu lle Leu Arg Arg Gly Tyr Glu Phe Gly Glu Ser Arg

155 160 165

GTC ATC TGC GGT GCG CAT TGG CAA AGC GAT GTA GAG GCT GGG CGT TTA 864 Val lle Cys Gly Ala His Trp Gln Ser Asp Val Glu Ala Gly Arg Leu

170 175 180 185

ATG GGA GCA TCG GTT GTT GCA GTA CTT CAT AAT ACA CCT GAA TTT ACC 912 Met Gly Ala Ser Val Val Ala Val Leu His Asn Thr Pro Glu Phe Thr

190 195 200

AAA AGC CTT AGC GAA GCC AAA AAA GAG TTT GAA GAA TTA AAT ACT CCT 960 Lys Ser Leu Ser Glu Ala Lys Lys Glu Phe Glu Glu Leu Asn Thr Pro

205 210 215

ACC AAT GAA CTG ACC CCA TAAAGCTGGA CAGCCTGTAT CAGGCTATGG 1008

Thr Asn Glu Leu Thr Pro

220

AGGGCCCATA GACAAATCTA CCCTATATGA GCATAGGAGG AGTCTATGGG CACACCACGT 1068 TTTACCCCTG AATTTAAGGG ATTACTGGAA AGGCTGGGAC ATATCCTCCG GCAGAAGCAG 1128 AAAAAG 1134

(2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 246 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Lys Thr Lys Asn Phe Leu Leu Phe Cys lle Ala Thr Asn Met lle

-23 -20 -15 -10

Phe lle Pro Ser Ala Asn Ala Leu Lys Ala Glu Gly Phe Leu Thr Gln

-5 1 5

Gln Thr Ser Pro Asp Ser Leu Ser lle Leu Pro Pro Pro Pro Ala Glu

10 15 20 25

Asn Ser Val Val Phe Gln Ala Asp Lys Ala His Tyr Glu Phe Gly Arg

30 35 40

Ser Leu Arg Asp Ala Asn Arg Val Arg Leu Ala Ser Glu Asp Ala Tyr

45 50 55

Tyr Glu Asn Phe Gly Leu Ala Phe Ser Asp Ala Tyr Gly Met Asp lle

60 65 70

Ser Arg Glu Asn Thr Pro lle Leu Tyr Gln Leu Leu Thr Gln Val Leu

75 80 85

Gln Asp Ser His Asp Tyr Ala Val Arg Asn Ala Lys Glu Tyr Tyr Lys

90 95 100 105

Arg Val Arg Pro Phe Val lle Tyr Lys Asp Ala Thr Cys Thr Pro Asp

110 115 120

Lys Asp Glu Lys Met Ala lle Thr Gly Ser Tyr Pro Ser Gly His Ala

125 130 135

Ser Phe Gly Trp Ala Val Ala Leu lle Leu Ala Glu lle Asn Pro Gln

140 145 150

Arg Lys Ala Glu lle Leu Arg Arg Gly Tyr Glu Phe Gly Glu Ser Arg

155 160 165

Val lle Cys Gly Ala His Trp Gln Ser Asp Val Glu Ala Gly Arg Leu

170 175 180 185

Met Gly Ala Ser Val Val Ala Val Leu His Asn Thr Pro Glu Phe Thr

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 (2) INFORMATION FOR SEQ ID NO : 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Shigella flexneri

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Leu Lys Ala Glu Gly Phe Leu Thr Gln Gln Thr Ser Pro Asp Ser Leu 1 5 10 15

Ser lle Leu Pro

20

(2) INFORMATION FOR SEQ ID NO : 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

AAACCATGGA AACCAAAAAC TTTCTTC 27

(2) INFORMATION FOR SEQ ID NO : 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5:

GCCGGATCCA GGCTGTCCAG C 21

Claims

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 (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.

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.

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 1-5, or a specific part thereof.

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.

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

10. 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:

(a) growing bacteria from a clinical sample in a

suitable culture medium;

(b) extracting the nucleic acids from the bacteria and optionally amplifying the nucleic acids by

Polymerase Chain Reaction;

(c) hybridization of nucleic acids obtained in step (b) to a labelled hybridization probe according to claim 6;

(d) capturing of the hybrids as obtained in step (c) with a second hybridization probe according to claim 6, said hybridization probe being coated on a solid support;

(e) 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 the protein.

15. 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. A virulence associated apyrase protein from

Shigella or EIEC strains in substantially pure form.

18. A process for obtaining the apyrase protein

according to claim 17 in substantially pure form comprising (a) anionic exchange chromatography of a cell extract from Shigella or EIEC; and (b) preparative gel electrophoresis of the sample from (a).

19. 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.

20. A method for protecting a host against Shigella and/or EIEC infection comprising interfering with the activity of the apyrase enzyme.

21. Use of an apyrase protein according to claim 17 as a target for therapy of Shigella and/or EIEC infection.

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

23. Use of a hybridization probe according to any one of claims 6 for the detection of virulent Shigella and/or EIEC strains.

24. Use of a polypeptide according to claim 13 for the detection of virulent Shigella and/or EIEC strains.

25. Use of an apyrase protein according to claim 17 for the detection of virulent Shigella and/or EIEC strains.