| UPDATE | 07.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 11.07.00 |
| PATENT TITLE |
Gene encoding invasion protein of campylobacter species |
| PATENT ABSTRACT |
A protein associated with adherence and invasion of Campylobacter spp. including C. jejuni and C. coli is provided. Methods are disclosed for detecting Campylobacter spp. including C. jejuni and C. coli in a biological sample by determining the presence of the protein or a nucleic acid molecule encoding the protein in the sample. Compositions for treatment of infections diseases and vaccines are also described. |
| PATENT INVENTORS | This data is not available for free |
| PATENT FILE DATE | 08.04.98 |
| PATENT REFERENCES CITED |
Pei et al, J Biological Chemistry, vol. 268, Sep. 5, pp. 18717-18725, 1993. Grant, CCR et al, Infection and Immunity, May 1993, vol. 61(5), pp. 1764-1771. Ketley, J.M., J. Med. Microbiol., vol. 42, pp. 312-327, 1995. Bourke, B. et al, Microbiology, vol. 141, pp. 2417-2424, 1995. Pesci, EC et al, Infection and Immunity, vol. 62, (7), Jul. 1994, pp. 2687-2694. Yao, R et al, Mol. Microbiol., vol.14(5), pp. 883-893, 1994. Boot, HJ et al, Mol. Microbiol. vol. 21(6), Sep. 1996, pp. 1117-1123. Beveridge, TJ et al, FEMS Microbiol. Reviews, vol. 20, pp. 99-149, 1997. Altschul, S. F. et al. J. Mol. Biol. 215:403-410, 1990. Bourke, B., et al., Microbiol. (UK) 141:2417-2424, 1995. Chan V.L. and Bingham H.L., Gene 101:51-58,1991. Chan, V. L., et al. Gene 164:25-31, 1995. Chang, N., and D. E. Taylor. J. Bacteriol. 172:5211-5217, 1990. Grant, C. C. R., et al., Infect. Immun. 61:1764-1771, 1993. Hani, E. K., and V. L. Chan. J. Bacteriol. 177:2396-2402, 1995. Hong, Y., et al., Microbiol. (UK) 141:2561-2567, 1995. Ketley, J. M. J. Med. Microbiol. 42:312-327, 1995. Kim, N.W. et al. Gene 164:101-106, 1995. Kim, N. W., et al. J. Bacteriol. 175:7468-7470, 1993. Kim, N. W., et al. J. Bacteriol. 174:3494-3498, 1992. Labigne-Roussel, A., et al. J. Bacteriol. 170:1704-1708, 1988. McClelland, H., et al. Nucl. Acids Res. 15:5985-6005, 1987. Mecsas, J., and E. J. Strauss. Emerg. Infect. Dis. 2:271-288, 1996. Pesci, E.C., et al. Infect. Immun. 62:2687-2694, 1994. Rosqvist, R., et al. EMBO J. 13:964-972, 1994. Sory, M. P., and G. R. Cornelis. Mol. Microbiol. 14:583-594, 1994. Yao, R., et al. Mol. Microbiol. 14:883-893, 1994. Walker, R.I. et al. Microbiol. Rev. 50(1):81-94, 1986. Wang, Y., and D. E. Taylor. J. Bacteriol. 172:949-955, 1990. Wassenaar, T. M., et al. Infect. Immun. 62:3901-3906, 1994. Watarai, M., et al. EMBO J. 14:2461-2470, 1995. Zierler, M. K., and J. E. Galan. J. Infect. Immun. 63:4024-4028, 1995. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A purified and isolated nucleic acid molecule encoding a protein associated with adherence and invasion of Campylobacter jejuni and having a nucleic acid sequence which comprises: (a) a nucleic acid sequence as shown in SEQ ID NO:1 wherein T can also be U; or (b) nucleic acid sequences complementary to (a). 2. A recombinant molecule adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in claim 1 and an expression control sequence operatively linked to the DNA segment. 3. A transformed host cell including the recombinant molecule as claimed in claim 2. 4. A method for preparing a Campylobacter invasion phenotype (CipA) protein associated with adherence and invasion of C. jejuni comprising culturing a host cell according to claim 3 under conditions which allow the expression of the protein and isolating the expressed protein. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The invention relates to novel nucleic acid molecules encoding a protein involved in the virulence of bacteria and more particularly of Campylobacter jejuni; the novel proteins encoded by the nucleic acid molecules; and, uses of the proteins and nucleic acid molecules. BACKGROUND OF THE INVENTION Campylobacter jejuni (C. jejuni), a gram-negative microaerophilic bacterium, is a leading cause of bacterial diarrhea and enterocolitis in children and adults in both developing and developed countries (Walker R I et al, Microbiol. Rev. 50(1): 81-94, 1986; Kim N W et al, J. Bacteriol. 174(11):3494-3498, 1992; Chan V L and Bingham H L, Gene 101:51-58, 1991). Clinical symptoms of Campylobacter infections range from watery diarrhea to inflammatory dysentery and bloody diarrhea (Cover T L and Blaser N J, Ann. Rev. Ned. 40:269-285, 1989; Walker R I et al, supra). Complications from C. jejuni infections have included Guillain-Barre syndrome, a neurological disease which may lead to respiratory paralysis and death, toxic megacolon, acute mesenteric adenitis syndrome, and reactive arthritis (Kaldor J and Speed B R, British Medical J. 288:1867-1870, 1984; Johnson K et al, Acta. Med. Scand. 214:165-168, 1983; Walker R I et al, supra). C. jejuni is commonly found in surface water, in animals such as cattle, sheep, goats, swine and poultry, in industrial wastes, and in many different types of foods including unpasteurized dairy products. Human pets such as dogs, cats and birds may also be infected with C. jejuni and may transmit the bacterium to humans. (Cover T L and Blaser M J, Ann. Rev. Med. 40:269-285, 1989; and Penner, J. L., Clin. Micro. Rev. 1:157-172, 1988). Despite recognition of C. jejuni as a major human enteropathogen, an understanding of both the genetic organization and virulence mechanisms of this organism remains rudimentary. Campylobacteria have small genomes with a low-percent G+C and high A+T content. For instance, the C. jejuni TGH9011 chromosome is 1812 kb in size with a G+C content of approximately 30%. Within this A+T-rich genome, restriction enzyme sites high in G+C such as SacII (CCGGCC) and SmaI (CCCGGG) are infrequently found (Kim, 1992). On the basis of its size and G+C content, A C. jejuni chromosome should contain approximately 110 SalI (GTCGAC) recognition sites (McClelland, 1987). Surprisingly, all of the C. jejuni isolates analyzed to date contain only 5-6 SalI sites (Chang & Taylor 1990; Kim et al., 1992), and three of these sites are located within 23S rRNA encoding sequences (Kim et al.,1993; Kim et al., 1995). The three recognition sites for the enzyme SalI are each located within a conserved rRNA operon. SUMMARY OF THE INVENTION The present inventors have identified and characterized a novel SalI site-containing a non-rRNA gene from C. jejuni TGH9011. The protein encoded by this gene is comprised of 464 amino acids with a predicted molecular weight of 55,651. No significant homology to other known proteins was found in a database search. Maxicell analysis revealed the synthesis of a cloned gene product with an apparent molecular mass of 55 kDa. A site-specific insertional mutation within the gene reduced the ability of C. jejuni to adhere to and invade the human intestinal cell line INT407. The gene was designated cipA (Campylobacter invasion phenotype). Mapping studies indicate that cipA is a linking gene for a previously unrecognized SalI PFGE restriction fragment (denoted Sall F) on the physical map of C. jejuni. Accordingly, in its broad aspect, the present invention provides a purified and isolated nucleic acid molecule comprising a sequence encoding a protein associated with invasion of virulent bacteria. In one embodiment, the present invention provides an isolated nucleic acid molecule having a sequence encoding a protein associated with adherence and invasion of Campylobacter spp. According to one embodiment, the present invention provides a purified and isolated nucleic acid molecule comprising a sequence encoding a protein associated with invasion by C. jejuni. According to another embodiment, the present invention provides a purified and isolated nucleic acid molecule comprising a sequence encoding a protein associated with invasion by C. coli. In a preferred embodiment, a purified and isolated nucleic acid molecule is provided having a sequence which encodes a protein associated with invasion by C. jejuni (this protein is encompassed within the terms "CipA protein" or "CipA proteins" used herein). The nucleic acid molecule having a nucleic acid sequence as shown in FIG. 1 and in the Sequence Listing as SEQ ID NO: 1 and the protein having an amino acid sequence as shown in FIG. 1 and in the Sequence Listing as SEQ ID No: 2. Most preferably, the purified and isolated nucleic acid molecule comprises: (a) a nucleic acid sequence as shown in SEQ ID NO: 1 and FIG. 1, wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are homologous to (a) or (b); or, (d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions. The invention also contemplates a purified, isolated nucleic acid molecule comprising a sequence encoding a truncation of a protein of the invention, an analog, or a homolog of a protein of the invention, or a truncation thereof (herein collectively referred to as "CipA protein" or "CipA proteins"). According to one embodiment, the invention provides a purified and isolated polypeptide having an amino acid sequence of a CipA protein associated with adherence and invasion of Campylobacter spp. Preferably the Campylobacter species is C. Jejuni or C. Coli. The nucleic acid molecules of the invention may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcription and translation of the inserted protein-coding sequence. Accordingly, recombinant DNA molecules adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention operatively linked to an expression control sequence. A transformant host cell including a recombinant molecule of the invention is also provided. Still further, this invention provides plasmids which comprise recombinant molecules of the invention. The present invention further relates to an avirulent strain of C. jejuni comprising an avirulent bacterial carrier strain transformed with a recombinant molecule of the invention, and a vaccine composition comprising a bacterial carrier strain transformed with a recombinant molecule of the invention. The invention also provides a method of preparing a CipA protein of the invention utilizing a nucleic acid molecule of the invention. In an embodiment a method for preparing a CipA protein of the invention is provided comprising: (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein. The invention still further provides a purified and isolated polypeptide having part or all of the primary structural conformation (ie., a continuous sequence of amino acid residues) and the activity of CipA. In a preferred embodiment the polypeptide has an amino acid sequence as shown in FIG. 1 and in the Sequence Listing as SEQ ID NO: I and SEQ ID NO: 2, or a sequence having between 40-50 percent homology thereto. The invention also includes truncations of such purified and isolated polypeptide and analogs, homologs, and isoforms of the polypeptide and truncations thereof (herein collectively, also included in the terms "CipA protein" or "CipA proteins"). According to a further embodiment the present invention provides a protein encoded by the purified and isolated nucleic acid molecule having a nucleic acid sequence as identified in SEQ ID NO: 1. The proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins. The invention also relates to an antibody specific for one or more epitopes of a protein of the invention, preferably a monoclonal antibody and methods for preparing the antibodies. A method for detecting Campylobacter spp. as well as C. jejuni in a sample is provided comprising assaying for CipA protein in the sample. In an embodiment of the invention the method comprises contacting the sample with an antibody of the invention which is capable of being detected after it becomes bound to CipA in the sample, and measuring the amount of antibody bound to CipA in the sample, or unreacted antibody. According to one embodiment the invention provides a monoclonal or polyclonal antibody specific for an epitope of the purified and isolated CipA polypeptide. An antibody provided in accordance with the invention can be one which binds a distinct epitope in an unconserved region of the polypeptide. A kit for detecting Campylobacter spp. as well as Campylobacter jejuni in a sample comprising an antibody of the invention, preferably a monoclonal antibody and directions for its use is also provided. The kit may also contain reagents which are required for binding of the antibody to a CipA protein in the sample. The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences in samples such as biological, food, or environmental samples. The nucleotide probes may be used to detect nucleotide sequences that encode polypeptides related to or analogous to the CipA polypeptide of the invention. Accordingly, the invention provides a method for detecting the presence of a nucleic acid molecule having a sequence encoding a polypeptide related to or analogous to a polypeptide of the invention, comprising contacting the sample with a nucleotide probe which hybridizes with the nucleic acid molecule, to form a hybridization product under conditions which permit the formation of the hybridization product, and assaying for the hybridization product. The invention further provides a kit for detecting the presence of a nucleic acid molecule having a sequence encoding a polypeptide related to or analogous to a polypeptide of the invention, comprising a nucleotide probe which hybridizes with the nucleic acid molecule, reagents required for hybridization of the nucleotide probe with the nucleic acid molecule, and directions for its use. The nucleic acid molecules of the invention also permit the identification and isolation, or synthesis, of nucleotide sequences which may be used as primers to amplify a nucleic acid molecule of the invention, for example in the polymerase chain reaction (PCR). Accordingly, the invention relates to a method of determining the presence of a nucleic acid molecule having a sequence encoding a CipA protein or a predetermined part of a CipA protein in a sample, comprising treating the sample with primers which are capable of amplifying the nucleic acid molecule, in a polymerase chain reaction to form amplified sequences, under conditions which permit the formation of amplified sequences, and, assaying for amplified sequences. The invention further relates to a kit for determining the presence of a nucleic acid molecule having a sequence encoding a CipA protein or a predetermined part of the protein in a sample, comprising primers which are capable of amplifying the nucleic acid molecule in a polymerase chain reaction to form amplified sequences, reagents required for amplifying the nucleic acid molecule thereof in an amplification reaction, preferably the polymerase chain reaction, means for assaying the amplified sequences, and directions for its use. The nucleic acid molecules of the invention may also be used to assay for a substance which inhibits adherence or invasion of Campylobacter spp. including C. jejuni. Accordingly, the invention provides a method for assaying for a substance that interferes with a CipA protein. The method may be used, for example, to assay for a substance which affects the growth or pathogenicity of C. jejuni. The substances identified using the method of the invention, antibodies, and antisense molecules may be used to reduce adherence and/or invasion of Campylobacter spp. including C. jejuni and accordingly may be used in the treatment of infectious diseases caused by Campylobacter spp. including C. jejuni. Accordingly, the substances may be formulated into pharmaceutical compositions for adminstration to subjects. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS The invention will now be described in relation to the drawings: FIG. 1 shows the sequence of the orfS (cipA) gene and its flanking regions obtained from C. jejuni genomic DNA library clone E3-8 and the deduced amino acid sequence is indicated in single letter code below the nucleotide sequence SEQ ID NO: 1 and SEQ ID NO: 2; FIG. 2 is a primer extension mapping of the transcription start site of the orfS (cipA) mRNA; FIG. 3 shows maxicell analysis for the elucidation of the plasmid-encoded proteins; FIG. 4 shows Southern blot analysis of genomic DNA from representative samples of various Campylobacteraceae; FIG. 5A is a gel showing the resolution of C. jejuni SalI-E and SalI-F fragments using pulsed-field gel electrophoresis; FIG. 5B is a Southern blot of the gel shown in FIG. 5A probed with plasmid pE3-8 indicating the contiguity of SalI-F and SalI-D on the physical map of C. jejuni TGH9011; and FIG. 5C is an updated physical map of C. jejuni TGH9011 showing the position of the newly recognized SalI-F fragment. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |