A polypeptide, designated as "Streptococcus uberis Adhesion Molecule" (SUAM), and fragments of SUAM, prevent internalization and adherence of Streptococcus uberis and other streptococcal pathogens to cells. The SUAM polypeptide and fragments may be used diagnostically and therapeutically. Nucleic acid sequences encoding the SUAM polypeptide and fragments are included in the invention.

Description of the Invention

In this application, the terms "Streptococcus uberis Adhesion Molecule" or "SUAM" is preferably used although the terms "Streptococcus Lactoferrin-binding Protein", "Lactoferrin Binding Protein" and "LBP" are also used to refer to the same polypeptide. The terms "Streptococcus uberis Adhesion Molecule" and "SUAM" are preferred so as not to confuse the polypeptide of the present invention with the protein identified as "Streptococcus uberis Lactoferrin-Binding Protein" in Jiang et al., WO 98/21231. The Jiang protein is a different protein than the SUAM of the present invention. Protein-nucleic acid TBLASTN (National Center for Biotechnology Information) and Swissprot amino acid data bank were used to align the SUAM N-terminal amino acid sequence with previously sequenced genes and proteins including S. uberis LBP described by Jiang et al. No similarities were found, thus indicating that the SUAM bacterial protein of the invention is novel.

Recently, it has been shown that S. uberis binds to purified bovine milk lactoferrin (LF) and that at least two proteins from S. uberis were involved in this binding. Fang and Oliver, FEMS Microbiol. Lett., 176:91 (1999). It has further been shown that LF appears to function as a bridging molecule between S. uberis and bovine mammary epithelial cells, facilitating adherence of this mastitis pathogen to host cells. Fang, et al., American Journal of Veterinary Research, 61:275 (2000). This research indicates that the S. uberis proteins that bind to LF influence adherence of S. uberis to mammary epithelial cells and internalization of S. uberis into bovine mammary epithelial cells.

Further research in our laboratory has provided the following discoveries.

(1) A 112 kDA protein from S. uberis that binds to LF was isolated and purified and an N-terminal amino acid sequence of this 112 kDa protein was determined. The sequence is that of a novel protein, which is referred to herein as Streptococcus uberis Adhesion Molecule or SUAM.

(2) SUAM-like proteins were identified in other Streptococci, including Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae.

(3) The SUAM-like proteins produced by S. dysgalactiae subsp. dysgalactiae bind to bovine LF in a manner similar to that which occurs with S. uberis.

(4) Antibodies against SUAM (whole protein) and to a synthetic peptide (pepSUAM) encompassing 15 amino acids near the N-terminus of SUAM have been produced.

(5) These antibodies cross-react with homologous proteins present in other strains of S. uberis demonstrating that SUAM was produced by all strains of S. uberis evaluated.

(6) Anti-pepSUAM and anti-SUAM antibodies cross-react with other streptococcal pathogens, including S. agalactiae, S. dysgalactiae subsp. dysgalactiae, and Streptococcus pyogenes.

(7) Antibodies directed against pepSUAM or SUAM inhibit adherence of S. uberis to, and internalization of S. uberis into, bovine mammary epithelial cells. This establishes that pepSUAM and SUAM are biologically active and are involved in adherence to and internalization of S. uberis into bovine mammary epithelial cells, indicating the importance of SUAM as a significant S. uberis virulence factor.

(8) A theoretical DNA sequence of SUAM was determined and confirmed by PCR and restriction digests.

(9) The "true" DNA sequence encoding for SUAM was elucidated and found to have 99% homology to the theoretically elucidated SUAM DNA.

It is conceived that this single virulence factor (SUAM) plays a critical role in the pathogenesis of streptococcal mastitis by facilitating bacterial adherence to bovine mammary epithelial cells. It is conceived that S. uberis expresses SUAM and uses LF in milk and/or on the epithelial cell surface to adhere to mammary epithelial cells. It is further conceived that antibodies that bind to SUAM or pepSUAM may be used to diagnose infections due to S. uberis or other streptococci or to treat infections due to S. uberis or other streptococci. It is further conceived that nucleic acid sequences that encode SUAM or pepSUAM may be used diagnostically or in the production of anti-streptococcal vaccines. It is further conceived that the SUAM and pepSUAM polypeptides of the invention may be used to in the production of antisera or vaccines to combat diseases due to S. uberis or other streptococci.

In one embodiment, the invention is a polypeptide comprising an amino acid sequence of at least 6 sequential amino acids of pepSUAM (MTTADQSPKLQGEEA), designated herein as Seq. ID No. 4, wherein an antibody that binds to the polypeptide inhibits adherence to and/or internalization of S. uberis into bovine mammary epithelial cells. For example the 6 sequential amino acids of the polypeptide of the invention may be amino acids 1 to 6, 2 to 7, 3 to 8, 4 to 9, 5 to 10, 6 to 11, 7 to 12, 8 to 13, 9 to 14, or 10 to 15 of Seq. ID No. 4 pepSUAM.

Preferably, the polypeptide of this embodiment of the invention comprises an amino acid sequence of more than 6 sequential amino acids of pepSUAM of Seq. ID No. 4, for example, 7, 8, 9, 10, 11, 12, 13, 14 sequential amino acids, or the entire 15 amino acid sequence of Seq. ID No. 4. The polypeptide of the invention may further contain additional amino acids to the amino terminal or carboxy terminal sides of the sequence that is a portion or all of pepSUAM. For example, the polypeptide of the invention may contain at its amino terminal end the amino acids DD, which are present at the amino terminal end of full-length SUAM.

The polypeptide may be used to elicit antibodies which may be used to diagnose infections due to SUAM-expressing organisms such as Streptococcus, like S. uberis. The polypeptide may also be used to elicit an immune response in an animal or human that is susceptible to infection by an organism that contains a surface antigen that will bind to an antibody that binds to the polypeptide of the invention. Thus, the polypeptide of the invention may be useful as a vaccine against infection due to Streptococcus, such as S. uberis, S. pyogenes, S. agalactiae, or S. dysgalactiae.

In another embodiment, the invention is an isolated SUAM protein preferably having the amino acid sequence shown in FIG. 4 or FIG. 6 (see Original Patent) and designated herein as Seq. ID No. 2 or Seq. ID No. 15, respectively.

In another embodiment, the invention is a polypeptide derived from SUAM protein, which may be isolated by the method described below and which comprises the sequence of amino acids MTTADQSPKLQGEEA, Seq. ID No. 4.

The invention also includes polypeptides that are substantially homologous with the pepSUAM polypeptide or SUAM protein and polypeptides derived therefrom, as described above. As used in this context, the term "substantially homologous" means that the amino acid sequence shares at least 50%, such as at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% amino acid identity with the pepSUAM or SUAM protein or polypeptides derived therefrom and wherein an antibody that binds to the polypeptide inhibits the adherence and/or the internalization of S. uberis to bovine mammary epithelial cells.

In another embodiment, the invention is an antibody that selectively binds to an amino acid sequence of any 6 to 15 sequential amino acids of pepSUAM, as described above. Preferably, the antibody inhibits the adherence and/or the internalization of S. uberis to bovine mammary epithelial cells. The antibody may be a monoclonal or polyclonal antibody and may be used diagnostically or therapeutically.

In another embodiment, the invention is an antibody that selectively binds to the SUAM polypeptides or proteins of the invention. Preferably, the antibody inhibits the adherence and/or the internalization of S. uberis to bovine mammary epithelial cells. The antibody may be a monoclonal or polyclonal antibody and may be used diagnostically or therapeutically.

In another embodiment, the invention is an isolated nucleic acid sequence that encodes the pepSUAM polypeptide. Preferably, the nucleic acid sequence comprises the sequence shown in underline and in bold in FIG. 3 (see Original Patent), and designated Seq. ID No. 5 -- see Original Patent.

In another embodiment, the invention is an isolated nucleic acid sequence that encodes the SUAM protein. Preferably, the nucleic acid sequence comprises either of the sequence shown in FIG. 3 or 5 (see Original Patent), designated Seq. ID No. 1 and Seq. ID No. 3, respectively. More preferably, the nucleic acid sequence comprises the sequence from nucleotide 317 to nucleotide 2836 of Seq. ID No. 1 or from nucleotide 289 to nucleotide 2808 of Seq. ID No. 3. Most preferably, the nucleic acid sequence comprises the sequence from nucleotide 311 to nucleotide 2836 of Seq. ID No. 1 or nucleotide 283 to nucleotide 2808 of Seq. ID No. 3.

Also included in the isolated nucleic acid sequences of the invention is a nucleic acid sequence that will hybridize under highly stringent conditions, for example at 3.times.SSC at 65.degree. C. and preferably at 6.times.SSC at 65.degree. C., to the complement of the above specifically described nucleic acid sequences.

In another embodiment, the invention is a method for immunizing an animal or human with an antigen against a bacterial organism. In accordance with the method of the invention, the polypeptide of the invention or the SUAM polypeptide is administered to an animal or human subject by any suitable means such as by injection or intramammary infusion and the subject is thereby caused to produce antibodies that selectively bind thereto, which antibodies inhibit bacteria that bind to lactoferrin from adhering and/or internalizing to cells and/or enhance clearance of bacterial pathogens. In this way, the ability of the microorganism to cause disease is reduced.

In another embodiment, the invention is a primer selected from the group of -- see Original Patent.

Preferably, the primers are grouped in pairs with primers (a) and (b) being paired as a forward and reverse PCR primer, respectively, primers (c) and (d) being paired as a forward and reverse PCR primer, respectively, and primers (e) and (f) being paired as a forward and reverse PCR primer, respectively.

The primers and primer pairs of the invention are useful, for example, in identifying microorganisms that produce SUAM or a polypeptide molecule having a high degree of homology to SUAM, such as 70% or more homology. As such, the primers of the invention may be used to diagnose the presence of an infection with a SUAM polypeptide, or SUAM-like polypeptide, producing microorganism. It is conceived that an animal or human patient that is diagnosed in this manner may be treated with administration of the polypeptide of the invention to induce an immune response against such microorganism.

Following is a list of possible applications of various embodiments of the invention. This list is not intended to be all inclusive as those skilled in the art will understand that additional uses exist for the invention.

I. Antibodies to SUAM and pepSUAM

A. Commercial Use

Diagnostic

Microbiology: immuno-fluorescence, card-test for preliminary confirmation (including cow-side rapid tests using milk from cows with mastitis) Serology: Agglutination/precipitation tests (cow-side rapid tests), ELISA Diagnostic enrichment of bacteria from crude samples Treatment/Prevention Therapy for cows with mastitis (systemic/intramammary) Prevention for new cows introduced to a farm with history of S. uberis infection Intramammary preparations for cows near parturition B. Research Use 1. Isolation/purification of SUAM 2. In vitro pathogenicity assays 3. Recombinant protein expression (monitoring and isolation) 4. Mutant detection 5. Immuno-histochemistry 6. Western blot 7. Immunoprecipitation for protein/protein interaction studies 8. Steric inhibition studies II. Suam Protein A. Commercial Use 1. Vaccine production 2. Antisera production 3. Protein as antigen component of multivalent vaccine B. Research Use 1. Antisera production 2. Experimental vaccination studies 3. Protein as antigen component of multivalent vaccine 4. Protein as ligand in affinity purification of bovine lactoferrin III. pepSUAM A. Commercial Use 1. Vaccine production 2. Antisera production 3. Peptide as antigen component of multivalent vaccine 4. Peptide as competitive inhibitor of adhesion/invasion in intramammary preps B. Research Use 1. Antisera production 2. Experimental vaccination studies 3. Peptide as component of multivalent vaccine 4. Peptide as ligand in affinity purification of Bovine Lactoferrin IV. Suad DNA Sequence A. Commercial Use Diagnostic 1. Probes 2. PCR (alternative primers design) 3. Cow-side rapid test (i.e., cantilever) Prevention of Mastitis 1. Recombinant expression for vaccine production (baculo-virus cloning and expression) 2. DNA vaccines (cloning into retro-virus vectors or Agrobacterium tumefaciens) 3. Cloning and expression in vitro for vaccine production B. Research Use 1. Probes 2. PCR (alternative primers design) 3. Real time PCR for selection and identification of strains 4. DNA microarrays/differential display (to identify and study factors that enhance or repress SUAM expression) 5. Site directed mutagenesis 6. Production of avirulent carrier strain for this or any other expressed protein vaccine. V. Suam PCR Primers A. Commercial Use Diagnostic 1. PCR amplification products detected by any means 2. Real time PCR (taq-man, beacons, etc.) 3. Probes B. Research Use 1. PCR Detection 2. Real time PCR (taq-man, beacons, etc.) 3. Probes for southerns, reverse transcriptase protection assays, etc. 4. Cloning and expression
 

Claim 1 of 2 Claims

1. A purified polypeptide having an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4.

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