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Compositions and methods for detecting pathogen infectionRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophageCompositions and methods for detecting pathogen infection description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060063149, Compositions and methods for detecting pathogen infection. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention generally features diagnostic and therapeutic compositions and methods derived from the characterization of the binding of a lysozyme polypeptide to a Treponema pallidum P17 polypeptide (Tp17) or Tp17-like polypeptides. In addition, the invention provides methods and compositions for increasing or decreasing the binding between a lysozyme and a TP-17 or a TP-17 like polypeptide. BACKGROUND OF THE INVENTION [0002] Syphilis is a disease caused by Treponema pallidum (hereinafter also referred to as "Tp") infection. The diagnosis of syphilis is generally made by an immunoassay of anti-Tp antibody in the blood using, for example, the Treponema pallidum Hemagglutination Test (TPHA), the Fluorescent Treponema Antibody Absorption Test (FTA-ABS) and/or the Treponema pallidum Immobilization Test (TPI), as well as enzyme-linked immuno sorbent assay (ELISA) and Western Blot systems. These tests detect antibodies that react with Tp or antigen preparations from Tp, such as the Tp antigens Tp15, Tp17, and Tp47, for example. The TPI test involves a microscopic assessment of the extent to which complement activating antibodies in a patient's serum inhibit the mobility of Tp. This test is not generally used as a diagnostic due to its high cost. The TPHA test involves agglutination of patient serum antibodies with erytirocytes to which the Tp sonicate antigen is bound. The FTA-ABS test involves indirect immunofluorescence microscopy to detect the binding of specific antibodies in patient serum to Tp attached to a solid support via a fluorescent-labelled secondary antibody. Although these assays are widely used, they lack the sensitivity required for detecting early stage or low level infection, when Tp antibody levels in the body fluids are very low. [0003] In addition, the popular use of individual recombinant or purified Tp antigens in existing immunoassays for syphilis does not account for the binding by some antibodies to complexes formed between Tp antigens and other antigens (i.e., Tp antigen binding partners) normally present in the subject's body fluid. Existing immunoassays thus fail to detect such antibodies in the absence of the Tp antigen binding partner, which results in some instances in low sensitivity or false negative assay results. A need therefore exists for pathogen diagnostic assays with improved sensitivity, which detect antibodies that bind to pathogen antigen/binding partner complexes. SUMMARY OF THE INVENTION [0004] The invention provides compositions and methods for the detection and treatment of a pathogen infection. The invention generally relates to the discovery that the Treponema pallidum polypeptide, Tp17, binds to lysozyme, an anti-microbial peptide produced by the host, and inhibits lysozyme's anti-microbial activity. The binding site on Tp17 for lysozyme and the binding site on lysozyme for Tp17 were identified. Comparisons of Tp17 sequences required for binding and inhibiting lysozyme with related proteins identified lysozyme binding consensus sequences that are conserved among many different pathogens, including, for example, Herpes Simplex viruses, Hepatitis C virus, and parasites such as amoebas. This discovery suggests that pathogens share a general mechanism for inhibiting a host immune response by producing a polypeptide containing a lysozyme binding motif that binds to lysozyme and inhibits its anti-microbial activity. Accordingly, the invention also provides for the identification of mutant lysozyme polypeptides containing mutations that interfere with or destabilize such binding. Such mutant lysozyme polypeptides, or fragments thereof, are useful in preventing or treating a pathogen infection. In addition, the invention provides for improved diagnostic assays based on the detection of antibodies that bind to a polypeptide-lysozyme complex, such as a Tp17-lysozyme complex. [0005] The identification of a lysozyme binding motif on Tp17, as well as the identification of lysozyme binding motif consensus sequences shared by other pathogen-derived proteins, also provides useful compositions and methods for the detection or inhibition of lysozyme, as well as the detection of specific pathogens and inhibition of the corresponding infections besides syphilis. In addition, pathogen-derived polypeptides are useful in the diagnosis, prophylaxis, and treatment of a number of diseases, including diseases related to aberrant lysozyme activity. Those polypeptides also provide a useful affinity tag for use in protein purification using inexpensive lysozyme affinity chromatography. [0006] In one aspect, the invention generally features a substantially pure polypeptide containing a lysozyme binding motif or a fragment thereof. In one embodiment, the invention provides a substantially pure Tp17-like polypeptide, or a fragment thereof, containing at least a lysozyme binding motif containing an amino acid sequence of Xaa.sub.n Pro His Xaa.sub.n (SEQ ID NO:1), where Xaa is any amino acid and n is at least one and the fragment is capable of binding a lysozyme polypeptide and where the motif is not CKPHDC (SEQ ID NO:24). In one embodiment, the fragment is between about 4 amino acids and about 200 amino acids (e.g., 5, 10, 25, 50, 75, 100, 125, 150, 175) of a Tp17-like polypeptide. In another embodiment, the fragment is less than about 30 or 100 amino acids of a Tp17-like polypeptide. In another embodiment, the polypeptide or the fragment is capable of substantially inhibiting an enzymatic activity or an anti-microbial activity of the lysozyme. In yet another embodiment, the lysozyme binding motif contains an amino acid consensus sequence CS1: Cys Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Pro His Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13 Cys (SEQ ID NO:2), where Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, Xaa.sub.4, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12, or Xaa.sub.13 is any amino acid, is absent, or is a peptide bond. Alternatively, the lysozyme binding motif contains an amino acid consensus sequence: Cys Xaa1 Xaa2 Arg Xaa3 Xaa4 Xaa5 Cys (SEQ ID NO:314), where Xaa1, Xaa2, Xaa3, Xaa4, or Xaa5 is any amino acid, is absent, or is a peptide bond. Accordingly, a lysozyme binding motif of the present invention typically contains at least 4 amino acids, 2 of which are selected from the group consisting of Cys, Pro, His, and Arg. [0007] In related aspects, the invention features a substantially pure nucleic acid molecule encoding the polypeptide or the fragment of the previous aspect, a vector containing the nucleic acid molecule, and a host cell containing such a vector. [0008] In another aspect, the invention features a substantially pure mutant lysozyme polypeptide containing at least one amino acid mutation that reduces binding between the mutant lysozyme polypeptide and a Tp17-like polypeptide (e.g., reduces binding by 5%, 10%, 25%, 50%, 75%, 85%, or 95% relative to wild-type binding), where the mutant lysozyme polypeptide retains at least 50%, 60%, 75%, 85%, or 95% of the anti-microbial activity of a corresponding wild-type lysozyme polypeptide. In one embodiment, the mutation reduces binding between the mutant lysozyme polypeptide and a Tp17-like polypeptide by at least 5% relative to wild-type lysozyme polypeptide binding. In another embodiment, the mutation affects a surface charge in the corresponding wild-type polypeptide. In other embodiments, the mutation is in a positively charged amino acid residue, is in a negatively charged amino acid residue, or is in a hydrophobic amino acid residue of a wild-type lysozyme polypeptide that contacts a Tp17-like polypeptide. In other embodiments, the mutation is of at least one, two, three, four, or more amino acid positions selected from the group consisting of Lys19, Arg23, Lys51, Gly 55, Asn57, Arg131, Asn132 and Arg133 of human lysozyme or is present at a corresponding position in a lysozyme derived from another species (e.g., prokaryotic, eukaryotic, mammalian). [0009] In a related aspect, the invention features a substantially pure nucleic acid molecule encoding the lysozyme polypeptide of the previous aspect, a vector containing such a nucleic acid molecule, and a host cell containing this vector. [0010] In yet another aspect, the invention features a composition containing a polypeptide with at least a lysozyme binding motif and a lysozyme polypeptide. For example, a preferred composition contains a substantially pure Tp17-like polypeptide and a substantially pure lysozyme polypeptide, where the Tp17-like polypeptide is not the Ivy polypeptide. In one embodiment, the lysozyme is exogenous. In one embodiment, the composition further contains carrier particles (e.g., red blood cells, polypeptide aggregate particles, polymeric particles, inorganic particles, paramagnetic particles, and yeast cells). [0011] In yet another aspect, the invention features a method for detecting an immune response against a pathogen in a subject. The method involves (a) contacting a biological sample from the subject with an exogenous lysozyme and a polypeptide containing a lysozyme binding motif (i.e., a Tp17-like polypeptide); and (b) detecting antibody binding to the polypeptide with a lysozyme binding motif or to a polypeptide-lysozyme complex. [0012] In yet another aspect, the invention features a method for enhancing the sensitivity of a diagnostic assay for detecting an immune response against a pathogen. The method involves adding a lysozyme polypeptide to the diagnostic assay (e.g., an agglutination assay), where the addition of the lysozyme polypeptide increases the assay's sensitivity by at least 5%. [0013] In various embodiments of the previous aspects, the lysozyme polypeptide is contacted with the Tp17-like polypeptide prior to, during, or after contacting the biological sample. In other embodiments, the assay is an immunoassay (e.g., enzyme-linked immunoabsorbent assay (ELISA), Western blot, immunoagglutination assay, radioimmunoassay, turbidimetric assay, nephelometric assay, immunochromatographic assay, chemiluminescent assay, and fluorescent assay). In other embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, tears, saliva, sputum, nasal fluid, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, perspiration, synovial fluid, ascites fluid, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, amniotic fluid, cerebrospinal fluid, bile, gastric fluid, semen, fecal material, upper airway fluid, peritoneal fluid, fluid harvested from a site of inflammation, fluid harvested from a pooled collection site, bronchial lavage, urine, biopsy material, aqueous humor, material from the forestomach of a ruminant animal, nucleated cell sample, any fluid associated with a mucosal surface, hair, and skin. In one preferred embodiment, the method is used to diagnose syphilis. [0014] In another aspect, the invention features a composition for detecting a pathogen in a sample, the composition containing a lysozyme polypeptide or a fragment thereof containing the amino acid sequence Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa Xaa Gln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa (SEQ ID NO:28), where Xaa is any amino acid or is absent, where the fragment is capable of binding to a Tp17-like polypeptide. [0015] In yet another aspect, the invention features a method for detecting or identifying a pathogen or pathogen polypeptide. The method involves (a) contacting a sample with a lysozyme polypeptide fragment containing the amino acid sequence: TABLE-US-00001 Xaa Xaa Xaa Xaa Xaa Xaa Cys (SEQ ID NO: 28) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa Xaa Gln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa, where Xaa is any amino acid or is absent, under conditions that permit binding; and (2) detecting binding of the lysozyme polypeptide to a polypeptide in the sample, where the binding indicates the presence of a pathogen or pathogen polypeptide in the sample. In another embodiment, the invention further involves determining the sequence of the polypeptide bound to the lysozyme polypeptide. In some embodiments, the lysozyme polypeptide contains a sequence derived from a human lysozyme polypeptide. [0016] In another aspect, the invention features a method for detecting lysozyme in a sample, the method involves (a) contacting the sample with a Tp17-like polypeptide bound to a solid support; and (b) detecting binding of the polypeptide to a polypeptide in the sample, where the binding indicates lysozyme is present in the sample (e.g., a biological fluid, a cell culture, a body sample, a water sample, a fluid sample, a food, a medicine, or a pathogen culture). In one embodiment, the method further involves removing lysozyme from the sample through the binding. [0017] In another aspect, the invention features a kit for detecting an immune response to a pathogen containing a polypeptide having a lysozyme binding motif and a lysozyme polypeptide. [0018] In another aspect, the invention features a kit for detecting a pathogen in a sample containing a labeled lysozyme polypeptide containing SEQ ID NO:28. [0019] In another aspect, the invention features a fusion polypeptide containing at least a lysozyme binding motif (e.g., a fragment of a Tp17-like polypeptide lysozyme binding motif) fused to a second amino acid sequence with which it is not naturally linked. In one embodiment, the fusion polypeptide is fixed to a solid support. In another embodiment, the motif is at a terminus of the fusion polypeptide. In another embodiment, the fusion polypeptide is capable of reducing a lysozyme anti-microbial activity. [0020] In another aspect, the invention features a method for isolating a fusion polypeptide, the method containing the steps of: (a) providing the fusion polypeptide of the previous aspect; (b) contacting the fusion polypeptide with a lysozyme polypeptide, where the lysozyme is affixed to a solid support, under conditions that permit binding of the fusion polypeptide to the lysozyme polypeptide; and (c) eluting the fusion polypeptide from the lysozyme polypeptide. Continue reading about Compositions and methods for detecting pathogen infection... Full patent description for Compositions and methods for detecting pathogen infection Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compositions and methods for detecting pathogen infection patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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