Lysin Agent and Method of Use for Diagnostic Testing

Abstract

Novel Bacillus lysin proteins have been identified and characterized. Methods to detect the presence or absence of bacteria in a sample, methods for lysing bacteria, and methods for decontaminating or disinfecting areas contaminated with bacteria that utilize these lysin proteins have also been developed.

1 - 22 . (canceled) 
     
     
         23 . A method for selecting a candidate lysin polypeptide for molecular diagnostic testing comprising:
 (a) culturing a host cell comprising a vector comprising a nucleic acid sequence encoding the candidate lysin polypeptide, wherein the nucleic acid sequence is from a library of polynucleotide fragments;   (b) expressing the candidate lysin polypeptide under conditions in which a nucleic acid molecule encoding the candidate lysin polypeptide is expressed;   (c) isolating the candidate lysin polypeptide;   (d) incubating the candidate lysin polypeptide with a bacteria; and   (e) selecting the nucleic acid sequence which encodes the candidate lysin polypeptide which exhibits peptidoglycan hydrolase activity against the bacteria.   
     
     
         24 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 50% identity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         25 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 50% similarity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         26 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 60% identity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         27 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 60% similarity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         28 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having between 70%-90% identity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         29 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having between 70%-90% similarity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         30 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 90% identity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         31 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 90% similarity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         32 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 95% identity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         33 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for nucleic acid sequences having at least 95% similarity with a nucleic acid sequence of a model lytic enzyme. 
     
     
         34 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 50% identity with an amino acid sequence of a model lytic enzyme. 
     
     
         35 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 50% similarity with an amino acid sequence of a model lytic enzyme. 
     
     
         36 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 60% identity with an amino acid sequence of a model lytic enzyme. 
     
     
         37 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 60% similarity with an amino acid sequence of a model lytic enzyme. 
     
     
         38 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having between 70%-90% similarity with an amino acid sequence of a model lytic enzyme. 
     
     
         39 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having between 70%-90% identity with an amino acid sequence of a model lytic enzyme. 
     
     
         40 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 90% identity with an amino acid sequence of a model lytic enzyme. 
     
     
         41 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 90% similarity with an amino acid sequence of a model lytic enzyme. 
     
     
         42 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 95% identity with an amino acid sequence of a model lytic enzyme. 
     
     
         43 . The method of  claim 23 , wherein the library of polynucleotides is derived from sequences identified in a genome database by searching for amino acid sequences encoded by the sequences having at least 95% similarity with an amino acid sequence of a model lytic enzyme. 
     
     
         44 . The method of  claim 23 , wherein the bacteria is a gram-positive bacteria. 
     
     
         45 . The method of  claim 44 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
         46 . The method of  claim 45 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
         47 . The method of  claim 23 , wherein the bacteria is a gram-negative bacteria. 
     
     
         48 . The method of  claim 47 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
         49 . The method of  claim 48 , wherein the  Yersinia  species is  Yersinia pestis.    
     
     
         50 . A method for decontaminating a surface or room or area or object contaminated with bacteria comprising contacting the surface or room or area or object with a composition comprising an isolated polypeptide having peptidoglycan hydrolase activity against the bacteria, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or a fusion thereof. 
     
     
         51 . The method of  claim 50 , wherein the bacteria is a gram-positive bacteria. 
     
     
         52 . The method of  claim 51 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
         53 . The method of  claim 52 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
         54 . The method of  claim 50 , wherein the bacteria is a gram-negative bacteria. 
     
     
         55 . The method of  claim 54 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
         56 . The method of  claim 55 , wherein the  Yersinia  species is  Yersinia pestis.    
     
     
         57 . A method for disinfecting a surface or room or area or object contaminated with bacteria comprising contacting the surface or room or area or object with a composition comprising an isolated polypeptide having peptidoglycan hydrolase activity against the bacteria, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or a fusion thereof. 
     
     
         58 . The method of  claim 57 , wherein the bacteria is a gram-positive bacteria. 
     
     
         59 . The method of  claim 58 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
         60 . The method of  claim 59 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
         61 . The method of  claim 57 , wherein the bacteria is a gram-negative bacteria. 
     
     
         62 . The method of  claim 61 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
         63 . The method of  claim 62 , wherein the  Yersinia  species is  Yersinia pestis.    
     
     
         64 . A method for treating a bacterial infection comprising administering to a subject in need of such treatment, a composition comprising an antibiotic that is effective against the bacteria and an isolated polypeptide having peptidoglycan hydrolase activity against the bacteria, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, or a fusion thereof. 
     
     
         65 . The method of  claim 64 , wherein the bacteria is a gram-positive bacteria. 
     
     
         66 . The method of  claim 65 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
         67 . The method of  claim 66 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
         68 . The method of  claim 64 , wherein the bacteria is a gram-negative bacteria. 
     
     
         69 . The method of  claim 68 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
         70 . The method of  claim 69 , wherein the  Yersinia  species is  Yersinia pestis.    
     
     
         71 . The method of  claim 64 , wherein the antibiotic is polymyxin. 
     
     
         72 . The method of  claim 71 , wherein the polymyxin is administered at a concentration that is non-toxic to the subject.