Streptococcus bacteriophage lysins for detection and treatment of gram positive bacteria

Abstract

The present invention provides methods, compositions and articles of manufacture useful for the prophylactic and therapeutic amelioration and treatment of gram-positive bacteria, including Streptococcus and Staphylococcus, and related conditions. The invention provides compositions and methods incorporating and utilizing Streptococcus suis derived bacteriophage lysins, particularly PlySs2 and/or PlySs1 lytic enzymes and variants thereof, including truncations thereof. Methods for treatment of humans are provided.

1 - 42 . (canceled) 
     
     
         43 . A nucleic acid capable of encoding a  S. suis  lysin polypeptide capable of killing gram positive bacteria, wherein the lysin polypeptide is capable of killing one or more strain of each of  Staphylococcus, Streptococcus, Enterococcus  and  Listeria  bacteria, including combinations of one or more species of Staphylococcus, Streptococcus, Enterococcus or Listeria bacteria, the polypeptide comprising the amino acid sequence of SEQ ID NO:3, or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill gram positive bacteria. 
     
     
         44 . The nucleic acid of  claim 43  comprising an encoding nucleic sequence set out in  FIG.  4    (SEQ ID NO:12). 
     
     
         45 . A recombinant DNA molecule comprising a nucleic acid or DNA sequence or degenerate variant thereof, which encodes an  S. suis  lysin polypeptide, or a fragment thereof, selected from the group consisting of:
 a) the nucleic acid of  claim 43 ;   b) the DNA sequence of  FIG.  4    (SEQ ID NO:12);   b) DNA sequences that hybridize to any of the foregoing nucleic acid or DNA sequences under standard hybridization conditions; and   c) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing nucleic acid or DNA sequences,   
       wherein said nucleic acid or DNA sequence is operatively linked to a heterologous sequence which is an expression control sequence. 
     
     
         46 . A unicellular host cell transformed with a recombinant DNA molecule of  claim 45 . 
     
     
         47 . A vector comprising the recombinant DNA molecule of  claim 45 . 
     
     
         48 . A unicellular host cell transformed with the vector of  claim 47 . 
     
     
         49 . A vector system for expressing a lysin polypeptide comprising the recombinant DNA molecule of  claim 45 , wherein the vector system is a cell free expression system. 
     
     
         50 . A method for producing a lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3, or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill gram positive bacteria comprising culturing a host cell of  claim 46  under conditions to express the polypeptide and recovering the polypeptide. 
     
     
         51 . A nucleic acid encoding a chimeric protein comprising the catalytic domain or binding domain of the isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3 or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill gram-positive bacteria, said catalytic domain or binding domain optionally operably linked or covalently attached to a heterologous protein or polypeptide, wherein the chimeric protein is biologically active to kill or bind Staphylococcus or Streptococcus bacteria. 
     
     
         52 . The nucleic acid of  claim 51 , wherein the catalytic domain of the chimeric protein comprises SEQ ID NO:4 or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:4 and biologically active to kill gram-positive bacteria. 
     
     
         53 . The nucleic acid of  claim 51 , wherein the binding domain comprises SEQ ID NO:5 or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:5 and biologically active to bind gram-positive bacteria. 
     
     
         54 . The nucleic acid of  claim 51 , wherein the chimeric protein is a chimeric lytic enzyme comprising the catalytic domain or binding domain of the isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3 or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill gram-positive bacteria, wherein said catalytic domain is operably linked to a binding domain of another lysin or wherein said binding domain is operably linked to a catalytic domain of another lysin. 
     
     
         55 . The nucleic acid of  claim 54 , wherein the catalytic domain is operably linked to a binding domain of a  Staphylococcus -specific lysin or of a  Streptococcus -specific lysin. 
     
     
         56 . The nucleic acid of  claim 54 , wherein the binding domain is operably linked to a catalytic domain of a  Staphylococcus -specific lysin or of a  Streptococcus -specific lysin. 
     
     
         57 . The nucleic acid of  claim 51 , wherein the chimeric protein is effective to kill  Staphylococcus  and  Streptococcus  bacteria. 
     
     
         58 . The nucleic acid of  claim 51 , wherein the chimeric protein is effective to kill antibiotic-resistant  Staphylococcus  or  Streptococcus  bacteria. 
     
     
         59 . A recombinant DNA molecule comprising the nucleic acid sequence of  claim 51 , wherein said nucleic acid sequence is operatively linked to a heterologous sequence which is an expression control sequence. 
     
     
         60 . A unicellular host cell transformed with a recombinant DNA molecule of  claim 59 . 
     
     
         61 . A vector comprising the recombinant DNA molecule of  claim 59 . 
     
     
         62 . A unicellular host cell transformed with the vector of  claim 60 . 
     
     
         63 . A method for producing a chimeric protein comprising the catalytic domain or binding domain of the isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3 or variants thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill gram-positive bacteria, said catalytic domain or binding domain optionally operably linked or covalently attached to a heterologous protein or polypeptide, comprising culturing a host cell of  claim 60  under conditions to express the chimeric protein and recovering the chimeric protein. 
     
     
         64 . A method of killing or reducing a population of  Staphylococcus  and  Streptococcus  bacteria comprising the step of contacting the bacteria with a lysin polypeptide produced by the method of  claim 50  and effective to kill  Staphylococcus  and  Streptococcus  bacteria in an amount of the lysin polypeptide effective to kill the  Staphylococcus  and  Streptococcus  bacteria. 
     
     
         65 . A method of killing or reducing a population of  Staphylococcus  and  Streptococcus  bacteria comprising the step of contacting the bacteria with a lysin polypeptide produced by the method of  claim 63  and effective to kill  Staphylococcus  and  Streptococcus  bacteria in an amount of the lysin polypeptide effective to kill the  Staphylococcus  and  Streptococcus  bacteria. 
     
     
         66 . A method for treating gram-positive bacterial infection caused by  Staphylococcus  strains,  Streptococcus  strains,  Listeria monocytogenes  or  Enterococcus faecalis  bacteria in a human comprising the step of administering to a human having a bacterial infection, an effective amount of a lysin polypeptide produced by the method of  claim 50  and effective to kill the  Staphylococcus, Streptococcus, Listeria  or  Enterococcus  bacteria, whereby the number of  Staphylococcus, Streptococcus, Listeria  or  Enterococcus  bacteria in the human is reduced and the infection is controlled. 
     
     
         67 . A method for treating gram-positive bacterial infection caused by  Staphylococcus  strains,  Streptococcus  strains,  Listeria monocytogenes  or  Enterococcus faecalis  bacteria in a human comprising the step of administering to a human having a bacterial infection, an effective amount of a lysin polypeptide produced by the method of  claim 63  and effective to kill the  Staphylococcus, Streptococcus, Listeria  or  Enterococcus  bacteria, whereby the number of  Staphylococcus, Streptococcus, Listeria  or  Enterococcus  bacteria in the human is reduced and the infection is controlled. 
     
     
         68 . A pharmaceutical composition for killing gram-positive bacteria comprising the isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3, effective to kill the gram-positive bacteria. 
     
     
         69 . The composition of  claim 68 , where the composition has killing activity against one or more bacteria strains selected from the group consisting of  Staphylococcus aureus, Listeria monocytogenes, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae  (GBS),  Streptococcus pyogenes  (GAS),  Streptococcus sanguinis, Streptococcus gordonii, Streptococcus dysgalactiae, Streptococcus  GES,  Enterococcus faecalis  and  Streptococcus pneumonia.    
     
     
         70 . A method of killing  Staphylococcus  and/or  Streptococcus  bacteria comprising the step of contacting the bacteria with a pharmaceutical composition of  claim 68 , the composition comprising an amount of the isolated lysin polypeptide effective to kill the  Staphylococcus  and  Streptococcus  bacteria. 
     
     
         71 . The method of  claim 70  wherein the bacteria is an antibiotic resistant bacteria and is selected from methicillin-resistant  Staphylococcus aureus  (MRSA), vancomycin intermediate-sensitivity  Staphylococcus aureus  (VISA), or vancomycin resistant  Staphylococcus aureus  (VRSA). 
     
     
         72 . A method for treating an antibiotic-resistant  Staphylococcus aureus  infection in a human comprising the step of administering to a human having an antibiotic-resistant  Staphylococcus aureus  infection, an effective amount of the composition of  claim 68 , whereby the number of  Staphylococcus aureus  in the human is reduced and the infection is controlled. 
     
     
         73 . The method of  claim 72 , wherein the infection is selected frorr endocarditis and bacteremia.