Protease stable cell wall lysing enzymes

Abstract

The present invention relates to a modified polypeptide with a biological activity to lyse cell walls of bacteria, wherein the polypeptide has no caspase, clostripain, enterokinase, factor Xa, granzyme B, staphylococcus peptidase I (V8 Protease), plasmin, streptopain, bacillolysin and/or thrombin cleavage site. The invention further relates to nucleic acids with a sequence encoding a polypeptide according to the present invention.

1 . A modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site. 
     
     
         2 . (canceled) 
     
     
         3 . The polypeptide of  claim 1 , wherein the polypeptide exhibits the biological activity of lysing cell walls of Gram-positive bacteria. 
     
     
         4 . The polypeptide of  claim 3 , wherein the Gram-positive bacteria are selected from the group consisting of clostridia, bacilli,  listeria , staphylococci, lactobacilli, enterococci, aerococci, pediococci, streptococci, mycoplasms and/or  leuconostoc.    
     
     
         5 . The polypeptide of  claim 1 , wherein the polypeptide is a endolysin, a bacteriophage tail protein, an autolysin, or a bacteriocin. 
     
     
         6 . A polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. 
     
     
         7 . A nucleic acid molecule comprising a nucleotide sequence coding for a polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site. 
     
     
         8 . An expression vector comprising a nucleic acid molecule according to  claim 7 . 
     
     
         9 . The nucleic acid of  claim 7 , further comprised within a host cell. 
     
     
         10 . A pharmaceutical composition comprising a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site. 
     
     
         11 . A method for prevention or therapy of a disease caused by Gram-positive bacteria, comprising administering to a subject in need thereof a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site. 
     
     
         12 . A method of inhibiting the growth of Gram-positive bacterium comprising contacting said bacterium with a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site. 
     
     
         13 . A method for the detection of a bacterium in a patient sample, an environmental sample, a food sample or a cosmetic sample comprising:
 (i) contacting said sample with a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a  staphylococcus  peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site; and   (ii) detecting the polypeptide bound to a bacterium in said sample.   
     
     
         14 . The expression vector of  claim 8 , further comprised within a host cell. 
     
     
         15 . The method of  claim 11 , wherein said disease is caused by Gram-positive bacteria selected from the group consisting of clostridia, bacilli,  listeria , staphylococci, lactobacilli, entrococci, aerococci, pediococci, streptococci, mycoplasmas,  leuconostoc , or a combination thereof. 
     
     
         16 . The method of  claim 13 , wherein said bacterium is a Gram-positive bacterium. 
     
     
         17 . The method of  claim 16 , wherein said Gram-positive bacterium is selected from the group consisting of clostridia, bacilli,  listeria , staphylococci, lactobacilli, entrococci, aerococci, pediococci, streptococci, mycoplasmas,  leuconostoc , or a combination thereof.