Toxin-phage bacteriocide antibiotic and uses thereof

Abstract

The invention provides intracellular peptide toxins capable of killing bacterial and eukaryotic cells when present within the cell, while substantially lacking the ability to kill such cells when present externally. The invention also provides recombinant bacteriophage containing nucleic acid sequences encoding intracellular peptide toxins, and methods of using such bacteriophage to kill bacteria. Furthermore, the invention provides compositions, including pharmaceutical compositions, which can be used to kill bacteria or inhibit the growth of bacteria both in vitro and in vivo. Methods of treating a bacterial infection in a subject are also provided by the invention.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2 and a bacterial or mammalian promoter sequence.  
     
     
         2 . The isolated nucleic acid molecule of  claim 1  wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1  wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.  
     
     
         4 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of the amino acid sequence SEQ ID NO:2 having 10 or fewer amino acid substitutions, single amino acid insertions or single amino acid deletions in the amino acid sequence of SEQ ID NO:2 and a bacterial or mammalian promoter sequence.  
     
     
         5 . The isolated nucleic acid molecule of  claim 4  wherein the polypeptide comprises a variant of the amino acid sequence SEQ ID NO:2 having 5 or fewer amino acid substitutions in the amino acid sequence of SEQ ID NO:2.  
     
     
         6 . The isolated nucleic acid molecule of  claim 4  wherein the polypeptide comprises a variant of the amino acid sequence of SEQ ID NO:2 having an amino acid substitution at amino acid 5 and at amino acid 34.  
     
     
         7 . The isolated nucleic acid molecule of  claim 1  further comprising a nucleotide sequence encoding a bacteriophage coat protein.  
     
     
         8 . The isolated nucleic acid molecule of  claim 1  wherein the bacterial promoter sequence directs transcription in gram positive bacteria.  
     
     
         9 . The isolated nucleic acid molecule of  claim 1  wherein the bacterial promoter sequence directs transcription in gram negative bacteria.  
     
     
         10 . A genetically engineered bacteriophage comprising a heterologous nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         11 . A method for providing a genetically engineered bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid of SEQ ID NO:2, the method comprising: 
 (a) providing an isolated nucleic acid molecule comprising a 5′ region, a central region and a 3′ region, the 5′ region and the 3′ region comprising nucleotide sequences present in a selected bacteriophage genome, the central region comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;    (b) contacting a bacterial cell lysogenic for the selected bacteriophage genome with the isolated nucleic acid molecule such that the isolated nucleic acid is introduced into the bacterial cell; and    (c) allowing the isolated nucleic acid molecule to recombine with the bacteriophage genome present in the bacterial cell, thereby producing a genetically engineered bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid of SEQ ID NO:2.    
     
     
         12 . A method for providing a genetically engineered bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid of SEQ ID NO:2, the method comprising: 
 (a) providing an isolated nucleic acid molecule comprising a 5′ region, a central region and a 3′ region, the 5′ region and the 3′ region comprising nucleotide sequences present in a selected bacteriophage genome, the central region comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;    (b) contacting a bacterial cell lysate with the isolated nucleic acid molecule and with an isolated DNA molecule comprising the selected bacteriophage genome; and    (c) allowing the isolated nucleic acid molecule to recombine with the isolated DNA molecule comprising the bacteriophage genome and subsequently form bacteriophage particles, thereby producing a genetically engineered bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid of SEQ ID NO:2.    
     
     
         13 . A method for producing bacteriophage for reducing the viability of a selected type of bacteria, the method comprising: 
 (a) identifying a bacteriophage that is capable of infecting a selected type of bacteria;    (b) preparing a genetically engineered bacteriophage genome comprising at nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 and the genome of the identified bacteriophage; and    (c) preparing bateriophage comprising the genetically engineered bacteriophage genome.    
     
     
         14 . A composition comprising a genetically engineered bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 and a pharmaceutically acceptable carrier.  
     
     
         15 . A method for treating a bacterial infection comprising administering to a patient having a bacterial infection a composition comprising a genetically modified bacteriophage, the genetically modified bacteriophage encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2 wherein the polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2 is toxic to a bacterial cell when present internally, but is not toxic to the bacterial cell when present externally.  
     
     
         16 . The method of  claim 15  wherein the composition is administered orally.  
     
     
         17 . The method of  claim 15  wherein the composition is administered topically.  
     
     
         18 . The method of  claim 15  wherein the composition is administered parenterally.  
     
     
         19 . A method for eliminating a selected type of bacteria present in an aqueous composition, the method comprising adding to the composition genetically modified bacteriophage comprising a heterologous nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         20 . A method for reducing the viability of an unwanted cell, the method comprising providing a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2 within the cell wherein the polypeptide is toxic to cell when present internally, but is not toxic when present externally.  
     
     
         21 . The method of  claim 20  wherein the cell is a bacterial cell.  
     
     
         22 . The method of  claim 20  wherein the cell is a mammalian cell.