Methods and Compositions for the Display of Polypeptides on the Pili of Gram-Positive Bacteria

Abstract

Provided herein are methods and compositions for the display of polypeptides of interest on the tip of pili of Gram-positive bacteria. According to the present invention, the polypeptide of interest is amino terminal to a Gram-positive bacterial pilus tip protein or an active variant or fragment thereof, wherein the active variant or fragment comprises a cleaved cell wall sorting signal (CWSS) motif. The Gram-positive bacterium displaying a polypeptide of interest on the tip of pili that are disclosed herein are useful, for example, in methods for immunizing a subject with an antigen and methods for removing contaminants from a composition.

1 . A method for producing a Gram-positive bacterium having at least one polypeptide of interest covalently attached to the tip of at least one pilus, wherein said method comprises:
 a) introducing into said Gram-positive bacterium a polynucleotide comprising a nucleotide sequence that encodes a chimeric polypeptide, said chimeric polypeptide comprising said polypeptide of interest and a  Streptococcus pyogenes  pilus tip protein, active variant or active fragment thereof, wherein said pilus tip protein or active variant or fragment thereof comprises a cell wall sorting signal (CWSS), and said pilus tip protein or fragment thereof is carboxyl to said polypeptide of interest, and wherein said Gram-positive bacterium expresses a tip sortase and a pilus shaft polypeptide; and   b) growing said Gram-positive bacterium under conditions wherein said chimeric polypeptide is expressed and said pilus is formed.   
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein said  Streptococcus pyogenes  pilus tip protein is selected from the group consisting of Cpa, protein F1, Spy0130, FctX, and FctB. 
     
     
         5 . The method of  claim 1 , wherein said active variant of said  Streptococcus pyogenes  pilus tip protein has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein said active fragment of said  Streptococcus pyogenes  pilus tip protein has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6. 
     
     
         8 .- 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein said CWSS comprises:
 a) a CWSS motif, wherein said CWSS motif has an amino acid sequence of X 1 X 2 PTG, wherein X 1  and X 2  is any amino acid;   b) a substantially hydrophobic domain carboxyl to said CWSS motif; and   c) a charged tail region carboxyl to said substantially hydrophobic domain.   
     
     
         12 .- 17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein said tip sortase has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7. 
     
     
         19 .- 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein said pilus shaft polypeptide has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein said Gram-positive bacterium further expresses a pilin chaperone polypeptide. 
     
     
         26 .- 28 . (canceled) 
     
     
         29 . The method of  claim 25 , wherein said pilin chaperone polypeptide has an amino sequence having at least 80% sequence identity to SEQ ID NO: 14. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein said nucleotide sequence encoding said chimeric polypeptide further comprises a nucleotide sequence encoding an amino terminal signal sequence. 
     
     
         32 . The method of  claim 1 , wherein said Gram-positive bacterium belongs to a genus selected from the group consisting of  Actinomyces, Bacillus, Bifidobacterium, Cellulomonas, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Mycobactenum, Nocardia, Staphylococcus, Streptococcus , and  Streptomyces.    
     
     
         33 .- 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein said polypeptide of interest is selected from the group consisting of an antigen, an enzyme, a biosorbent, or an antibody or fragment thereof. 
     
     
         36 . (canceled) 
     
     
         37 . A Gram-positive bacterium comprising at least one polypeptide of interest covalently attached to the tip of at least one pilus, wherein said polypeptide of interest is amino terminal to a  Streptococcus pyogenes  pilus tip protein or an active variant or fragment thereof, wherein said pilus tip protein or active variant or active fragment thereof comprises a cleaved cell wall sorting signal (CWSS) motif. 
     
     
         38 .- 48 . (canceled) 
     
     
         49 . The Gram-positive bacterium of  claim 37 , wherein said cleaved CWSS motif has an amino acid sequence of X 1 X 2 PT, wherein said X 1  is any amino acid except leucine, and wherein X 2  is any amino acid except proline. 
     
     
         50 .- 52 . (canceled) 
     
     
         53 . The method of  claim 37 , wherein said pilus comprises a pilus shaft polypeptide, wherein said pilus shaft polypeptide has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12. 
     
     
         54 .- 55 . (canceled) 
     
     
         56 . The Gram-positive bacterium of  claim 37 , wherein said polypeptide of interest is selected from the group consisting of an antigen, an enzyme, a biosorbent, or an antibody or fragment thereof. 
     
     
         57 . A method for inducing an immunological response comprising administering to a subject a composition comprising a Gram-positive bacterium of  claim 37 , wherein said polypeptide of interest comprises an antigen. 
     
     
         58 . The method of  claim 57 , wherein said Gram-positive bacterium comprises an attenuated pathogenic bacterium or a non-pathogenic bacterium. 
     
     
         59 . The method of  claim 57 , wherein said Gram-positive bacterium is selected from the group consisting of  Streptococcus gordinii, Lactococcus lactis, Staphylococcus xylosus , and  Staphylococcus carnosus.