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