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 recombinant nucleic acid encoding a pilus tip fusion protein comprising an antigen fused to a carboxy-terminus of a fragment of Cpa (SEQ ID NO: 101) comprising a cell wall sorting signal (CWSS) motif, wherein the CWSS is VPPTG (SEQ ID NO: 10) and wherein the pilus tip fusion protein comprises an amino-terminal sec-dependent signal sequence.
2. The recombinant nucleic acid of claim 1 wherein the fragment of Cpa comprising the CWSS motif comprises is a polypeptide having at least 50% sequence identity to SEQ ID NO: 101.
3. The recombinant nucleic acid of claim 1 wherein the amino-terminal dec-dependent signal sequence is a polypeptide having at least 40% sequence identity to SEQ ID NO: 100.
4. The recombinant nucleic acid of claim 1 further comprising genes sipA2, tee3, and srtC2.
5. An expression cassette comprising the recombinant nucleic acid of claim 1 .
6. A vector comprising the expression cassette of claim 5 .
7. The expression cassette of claim 5 further comprising genes sipA2, tee3, and srtC2.
8. A pili expressing bacteria comprising the recombinant nucleic acid of claim 1 and a nucleic acid comprising genes sipA2, tee3, and srtC2, wherein expression of the genes and the pilus tip fusion protein localizes the pilus tip fusion protein to the tip of pili.
9. The recombinant nucleic acid of claim 1 wherein the fragment of Cpa is SEQ ID NO: 4.