Methods for making and using antimicrobial peptides
Provided herein are genetically modified microbes. In one embodiment, a genetically modified microbe includes an exogenous polynucleotide that includes a pheromone-responsive region. In one embodiment, the pheromone-responsive region is derived from a conjugative plasmid from a member of the genus Enterococcus spp. The pheromone-responsive region includes a pheromone-responsive promoter and an operably linked coding region encoding an antimicrobial peptide. In one embodiment, a genetically modified microbe includes an exogenous polynucleotide that includes a promoter and an operably linked coding sequence encoding an antimicrobial peptide, where expression of the coding region is controlled by a modulator polypeptide and is altered by a modulating agent, and where the coding region encodes an antimicrobial peptide. Also provided herein are methods of using the genetically modified microbes, including methods for inhibiting growth of an Enterococcus spp., a pathogenic E. coli , or a pathogenic Salmonella spp., for treating a subject, and for modifying a subject's gastrointestinal microflora.
1. A method for treating a subject, the method comprising:
administering to the subject in need thereof an amount of a composition comprising a genetically modified microbe, wherein the genetically modified microbe is a lactic acid bacteria, a member of a the family Clostridiaceae, a member of a the family Bifidobacteriaceae, or an Enterobacteria, wherein the administering comprises depositing the genetically modified microbe in the subject's gastrointestinal tract, and wherein the genetically modified microbe comprises
a first coding sequence and a first promoter operably linked to the first coding sequence, wherein the first coding sequence encodes an antimicrobial peptide, wherein expression of the first coding sequence by the first promoter is inhibited by a modulator polypeptide bound to the first promoter and preventing expression of the operably linked first coding sequence encoding the antimicrobial peptide, and wherein transcription of the first coding sequence is activated by a modulating agent compared to a genetically unmodified microbe; and
a second coding sequence and a second operably linked promoter, wherein the second coding sequence encodes the modulator polypeptide.
2. The method of claim 1 wherein the subject has or is at risk for having an antibiotic resistant Enterococcus spp.
3. The method of claim 2 wherein the Enterococcus spp. is E. faecalis or E. faecium.
4. The method of claim 1 wherein the subject has or is at risk for having a pathogenic E. coli or pathogenic Salmonella spp.
5. The method of claim 4 wherein the E. coli is E. coli O157:H7.
6. The method of claim 1 wherein the first promoter is an inducible promoter.
7. The method of claim 6 wherein the inducible promoter is repressed by a tet-repressor polypeptide, a lac-repressor polypeptide, or a xyl-repressor polypeptide.
8. The method of claim 1 wherein the antimicrobial peptide is selected from SEQ ID NO:11-31 or differs from the antimicrobial peptide selected from SEQ ID NO:25 or 26 at 1 amino acid residue, from SEQ ID NO:15, 21, 22, 23, or 24 at 1 or 2 amino acid residues, from SEQ ID NO:14 or 16 at 1 to 3 amino acid residues, from SEQ ID NO:17 or 18 at 1 to 4 amino acid residues, from SEQ ID NO:12 or 13 at 1 to 8 amino acid residues, from SEQ ID NO:11 at 1 to 9 amino acid residues, or from SEQ ID NO:19, 20, 27, 28, 29, 30, or 31 at 1 to 12 amino acid residues, and wherein the antimicrobial peptide has antimicrobial activity.
9. The method of claim 1 wherein the second promoter is a constitutive promoter.
10. The method of claim 9 wherein the constitutive promoter is P23.
11. The method of claim 1 wherein the modulator polypeptide encoded by the second coding sequence is a tet-repressor polypeptide, a lac-repressor polypeptide, or a xyl-repressor polypeptide.
12. The method of claim 1 wherein the antimicrobial peptide further comprises a secretion signaling polypeptide.
13. The method of claim 1 wherein the first coding sequence and second coding sequence are present on a polynucleotide, wherein the polynucleotide is integrated in the genomic DNA of the genetically modified microbe.
14. The method of claim 1 wherein the first coding sequence and second coding sequence are present on a polynucleotide, wherein the polynucleotide is not integrated in the genomic DNA of the genetically modified microbe.
15. The method of claim 1 wherein the lactic acid bacterium is a Lactococcus spp.
16. The method of claim 15 wherein the Lactococcus spp. is L. lactis.
17. The method of claim 1 wherein the lactic acid bacterium is a Lactobacillus spp.
18. The method of claim 17 wherein the Lactobacillus spp. is Lb. acidophilus, Lb. bulgaricus, Lb. reuteri , or Lb. plantarum.