Genetically modified
The present invention relates to efficient delivery of anti-infective activity, immunomodulatory factors, or growth-promoting biomolecules directly to the digestive tract of an animal via a live delivery platform. The live delivery platform can be a genetically modified microorganism. Delivery can be accomplished with a Lactobacillus sp which colonizes the gastrointestinal tract. The anti-infective activity can be a bacteriocidal or bacteriostatic peptide, an antibody or fragment thereof which specifically recognizes a pathogen, or a phage, or a lytic peptide from a phage which specifically targets a certain pathogen.
1 . An expression cassette comprising a promoter for transcriptional expression and at least one heterologous coding region encoding a biomolecule;
wherein the biomolecule is a bacterial peptide, an enzyme, a lysin, or a single chain antibody and wherein:
(a) the bacterial peptide is selected from mersacidin-E1 and E-2 molecules comprising sequences of SEQ ID NO: 2 and SEQ ID NO: 4, or Bacillus bacteriocin comprising a sequence selected from SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:47, and SEQ ID NO: 48;
(b) the enzyme is selected from SEQ ID NO: 5 or SEQ ID NO: 6;
(c) the lysin is selected from SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or
(d) the single chain antibody is directed against pathogenic bacterium Clostridium perfringens , specifically recognizes the bacterial protein C. perfringens alpha toxin or C. perfringens NetB toxin, and selected from SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 50;
wherein the promoter comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43.
2 . The expression cassette of claim 1 , wherein the cassette comprises an origin of replication comprising the sequence of SEQ ID NO: 8.
3 . The expression cassette of claim 1 , wherein the expression cassette is located on a plasmid or suicide vector or is located on or integrated into a bacterial chromosome.
4 . The expression cassette of claim 3 , wherein the expression cassette is integrated into a bacterial chromosome and is inserted into a transposase locus an Uracil phosphoribosyl (URP) transferase locus, or a pyrE locus of a bacterial chromosome.
5 . The expression cassette of claim 1 , wherein the single chain antibody directed against pathogenic bacterium Clostridium perfringens comprises one or more sequence selected from SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 49, and SEQ ID NO: 50.
6 . The expression cassette of claim 1 , wherein the promoter comprises the sequence of SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43.
7 . The expression cassette of claim 1 , wherein the expression cassette further comprises a nucleic acid sequence encoding a signal sequence for secretion.
8 . The expression cassette of claim 1 , wherein the biomolecule is a single chain antibody directed against pathogenic bacterium Clostridium perfringens and comprises the sequence of SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO:30, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 50 and wherein the promoter comprises the sequence of SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43.
9 . The expression cassette of claim 8 , wherein the expression cassette is selected from the expression cassette of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.
10 . A genetically-modified microorganism comprising the expression cassette of claim 1 , wherein the genetically-modified microorganism is a bacterium selected from the group consisting of Bacillus, Lactobacillus, Lactococcus , and Entercoccus.
11 . The genetically-modified microorganism of claim 10 , wherein the genetically-modified microorganism is a Lactobacillus reuteri strain.
12 . The genetically-modified microorganism of claim 11 , wherein the expression cassette is integrated into a Lactobacillus reuteri strain bacterial chromosome.
13 . The genetically-modified microorganism of claim 12 , wherein the expression cassette is inserted into a transposase locus. 32 , an Uracil phosphoribosyl (URP) transferase locus, or a pyrE locus.
14 . The genetically-modified microorganism of claim 11 , wherein the Lactobacillus reuteri strain is selected from strain 3632 ATCC PTA-126788 and strain 3630 ATCC PTA-126787.
15 . The genetically-modified microorganism of claim 10 , wherein the genetically-modified microorganism is a Lactobacillus bacterium and wherein the microorganism comprises an expression cassette wherein the biomolecule is a single chain antibody directed against pathogenic bacterium Clostridium perfringens and comprises the sequence SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30 , SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 50, and wherein the promoter comprises the sequence SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
16 . The genetically-modified microorganism of claim 15 , wherein the Lactobacillus bacterium is a Lactobacillus reuteri strain and is selected from strain 3632 ATCC PTA-126788 and strain 3630 ATCC PTA-126787.
17 . A method of reducing colonization of an animal by a pathogenic bacterium, the method comprising treating an animal with the genetically-modified microorganism of claim 14 .
18 . The method of claim 17 , wherein the pathogenic bacterium is selected from the group consisting of Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus , and an E. coli bacterium.
19 . The method of claim 17 , wherein the animal is a bird, a human, or a non-human animal.