Anthelmintic probiotic compositions and methods
Compositions and methods for treating or reducing the severity or likelihood of occurrence of a parasitic worm or helminth infection in a subject are described. The methods include administering to the subject a therapeutically effective amount of a recombinant bacterium expressing a crystal protein such as a Bacillus thuringiensis crystal protein (Cry). The crystal proteins may be full length, truncated, variant, or sub-variant Cry proteins. Examples of crystal proteins include Cry5B, Cry21, Cry14A, Cry6A, and Cry13A. The recombinant bacterium may be, for example, a Bacillus subtilis or other Gram-positive bacterium, for instance, a lactic acid fermenting bacterium such as Lactococcus or Lactobacillus . Related compositions and recombinant microorganisms are also described.
1 . An anthelmintic composition comprising:
a non-invasive or non-pathogenic bacterium expressing one or more heterologous anthelmintic crystal proteins,
wherein the non-invasive or non-pathogenic bacterium has a nucleic acid construct or vector comprising at least one promoter operably linked to coding sequences for expression of the one or more heterologous anthelmintic crystal proteins,
wherein the at least one promoter is selected from the group consisting of a phage promoter, a hup gene promoter, a gap gene promoter, an mbg gene promoter, a pgm gene promoter, a thyA gene promoter, a nisin inducible promoter, and an arabinose inducible promoter,
wherein the non-invasive or non-pathogenic bacterium is present in an amount effective to reduce an intestinal worm burden in a mammal, and
wherein the non-invasive or non-pathogenic bacterium is suitable for oral administration to a mammal.
2 . The anthelmintic composition of claim 1 , wherein the one or more heterologous anthelmintic crystal proteins is selected from the group consisting of Cry5B, Cry21A, Cry14A, Cry13A, and Cry6A.
3 . The anthelmintic composition of claim 1 , wherein the one or more heterologous anthelmintic crystal proteins is a truncated crystal protein.
4 . The anthelmintic composition of claim 3 , wherein the truncated anthelmintic crystal protein is truncated after a conserved amino acid sequence of block 5, wherein the conserved amino acid sequence of block 5 is DRIEF (SEQ ID NO: 23) or DRLEF (SEQ ID NO: 24).
5 . The anthelmintic composition of claim 3 , wherein the truncated anthelmintic crystal protein has toxic activity that is at least 10% or more of the toxic activity of a corresponding full-length protein.
6 . The anthelmintic composition of claim 1 , wherein at least one of the one or more heterologous anthelmintic crystal proteins is:
(a) a Cry5B comprising at least amino acids 1 through about 693 of SEQ ID NO:1,
(b) a Cry13A comprising at least amino acids 1 through about 688 of SEQ ID NO:2,
(c) a Cry14A comprising at least amino acids 1 through about 675 of SEQ ID NO:3,
(d) a Cry21A comprising at least amino acids 30 through about 685 of SEQ ID NO:4,
(e) a Cry21A comprising at least amino acids 30 through about 685 of SEQ ID NO:5, or
(f) a Cry6A comprising the amino acid sequence set forth in SEQ ID NO:6 or comprising at least amino acids 30 through about 395, 415 or 435 of SEQ ID NO:6.
7 . The anthelmintic composition of claim 1 , wherein the non-invasive or non-pathogenic bacterium is a lactic acid fermenting bacterium.
8 . The anthelmintic composition of claim 1 , wherein the non-invasive or non-pathogenic bacterium is selected from the group consisting of B. subtilis, B. subtilis PY79, B. subtilis natto, B. cereus, B. cereus var. Toyoi (Toyocerin), B. toyonensis, Lactobacillus rhamnosus, Lactobacillus casei , and Lactococcus lactis.
9 . The anthelmintic composition of claim 1 , wherein the one or more heterologous anthelmintic crystal proteins is a variant crystal protein.
10 . The anthelmintic composition of claim 3 , wherein the truncated crystal protein is truncated after a conserved amino acid sequence of block 5.
11 . The anthelmintic composition of claim 3 , wherein the truncated crystal protein is missing the last 10 amino acids of the C-terminus.
12 . The anthelmintic composition of claim 3 , wherein the truncated crystal protein is truncated between the end of a conserved amino acid sequence of block 5 and the C-terminus of a full length crystal protein.
13 . The anthelmintic composition of claim 5 , wherein the truncated crystal protein is truncated at the N-terminus.
14 . The anthelmintic composition of claim 5 , wherein the truncated crystal protein does not contain the first 5 amino acids of the N-terminus.
15 . The anthelmintic composition of claim 5 , wherein the truncated crystal protein is truncated at the C-terminus.
16 . The anthelmintic composition of claim 7 , wherein the lactic acid fermenting bacterium is a Lactococcus or Lactobacillus species.
17 . The anthelmintic composition of claim 16 , wherein the Lactococcus species is Lactococcus lactis.
18 . The anthelmintic composition of claim 16 , wherein the Lactobacillus species is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus johnsonii , and Lactobacillus reuteri.
19 . The anthelmintic composition of claim 1 , wherein the non-invasive or non-pathogenic bacterium is present in an amount effective to reduce an intestinal worm burden in a mammal by at least about 90%.
20 . A pharmaceutical composition comprising:
a non-invasive or non-pathogenic bacterium expressing one or more heterologous anthelmintic crystal proteins; and
a pharmaceutically acceptable carrier,
wherein the non-invasive or non-pathogenic bacterium has a nucleic acid construct or vector comprising at least one promoter operably linked to coding sequences for expression of the one or more heterologous anthelmintic crystal proteins,
wherein the at least one promoter is selected from the group consisting of a phage promoter, a hup gene promoter, a gap gene promoter, an mbg gene promoter, a pgm gene promoter, a thyA gene promoter, a nisin inducible promoter, and an arabinose inducible promoter,
wherein the non-invasive or non-pathogenic bacterium is present in an amount effective to reduce an intestinal worm burden in a mammal, and
wherein the non-invasive or non-pathogenic bacterium is suitable for oral administration to a mammal.
21 . An anthelmintic composition comprising:
a non-invasive or non-pathogenic bacterium expressing Cry5B,
wherein the non-invasive or non-pathogenic bacterium has a nucleic acid construct or vector comprising at least one promoter operably linked to coding sequences for expression of Cry5B,
wherein the at least one promoter is selected from the group consisting of a phage promoter, a hup gene promoter, a gap gene promoter, an mbg gene promoter, a pgm gene promoter, a thyA gene promoter, a nisin inducible promoter, and an arabinose inducible promoter,
wherein the non-invasive or non-pathogenic bacterium is present in an amount effective to reduce an intestinal worm burden in a mammal, and
wherein the non-invasive or non-pathogenic bacterium is suitable for oral administration to a mammal.