Patent Application
20180280452
The text file Sequences_001_ST25.txt of size 301 KB created Apr. 2, 2018, filed herewith, is incorporated in its entirety by reference herein.
The disclosure generally relates to microorganisms, methods, and compositions for reducing methionine content in a diet of a subject or extending a lifespan of the subject and processes for preparing the microorganisms and compositions.
The question of how animals age, and how the aging process can be slowed, is of paramount interest. The use of model organisms, especially Caenorhabditis elegans and Drosophila melanogaster have helped us to better understand mammalian and human aging. For example, such models have helped reveal that dietary restriction, reduction in oxidative stress, and reductions in inflammation can all promote a long and healthy lifestyle (MCISAAC, '16). In particular, dietary restriction extends longevity across the tree of life, underscoring its potential as a therapeutic target. In some cases, restriction of individual nutrients through nutrient allocation or acquisition can fully or partially reproduce the longevity-promoting benefits of total calorie restriction.
The disclosure generally relates to microorganisms, methods, and compositions for reducing methionine content of a diet of a subject or extending a lifespan of the subject and processes for preparing the microorganisms and compositions. The disclosure also relates to populating an intestinal microbiome with probiotic microorganisms modified to have enhanced rates of methionine to cysteine conversions or reduced rates of methionine recycling relative to the modified organisms devoid of the modifications.
In various embodiments are disclosed probiotic bacteria including a heterologous polynucleotide encoding a cystathionine-β-synthase or a cystathionine-γ-lyase and operably linked to a promoter polynucleotide, wherein the probiotic bacterium is derived from a bacteria strain incapable of reverse transsulfurylation.
In various embodiments are disclosed probiotic bacteria including a constitutive promoter polynucleotide operably linked to a polynucleotide encoding a cystathionine-γ-synthase, a cystathionine-β-lyase, or a adenosylhomocysteine nucleosidase.
In various embodiments are disclosed probiotic bacteria including a mutation in a homologous polynucleotide that encodes a protein selected from at least one of a S-ribosylhomocysteine lyase, methionine synthase, homoserine O-acetyltransferase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, or a cobalamin synthase W domain-containing protein 1 or in a homologous promoter polynucleotide operably linked to the homologous polynucleotide, wherein the mutation reduces activity or expression of the protein.
In various embodiments are disclosed methods for reducing methionine content of a diet of a subject or enhancing the lifespan of the subject including administering a composition having an amount of probiotic bacteria in a range from about 103 to about 1015 colony forming units (cfu) per gram of the composition to a subject, wherein a bacterium of the probiotic bacteria, colonizing an intestinal microbiome of the subject, metabolizes methionine at a rate greater than a rate the bacterium synthesizes or recycles methionine when the subject digests a methionine containing substance.
For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein
SEQ ID NO: 1 sets forth an amino acid sequence of a cystathionine-β-synthase from Klebsiella variicola.
SEQ ID NO: 2 sets forth a polynucleotide sequence encoding SEQ ID NO: 1.
SEQ ID NO: 3 sets forth an amino acid sequence of a cystathionine-γ-lyase from Klebsiella variicola.
SEQ ID NO: 4 sets forth a polynucleotide sequence encoding SEQ ID NO: 3.
SEQ ID NO: 5 sets forth an amino acid sequence of a cystathionine-γ-synthase from Klebsiella variicola.
SEQ ID NO: 6 sets forth a polynucleotide sequence encoding SEQ ID NO: 5.
SEQ ID NO: 7 sets forth an amino acid sequence of a cystathionine-β-lyase from Klebsiella variicola.
SEQ ID NO: 8 sets forth a polynucleotide sequence encoding SEQ ID NO: 7.
SEQ ID NO: 9 sets forth an amino acid sequence of an adenosylhomocysteine nucleosidase from Klebsiella variicola.
SEQ ID NO: 10 sets forth a polynucleotide sequence encoding SEQ ID NO: 9.
SEQ ID NO: 11 sets forth a polynucleotide sequence of an operon including SEQ
ID NO: 2 (nt 1 to nt 1371) and SEQ ID NO: 4 (nt 1393 to nt 2531).
SEQ ID NO: 12 and SEQ ID NO: 13 are forward and reverse primers with restriction enzyme recognition sequences for cloning SEQ ID NO: 11.
SEQ ID NO: 14 is a sequencing primer that is upstream from the operon (SEQ ID NO: 11) from Klebsiella variicola.
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 are sequencing primers specific to the operon (SEQ ID NO: 11) from Klebsiella variicola.
SEQ ID NO: 20 sets forth an amino acid sequence of a cystathionine-β-synthase like from Homo sapiens.
SEQ ID NO: 21 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 20.
SEQ ID NO: 22 sets forth an amino acid sequence of a cystathionine-β-synthase from Homo sapiens.
SEQ ID NO: 23 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 22.
SEQ ID NO: 24 sets forth an amino acid sequence of a cystathionine-γ-lyase from Homo sapiens.
SEQ ID NO: 25 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 24.
SEQ ID NO: 26 sets forth an amino acid sequence of a cystathionine-β-synthase from Mus musculus.
SEQ ID NO: 27 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 26.
SEQ ID NO: 28 sets forth an amino acid sequence of a cystathionine-γ-lyase from Mus musculus.
SEQ ID NO: 29 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 28.
SEQ ID NO: 30 sets forth an amino acid sequence of a cystathionine-β-synthase from Pseudomonas aeruginosa (PAO1).
SEQ ID NO: 31 sets forth a polynucleotide sequence encoding SEQ ID NO: 30.
SEQ ID NO: 32 sets forth an amino acid sequence of a cystathionine-γ-lyase from Pseudomonas aeruginosa (PAO1).
SEQ ID NO: 33 sets forth a polynucleotide sequence encoding SEQ ID NO: 32.
SEQ ID NO: 34 sets forth an amino acid sequence of a cystathionine-β-synthase from Streptomyces venezuelae.
SEQ ID NO: 35 sets forth a polynucleotide sequence encoding SEQ ID NO: 34.
SEQ ID NO: 36 sets forth an amino acid sequence of a cystathionine-β-synthase from Streptomyces venezuelae.
SEQ ID NO: 37 sets forth a polynucleotide sequence encoding SEQ ID NO: 36.
SEQ ID NO: 38 sets forth an amino acid sequence of a cystathionine-γ-lyase from Streptomyces venezuelae.
SEQ ID NO: 39 sets forth a polynucleotide sequence encoding SEQ ID NO: 38.
SEQ ID NO: 40 sets forth an amino acid sequence of a cystathionine-γ-lyase from Streptomyces venezuelae.
SEQ ID NO: 41 sets forth a polynucleotide sequence encoding SEQ ID NO: 40.
SEQ ID NO: 42 sets forth an amino acid sequence of a cystathionine-β-lyase from Streptomyces venezuelae.
SEQ ID NO: 43 sets forth a polynucleotide sequence encoding SEQ ID NO: 42.
SEQ ID NO: 44 sets forth an amino acid sequence of a cystathionine-γ-synthase from Streptomyces venezuelae.
SEQ ID NO: 45 sets forth a polynucleotide sequence encoding SEQ ID NO: 44.
SEQ ID NO: 46 sets forth an amino acid sequence of an adenosylhomocysteine nucleosidase from Streptomyces venezuelae.
SEQ ID NO: 47 sets forth a polynucleotide sequence encoding SEQ ID NO: 46.
SEQ ID NO: 48 sets forth an amino acid sequence of a cystathionine-β-lyase from Helicobacter pylori.
SEQ ID NO: 49 sets forth a polynucleotide sequence encoding SEQ ID NO: 48.
SEQ ID NO: 50 sets forth an amino acid sequence of a cystathionine-β-synthase from Aspergillus nidulans.
SEQ ID NO: 51 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 50.
SEQ ID NO: 52 sets forth an amino acid sequence of a cystathionine-γ-lyase from Aspergillus nidulans.
SEQ ID NO: 53 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 52.
SEQ ID NO: 54 sets forth an amino acid sequence of a cystathionine-β-lyase from Aspergillus nidulans.
SEQ ID NO: 55 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 54.
SEQ ID NO: 56 sets forth an amino acid sequence of a cystathionine-γ-synthase from Aspergillus nidulans.
SEQ ID NO: 57 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 56.
SEQ ID NO: 58 sets forth an amino acid sequence of a cystathionine-β-synthase from Leishmania major.
SEQ ID NO: 59 sets forth a polynucleotide sequence encoding SEQ ID NO: 58.
SEQ ID NO: 60 sets forth an amino acid sequence of a cystathionine-β-lyase from Leishmania major.
SEQ ID NO: 61 sets forth a polynucleotide sequence encoding SEQ ID NO: 60.
SEQ ID NO: 62 sets forth an amino acid sequence of a cystathionine-β-lyase from Leishmania major.
SEQ ID NO: 63 sets forth a polynucleotide sequence encoding SEQ ID NO: 62.
SEQ ID NO: 64 sets forth an amino acid sequence of a cystathionine-β-lyase from Oryza sativa.
SEQ ID NO: 65 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 64.
SEQ ID NO: 66 sets forth an amino acid sequence of a cystathionine-β-lyase from Oryza sativa.
SEQ ID NO: 67 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 66.
SEQ ID NO: 68 sets forth an amino acid sequence of a cystathionine-γ-synthase from Oryza sativa.
SEQ ID NO: 69 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 68.
SEQ ID NO: 70 sets forth an amino acid sequence of a cystathionine-γ-synthase from Oryza sativa.
SEQ ID NO: 71 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 70.
SEQ ID NO: 72 sets forth an amino acid sequence of a cystathionine-γ-synthase from Oryza sativa.
SEQ ID NO: 73 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 72.
SEQ ID NO: 74 sets forth an amino acid sequence of a cystathionine-β-synthase from Saccharomyces cerevisiae.
SEQ ID NO: 75 sets forth a polynucleotide sequence encoding SEQ ID NO: 74.
SEQ ID NO: 76 sets forth an amino acid sequence of a cystathionine-γ-lyase from Saccharomyces cerevisiae.
SEQ ID NO: 77 sets forth a polynucleotide sequence encoding SEQ ID NO: 76.
SEQ ID NO: 78 sets forth an amino acid sequence of a cystathionine-β-lyase from Saccharomyces cerevisiae.
SEQ ID NO: 79 sets forth a polynucleotide sequence encoding SEQ ID NO: 78.
SEQ ID NO: 80 sets forth an amino acid sequence of a cystathionine-β-lyase from Saccharomyces cerevisiae.
SEQ ID NO: 81 sets forth a polynucleotide sequence encoding SEQ ID NO: 80.
SEQ ID NO: 82 sets forth an amino acid sequence of a cystathionine-γ-synthase from Saccharomyces cerevisiae.
SEQ ID NO: 83 sets forth a polynucleotide sequence encoding SEQ ID NO: 82.
SEQ ID NO: 84 sets forth an amino acid sequence of a cystathionine-γ-synthase from Saccharomyces cerevisiae.
SEQ ID NO: 85 sets forth a polynucleotide sequence encoding SEQ ID NO: 84.
SEQ ID NO: 86 sets forth an amino acid sequence of a cystathionine-γ-synthase from Saccharomyces cerevisiae.
SEQ ID NO: 87 sets forth a polynucleotide sequence encoding SEQ ID NO: 86.
SEQ ID NO: 88 sets forth an amino acid sequence of a cystathionine-β-lyase from Arabidopsis thaliana.
SEQ ID NO: 89 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 88.
SEQ ID NO: 90 sets forth an amino acid sequence of a cystathionine-γ-synthase from Arabidopsis thaliana.
SEQ ID NO: 91 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 90.
SEQ ID NO: 92 sets forth an amino acid sequence of a cystathionine-γ-synthase from Arabidopsis thaliana.
SEQ ID NO: 93 sets forth a cDNA polynucleotide sequence encoding SEQ ID NO: 92.
SEQ ID NO: 94 sets forth an amino acid sequence of a cystathionine-β-lyase from Klebsiella variicola.
SEQ ID NO: 95 sets forth a polynucleotide sequence encoding SEQ ID NO: 94.
SEQ ID NO: 96 sets forth an amino acid sequence of an adenosylhomocysteine nucleosidase from Klebsiella variicola.
SEQ ID NO: 97 sets forth a polynucleotide sequence encoding SEQ ID NO: 96.
SEQ ID NO: 98 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus plantarum.
SEQ ID NO: 99 sets forth a polynucleotide sequence encoding SEQ ID NO: 98.
SEQ ID NO: 100 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus plantarum.
SEQ ID NO: 101 sets forth a polynucleotide sequence encoding SEQ ID NO: 100.
SEQ ID NO: 102 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus plantarum.
SEQ ID NO: 103 sets forth a polynucleotide sequence encoding SEQ ID NO: 102.
SEQ ID NO: 104 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus plantarum.
SEQ ID NO: 105 sets forth a polynucleotide sequence encoding SEQ ID NO: 104.
SEQ ID NO: 106 sets forth an amino acid sequence of a cystathionine-γ-synthase from Lactobacillus plantarum.
SEQ ID NO: 107 sets forth a polynucleotide sequence encoding SEQ ID NO: 106.
SEQ ID NO: 108 sets forth an amino acid sequence of an adenosylhomocysteine nucleosidase from Lactobacillus plantarum.
SEQ ID NO: 109 sets forth a polynucleotide sequence encoding SEQ ID NO: 108.
SEQ ID NO: 110 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus rhamnosus.
SEQ ID NO: 111 sets forth a polynucleotide sequence encoding SEQ ID NO: 110.
SEQ ID NO: 112 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus rhamnosus.
SEQ ID NO: 113 sets forth a polynucleotide sequence encoding SEQ ID NO: 112.
SEQ ID NO: 114 sets forth an amino acid sequence of a cystathionine-β-lyase from Lactobacillus rhamnosus.
SEQ ID NO: 115 sets forth a polynucleotide sequence encoding SEQ ID NO: 114.
SEQ ID NO: 116 sets forth an amino acid sequence of an adenosylhomocysteine nucleosidase from Lactobacillus rhamnosus.
SEQ ID NO: 117 sets forth a polynucleotide sequence encoding SEQ ID NO: 116.
As required, detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary and may be embodied in various and alternative forms. The figures are not necessarily to scale; some features may be exaggerated or minimized to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art.
Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word “about”. The first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.
Unless indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.
It is also to be understood that this disclosure is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for describing particular embodiments and is not intended to be limiting in any way.
It must also be noted that, as used in the specification and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.
The terms “or” and “and” can be used interchangeably and can be understood to mean “and/or”.
The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.
The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
The terms “comprising”, “consisting of”, and “consisting essentially of” can be alternatively used. When one of these three terms is used, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
The term “microbiome” refers to the totality of microbes (bacteria, fungae, protists), their genetic elements (genomes) in a defined environment. The microbiome may be a gut microbiome (i.e. intestinal microbiome).
The term “bacterium”, “bacteria”, and “strain” are used interchangeably and refers to microorganism(s) having its conventional meaning as used in the art, that is, generally, a low taxonomic rank indicating a genetic variant or subtype of a microorganism (within a defined species). Further, it can be also understood that “bacterium” and “bacteria” are genetically modified bacterium or bacteria.
The term “probiotic” is understood to mean probiotic bacteria that impart benefits to a subject when colonizing the microbiome of the subject. For example, a probiotic bacterium can express competitive exclusion effect against pathogenic microorganisms or intensify disease-resistant properties of subjects through suppressive action of metabolites.
The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably in this disclosure. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
The term “heterologous” nucleic acid can refer to a nucleic acid that is not normally or naturally found in or produced by a given bacterium, organism, or cell in nature. The term “homologous” nucleic acid can refer to a nucleic acid that is normally found in or produced by a given bacterium, organism, or cell in nature.
The term “recombinant” is understood to mean that a particular nucleic acid (DNA or RNA) or protein is the product of various combinations of cloning, restriction, or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
The terms “amino acid sequence” or “amino acid” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.
The terms “peptide” or “protein” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules.
The terms “construct”, “cassette”, “expression cassette”, “plasmid”, “vector”, or “expression vector” is understood to mean a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression or propagation of a nucleotide sequence(s) of interest, or is to be used in the construction of other recombinant nucleotide sequences.
The term “promoter” or “promoter polynucleotide” is understood to mean a regulatory sequence/element or control sequence/element that is capable of binding/recruiting a RNA polymerase and initiating transcription of sequence downstream or in a 3′ direction from the promoter. A promoter can be, for example, constitutively active or always on or inducible in which the promoter is active or inactive in the presence of an external stimulus. Example of promoters include T7 promoters or lactose (lac) promoters.
The term “operably linked” can mean the positioning of components in a relationship which permits them to function in their intended manner. For example, a promoter can be linked to a polynucleotide sequence to induce transcription of the polynucleotide sequence.
The terms “sequence identity” or “identity” refers to a specified percentage of residues in two nucleic acid or amino acid sequences that are identical when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection, wherein the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
The term “comparison window” refers to a segment of at least about 20 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. In a refinement, the comparison window is from 15 to 30 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. In another refinement, the comparison window is usually from about 50 to about 200 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
The terms “complementarity” or “complement” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 4, 5, and 6 out of 6 being 66.67%, 83.33%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 40%, 50%, 60%, 62.5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, or percentages in between over a region of 4, 5, 6, 7, and 8 nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
The term “subject(s)” refers to subjects of any mammalian subject(s) of any mammalian species such as, but not limited to, humans, dogs, cats, horses, rodents, any domesticated animal, or any wild animal.
The disclosure generally relates to microorganisms, methods, and compositions for reducing methionine content of a diet of a subject or extending a lifespan of the subject and processes for preparing the microorganisms and compositions. The disclosure also relates to populating an intestinal microbiome with probiotic microorganisms modified to have enhanced rates of methionine to cysteine conversions or reduced rates of methionine recycling relative to the modified organisms devoid of the modifications.
In various embodiments are disclosed probiotic bacteria including a heterologous polynucleotide encoding a cystathionine-β-synthase or a cystathionine-γ-lyase and operably linked to a promoter polynucleotide, wherein the probiotic bacterium is derived from a bacteria strain incapable of reverse transsulfurylation. In reverse transsulfurylation, an 1-allo-cystathionine intermediate is formed when homocysteine donates its sulfhydryl group to activated serine through a cystathionine-β-synthase; cystathionine is cleaved by cystathionine-γ-lyase to yield cysteine, α-keto-butyrate, and ammonia. The probiotic bacterium of various embodiments can further include a mutation in a homologous polynucleotide that encodes a protein selected from at least one of a S-ribosylhomocysteine lyase, methionine synthase, homoserine O-acetyltransferase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and a cobalamin synthase W domain-containing protein 1 or in a homologous promoter polynucleotide operably linked to the homologous polynucleotide, wherein the mutation reduces activity or expression of the protein.
The promoter polynucleotide of various embodiments is a constitutive promoter polynucleotide or an inducible promoter polynucleotide. Transcription of proteins from heterologous polynucleotides are normally regulated and initiated by a promoter polynucleotide. The constitutive promoter polynucleotide of various embodiments is capable of expressing proteins at high concentration. In various embodiments, the transcript level of the constitutive promoter polynucleotide is about or is at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, 12.5-fold, 13-fold, 13.5-fold, 14-fold, 14.5-fold, 15-fold, 15.5-fold, 16-fold, 16.5-fold, 17-fold, 17.5-fold, 18-fold, 18.5-fold, 19-fold, 19.5-fold, 20-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, 4000-fold, 4500-fold, 5000-fold, 5500-fold, 6000-fold, 6500-fold, 7000-fold, 7500-fold, 8000-fold, 8500-fold, 9000-fold, 9500-fold, or 10000-fold higher than a transcript level of a native promoter for an operon encoding cystathionine-β-synthase or cystathionine-γ-lyase operon. In various embodiments, the transcript level of the constitutive promoter polynucleotide is a range between any two levels listed above. In various embodiments, the transcript level of the constitutive promoter polynucleotide is about 10000-fold or higher than a transcript level of a native promoter for an operon encoding cystathionine-β-synthase or cystathionine-γ-lyase operon. Examples of constitutive promoter polynucleotides or inducible promoter polynucleotides include lac, T7, T7Lac, Sp6, AraBAD, trp, Ptac, pL, λpL, λpR, T6, recA, gal, ara, or hut.
The heterologous polynucleotide operably linked to a promoter polynucleotide of various embodiments can be within a cassette of an expression vector introduced into the probiotic bacteria or stably incorporated within the probiotic bacteria or into a genome of the probiotic bacteria.
In various embodiments, the cystathionine-β-synthase has an amino acid sequence that is or that is at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 58, or SEQ ID NO: 74. In various embodiments, the percent identity is a range between any two percentages listed above. In various embodiments, cystathionine-β-synthase includes cystathionine-β-synthase like.
In various embodiments, the heterologous polynucleotide encoding cystathionine-β-synthase has a nucleotide sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 51, SEQ ID NO: 59, or SEQ ID NO: 75. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the cystathionine-y-lyase has an amino acid sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 52, or SEQ ID NO: 76. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the heterologous polynucleotide encoding cystathionine-γ-lyase has a nucleotide sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 53, or SEQ ID NO: 77. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments are disclosed probiotic bacteria including a constitutive promoter polynucleotide operably linked to a polynucleotide encoding a cystathionine-γ-synthase, a cystathionine-β-lyase, or a adenosylhomocysteine nucleosidase. The probiotic bacteria of various embodiments can further include a heterologous polynucleotide encoding a cystathionine-γ-synthase or a cystathionine-γ-lyase operably linked to the constitutive promoter polynucleotide or a mutation in a homologous polynucleotide that encodes a protein selected from at least one of a S-ribosylhomocysteine lyase, methionine synthase, homoserine O-acetyltransferase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and a cobalamin synthase W domain-containing protein 1 or in a homologous promoter polynucleotide operably linked to the homologous polynucleotide, wherein the mutation reduces activity or expression of the protein.
In various embodiments, the transcript level of the constitutive promoter polynucleotide is about or is at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, 12.5-fold, 13-fold, 13.5-fold, 14-fold, 14.5-fold, 15-fold, 15.5-fold, 16-fold, 16.5-fold, 17-fold, 17.5-fold, 18-fold, 18.5-fold, 19-fold, 19.5-fold, 20-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, 4000-fold, 4500-fold, 5000-fold, 5500-fold, 6000-fold, 6500-fold, 7000-fold, 7500-fold, 8000-fold, 8500-fold, 9000-fold, 9500-fold, or 10000-fold higher than a transcript level of a native promoter for an operon encoding cystathionine-β-synthase or cystathionine-γ-lyase. In various embodiments, the transcript level of the constitutive promoter polynucleotide is about 10000-fold or higher than a transcript level of a native promoter for an operon encoding cystathionine-β-synthase or cystathionine-γ-lyase operon. In various embodiments, the transcript level of the constitutive promoter polynucleotide is a range between any two levels listed above. Examples constitutive promoter polynucleotides or inducible promoter polynucleotides include lac, T7, T7Lac, Sp6, AraBAD, trp, Ptac, pL, λpL, λpR, T6, recA, gal, ara, or hut.
In various embodiments, the constitutive promoter is a native promoter driving expression of homologous polynucleotides encoding cystathionine-γ-synthase, cystathionine-β-lyase, or adenosylhomocysteine nucleosidase, where the native promoter has been modified to enhance expression levels of cystathionine-γ-synthase, cystathionine-β-lyase, or adenosylhomocysteine nucleosidase. Examples of such modifications can include removal of regulatory sequences such that a base promoter remains or introducing a constitutive promoter at a position within the genome of the bacterium to drive expression of cystathionine-γ-synthase, cystathionine-β-lyase, or adenosylhomocysteine nucleosidase.
In various embodiments, the polynucleotide encoding cystathionine-γ-synthase, cystathionine-β-lyase, or adenosylhomocysteine nucleosidase is a heterologous polynucleotide. The heterologous polynucleotide of various embodiments can be within a cassette of an expression vector introduced into the probiotic bacteria or stably incorporated within the probiotic bacteria or into a genome of the probiotic bacteria.
In various embodiments, the heterologous polynucleotide encoding cystathionine-γ-synthase has a nucleotide sequence that is about or that is least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 6, SEQ ID NO: 45, SEQ ID NO: 57, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 93, or SEQ ID NO: 107. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the cystathionine-γ-synthase has an amino acid sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 5, SEQ ID NO: 44, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 106. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the heterologous polynucleotide encoding cystathionine-β-lyase has a nucleotide sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 8, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 89, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO: 115. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the cystathionine-β-lyase has an amino acid sequence that is or that is about at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 7, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the heterologous polynucleotide encoding adenosylhomocysteine nucleosidase has a nucleotide sequence that is about or that is at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 10, SEQ ID NO: 47, SEQ ID NO: 97, SEQ ID NO: 109, or SEQ ID NO: 117. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the adenosylhomocysteine nucleosidase has an amino acid sequence that is or that is about at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of SEQ ID NO: 9, SEQ ID NO: 46, SEQ ID NO: 96, SEQ ID NO: 108, or SEQ ID NO: 116. In various embodiments, the percent identity is a range between any two percentages listed above.
In various embodiments, the heterologous polynucleotide encoding cystathionine-γ-synthase, cystathionine-β-lyase, or adenosylhomocysteine nucleosidase is operably linked to the constitutive promoter polynucleotide of a second promoter polynucleotide. The second promoter nucleotide of various embodiments can include a constitutive promoter nucleotide polynucleotide or an inducible promoter polynucleotide.
In various embodiments are disclosed probiotic bacteria including a mutation in a homologous polynucleotide that encodes a protein selected from at least one of a S-ribosylhomocysteine lyase, methionine synthase, homoserine O-acetyltransferase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and a cobalamin synthase W domain-containing protein 1 or in a homologous promoter polynucleotide operably linked to the homologous polynucleotide, wherein the mutation reduces activity or expression of the protein.
The probiotic bacterium or the bacteria strain incapable of reverse transsulfurylation of any embodiment can belong(s) to a genus Lactobacillus, Bifidobacterium, Escherichia, Enterococcus, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, or Saccharomyces or belong(s) to an order Lactobacillales.
In various embodiments are disclosed compositions for reducing methionine content of a diet of a subject or extending a lifespan of the subject including a probiotic bacteria of any embodiment and a pharmaceutically acceptable excipient. In various embodiments, the composition includes a plurality of different probiotic bacteria of any embodiment, wherein each of the different probiotic bacteria belongs to a different strain, species, or genus. The plurality of different probiotic bacteria can include or can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different probiotic bacteria. In various embodiments, the number of different probiotic bacteria in the composition is a range between any two numbers listed above. In various embodiments, the plurality of different probiotic bacteria can include 20 or more different bacteria.
In various embodiments, the amount of the probiotic bacteria in the composition is about or is at least about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 colony forming units (cfu) per gram of the composition. In various embodiments, the amount of the probiotic bacteria in the composition is a range between any two cfu per gram listed above. In various embodiments, the amount of the probiotic bacteria in the composition is about 1015 cfu or more per gram of the composition.
In various embodiments, the pharmaceutically acceptable excipient is a carrier suitable for oral consumption. Examples of carriers include silicon dioxide (silica, silica gel), carbohydrates or carbohydrate polymers (polysaccharides), cyclodextrins, starches, degraded starches (starch hydrolysates), chemically or physically modified starches, modified celluloses, gum arabic, ghatti gum, tragacanth, karaya, carrageenan, guar gum, locust bean gum, alginates, pectin, inulin or xanthan gum, or hydrolysates of maltodextrins and dextrins. In various embodiments, the bacteria is dispersed throughout the carrier.
In various embodiments, the pharmaceutically acceptable excipient is a carrier suitable for oral consumption and the probiotic bacteria is dispersed throughout the carrier. The pharmaceutically acceptable excipient of various embodiments is capable of delivering at least a portion of the amount of the probiotic bacteria to the intestinal microbiome of the subject in an active state.
In various embodiments, the pharmaceutically acceptable excipient includes an extended release phase capable of releasing at least a portion of the amount of the probiotic bacteria to the intestinal microbiome of the subject over a period of time. In other embodiments, the pharmaceutically acceptable excipient includes an immediate release phase capable of substantially immediately releasing at least a portion of the amount of the probiotic bacteria to the intestinal microbiome of the subject.
In various embodiments, the composition can include other prebiotic compounds or probiotic strains. Examples of such probiotic strains include Lactobacillus such as L. plantarum, L. paracasei, L. acidophilus , L. casei, L. rhamnosus, L. crispatus, L. gasseri , L. reuteri, L. bulgaricus; Bifidobacterium such as B. longum, B. catenulatum , B. breve, B. animalis, B. bifidum; Streptococcus such as S. sanguis, S. oxalis, S. mitis, S. thermophilus, S. salivarius; Bacillus such as B. coagulans, B. subtilis, B. laterosporus; Lactococcus such as L. lactis; Enterococcus such as E. faecium; Pediococcus such as P. acidilactici; Propionibacterium suchas P. jensenii, P. freudenreichii; Peptostreptococcus such as P. productus; and Saccharomyces such as S. boulardii.
In various embodiments, the probiotic bacteria of any embodiment are prepared in any manner to be incorporated as a component of a pharmaceutical, food stuff, feed additive, or liquid additive and can be in various forms such as, for example, a liquid state or a dried state including as a powder. In other examples, the probiotic bacteria of various embodiments are dried by air drying method, natural drying method, a spray drying method, a freeze-drying method, or the like. The preparation of the probiotic bacteria can also serve to enhance the properties of the composition including stability. The term “foodstuff” is understood to be any substance or product which in the processed, partially processed, or unprocessed state are intended to be, or reasonably expected to be, ingested by humans. “Foodstuff” can also include drinks, chewing gum, and any substance--including water-intentionally added to the foodstuff during its manufacture, preparation or treatment. The term “feed” is understood to cover all forms of animal food. Foodstuffs can also be used as feeds. The term “pharmaceutical” is understood to cover substances or substance compositions which are intended as agents having properties for curing or for preventing human or animal diseases or which can be used in or on the human or animal body or administered to a human or animal in order to restore, correct or influence either human or animal physiological functions by a pharmacological, immunological or metabolic action, or to produce a medical diagnosis. Pharmaceuticals can be used for non-therapeutic, in particular cosmetic, purposes.
In various embodiments are disclosed pharmaceutical compositions including a compound of any embodiments wherein the pharmaceutical composition is a gel capsule, tablet, pill, lozenge, capsule, microcapsule, liquid, or syrup.
In various embodiments are disclosed orally consumable products including a composition of any embodiment, wherein an orally consumable product is a semi-solid food, solid food, a semi-solid or solid spoonable food, confectionary, drink, or dairy product. The dairy product of various embodiments is ice cream, milk, milk powder, yogurt, kefir, or quark.
In various embodiments are disclosed methods for reducing methionine content of a diet of a subject or enhancing the lifespan of the subject including administering a composition having an amount of probiotic bacteria in a range from about 103 to about 1015 cfu per gram of the composition to a subject, wherein a bacterium of the probiotic bacteria, colonizing an intestinal microbiome of the subject, metabolizes methionine at a rate greater than a rate the bacterium synthesizes or recycles methionine when the subject digests a methionine containing substance. In various embodiments, the bacterium substantially always metabolizes methionine at a rate greater than a rate the bacterium synthesizes methionine when the subject digests a methionine containing substance. In other embodiments, the bacterium is unable to synthesize or recycle methionine.
In various embodiments, the rate that the bacterium of the probiotic bacteria, colonizing an intestinal microbiome of the subject, metabolizes methionine is about or is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, or 5000% greater than the rate the bacterium synthesizes or recycles methionine when the subject digests a methionine containing substance. In various embodiments, the rate is a range between any two percentages listed above. In various embodiments, the rate that the bacterium of the probiotic bacteria, colonizing an intestinal microbiome of the subject, metabolizes methionine is about 5000% or more than the rate the bacterium synthesizes or recycles methionine when the subject digests a methionine containing substance.
In various embodiments, the administering of the composition can be repeated daily for an undetermined period of time. In various embodiments, the probiotic bacteria colonizing the intestinal microbiome of the subject is capable of reducing the methionine content of the diet of the subject by about or by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% generally or within a given time period. In various embodiments, the reduction is a range between any two percentages listed above.
In various embodiments, the administering of the composition is capable of extending the lifespan of the subject by about or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% as compared to a subject no receiving the composition. In various embodiments, the lifespan extension is a range between any two percentages listed above.
In various embodiments are disclosed methods of preparing the probiotic bacteria of any embodiment. The method of various embodiments can include the step of transfecting or transducing bacteria with a heterologous polynucleotide of any embodiment. In other embodiments, the method can include recombinantly modifying homologous polynucleotides or native promoters to increase or reduce expression of a protein.
The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims.
The effect of dietary restriction on Drosophila melanogaster lifespan has been intensely studied. More recently, interest has grown in the microbiome's interaction with its host. This interaction has been shown to influence lifespan but the pathway by which this takes place is not known. Lifespans of flies mono-associated with 41 bacterial strains were studied. Running a metagenome wide association study allowed for the prediction of bacterial genes that were causing lifespan effects. After testing Escherichia coli mutants, the prediction that microbes were influencing Drosophila melanogaster lifespan by altering flux through the transsulfuration pathway was confirmed.
Methionine restriction is an established paradigm for lifespan extension across the tree of life, underscoring its potential as a therapeutic target (ORENTREICH, '93, KOZIEL, '14, LEE, '14). Methionine restriction as a means for lifespan extension is well documented in the fruit fly Drosophila melanogaster, and differential regulation of fruit fly methionine metabolism genes can extend lifespan (KABIL, '11, PARKHITKO, '16). It has also been shown that long-lived Ames dwarf mice lack growth hormone, prolactin, and thyroid stimulating hormone have an enhanced methionine metabolism (UTHUS, '06). In one of the best studied models for aging, fruit fly longevity can be promoted either by restricting the methionine content of the diet, or by differentially expressing genes that decrease the accumulation of methionine-cycle intermediates (TROEN, '07, GRANDISON, '09, KABIL, '11, LEE, '14, OBATA, '15, PARKHITKO, '16). Previous work, primarily in fruit flies and mice, suggests that altering an animal's metabolism to minimize the abundance of methionine cycle metabolites by increasing bacterial transsulfuration flux through transsulfuration decreases the available pool of methionine cycle metabolites (methionine, SAM, SAH, and homocysteine,
Despite our understanding of some of the genes involved, the mechanistic basis for Drosophila responses to dietary and methionine restriction is not fully understood and is complex. For example, the paradox that naturally long-lived flies display increased methionine contents in early life has been explained as an increased flux through the methionine cycle that decreases accumulation of SAM and SAH intermediates (PARKHITKO, '16). This idea is consistent with the explanation that increased SAM catabolism or transsulfuration also extends fruit fly lifespan (KABIL, '11, OBATA, '15). Additionally, some byproducts of methionine metabolism, such as cysteine and cystathionine, restrict lifespan. Finally, we note that in some cases, methionine supplementation is actually helpful for organismal development. These varied findings underscore the complexity of interactions between methionine metabolism and aging.
In addition to dietary and genetic intervations, associated microorganisms (‘microbiota’) are a powerful influence on organismal longevity. In Drosophila melanogaster numerous studies have revealed a positive, neutral, or antagonistic role for the microbiota in animal lifespan. A unifying explanation for the varied influence of bacteria on Drosophila melanogaster lifespan is that the influences of the microbiota are interactive with diet (BRUMMEL, '04, COX, '07, DESHPANDE, '15, YAMADA, '15, YAMADA, '17). For example, when flies are reared on a nutrient poor diet, the microbes may provision nutrients that aid in healthy growth and development, and promote longevity. Conversely, rearing on a nutrient-rich diet may spare the flies of a microbial dependence, and other (currently unknown but possibly pathogenic) influences of the microbiota may prevail. In addition to direct dietary influence on lifespan, there is diet-dependent variation in traits that presumably draw resources away from somatic maintenance, such as immunity (UNCKLESS, '15). Also, the influence of the microbiota on the duration of animal development, a trait that is positively correlated with lifespan in many natural fly populations, is dramatically influenced by changes in absolute and relative nutrient contents in the fly diet (WONG, '14).
Drosophila melanogaster is a well-established model organism for microbiome studies (BRODERICK, '14). As a model for basic and applied aging research, findings in Drosophila are relevant to human studies: human genes that are central to lifespan were first identified in fruit flies, and models for lifespan extension through calorie and methionine restriction were developed in fruit flies (HELFAND, '03, SHAW, '08, BUSHEY, '10). Both mammalian and Drosophila melanogaster lifespan are influenced by the microbiota (BRUMMEL, '04, OTTAVIANI, '11, ZHANG, '13). The fly's digestive tract is similar to humans in physiology and anatomy, making it a key model for studying the microbiome (BRODERICK, '14). Drosophila melanogaster is also an established model for studying the microbiota generally, and flies are readily produced that are reared bacteria-free or with defined microbial communities (including bacterial mutants). Also, host-microbiome interactions first seen in Drosophila have been later observed in mammals, underscoring the broad applicability of many key findings (WONG, '13, ROGERS, '14). Drosophila melanogaster 's simple microbiome also makes them an appealing model. Lab strains typically have between 2-20 bacterial species—being dominated by five or fewer (BRUMMEL, '04, COX, '07, CHANDLER, '11, WONG, '11). Of these five dominant species, the majority are Acetobacter and Lactobacillus species. Furthermore, the microbiota are readily eliminated or inoculated in defined ratios, enabling mechanistic dissection of the varied influences of different microbes on animal traits (CHASTON, '14, NEWELL, '14). Taken together, these tools and a rich history as a model for aging make Drosophila melanogaster an ideal model for understanding host-microbe interactions during aging.
In this work, we investigate the relationship between Drosophila melanogaster, it's lifespan, and it's microbiota and show that bacterial methionine metabolism is important in organismal aging. We also confirmed these predictions by mutant analysis. Finally, we extend these predictions by showing that a microbe engineered to drive flux from the methionine cycle extends organismal lifespan. These results show the influence on Drosophila melanogaster lifespan with a nutrient-rich diet simply by altering their microbes, which is applicable to using such microbes with mammals.
All experiments were conducted with Drosophila melanogaster. They were obtained and cultured at 25° C. on a 12-hr light-dark cycle. Drosophila melanogaster were fed on a yeast-glucose diet (1 liter H2O, 100 g inactive brewer's yeast, 100 g glucose, 1.2% agar, 0.84% propionic acid, and 0.08% phosphoric acid) as in our previous work (NEWELL, '14).
The bacterial strains listed in Table 1 were cultured as in our previous work on Glade-specific media: modified MRS medium (mMRS; 1.25% peptone, 0.75% yeast extract, 2% glucose, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.02% magnesium sulfate heptahydrate, 0.005% manganese sulfate tetrahydrate, 1.2% agar (NEWELL, '14)), potato medium (pot; 0.5% glucose, 1% yeast extract, 1% peptone, 0.8% potato extract), lysogeny broth (LB; 1% tryptone, 0.5% yeast extract, 0.5% sodium choloride), and brain heart infusion broth (BHI). Growth of auxotrophs was confirmed on M9 medium. Escherichia coli were grown at 37° C., and all other strains were grown at 30° C. Transposon insertion- or ectopic expression mutants were cultured with antibiotics: 50 mg/ml kanamycin for Escherichia coli transposon insertion mutants; and 20 μg/ml chlortetracycline for Acetobacter 54 ectopic expression strains. Strains grown in oxic conditions were grown in liquid culture with shaking or with no atmospheric treatment in solid culture. Strains grown under microoxic conditions were grown statically (liquid) or in a sealed, CO2-flooded chamber (solid).
Escherichia coli BW25113, CGSC wild-type;
Acetobacter aceti NBRC 14818; Accession
Acetobacter sp. DmW_043; Accession
Acetobacter
fabarum DsW_054-pAH1; Accession
A. fabarum DsW_054 expressing pAH1; TcR; this
Acetobacter indonesiensis DmW_046; Accession
Acetobacter malorum DsW_057; Accession
Acetobacter malorum DmCS_005; Accession
Acetobacter orientalis DmW_045; Accession
Acetobacter pasteurianus 3P3; Accession
Acetobacter pasteurianus NBRC 101655; Accession
Acetobacter pasteurianus NBRC 106471 or
Acetobacter pomorum DmCS_004; Accession
Acetobacter tropicalis DmCS_006; Accession
Acetobacter tropicalis NBRC 101654; Accession
Acetobacter tropicalis DmW_042; Accession
Bacillus subtilis subsp. subtilis str.168; Accession
Escherichia coli str. K-12 substr. MG1655;
Enterococcus faecalis V583; Accession
Enterococcus faecalis OG1RF; Accession
Enterobacter hormaechei ATCC 49162; Accession
Gluconobacter sp DsW_056; Accession
Gluconacetobacter europeus 5p3; Accession
Gluconobacter frateurii NBRC 101659; Accession
Gluconacetobacter hansenii ATCC 23769; Accession
Gluconacetobacter oboediens 174Bp2; Accession
Gluconacetobacter xylinus NBRC 3288; Accession
Lactobacillus brevis subsp. gravesensis ATCC
Lactobacillus brevis DmCS_003; Accession
Lactobacillus buchneri NRRLB-30929; Accession
Leuconostoc fallax KCTC 3537; Accession
Lactobacillus fermentum ATCC 14931; Accession
Lactobacillus fructivorans DmCS_002; Accession
Lactobacillus fructivorans KCTC 3543; Accession
Lactococcus lactis BPL1; Accession
Lactobacillus mali KCTC 3596 = DSM 20444;
Lactobacillus plantarum DmCS_001; Accession
Lactobacillus plantarum WCFS1; Accession
Lactobacillus rhamnosus GG; Accession
Acetobacter fabarum DsW_054; Accession na
Klebsiella variicola ATCC BAA-830; Accession na
Providencia burhodogranariea DSM 19968;
Pseudomonas putida F1; Accession NC_009512.1
Flies were reared under axenic or gnotobiotic conditions as in our previous work (KOYLE, '16). Briefly, less than 20 hour-old eggs laid on grape juice agar plates were collected from Drosophila melanogaster and surface sterilized with a 0.6% sodium hypochlorite solution in two 2.5 minute washes. Hypochlorite was washed away by three rinses with sterile water, and 30-60 eggs, qualitatively estimated, were aseptically transferred to sterile diet in a sterile biosafety cabinet. 7.5 ml of sterile yeast-glucose diet (omitting the acid) were inoculated into 50-ml conical tubes, autoclaved, and allowed to cool before transferring sterile eggs. To rear under axenic conditions the eggs were left left undisturbed. To rear with defined bacterial species the sterile eggs were inoculated with 50 μl of bacterial cultures normalized to OD600 0.1. If more than one species was added, the multiple strains were normalized as above and pooled in equal ratios before inoculating the flies with a 50 μl volume of bacteria. For each analysis, we sought to collect data from triplicate vials of flies in each of three separate experiments; however, after 6 separate experiments, some treatments could not be collected at this level of replication and all data were used regardless of replication (after discarding contaminants).
Drosophila melanogaster lifespan was measured by recording the number and sex of dead flies and transferring surviving flies to fresh sterile diet every 2-3 days until all flies in a vial were dead. The spent vials were incubated at room temperature (˜22° C.) until eggs laid during the 2-3 day interval grew to adulthood. For every P generation fly vial transferred to fresh diet, one F1 vial from each week of transfers was selected and homogenized to check for bacterial persistence and contamination during transfer. A pool of five mixed sex flies from each vial was homogenized in 125 μl homogenization buffer (10 mM Tris, pH 8, 1 mM EDTA, 0.1% Triton X-100 as in (CHASTON, '14)) with 125 μl Lysing Matrix D ceramic beads (MP Biomedicals 116540434) by shaking for 30-60 s at 4.0 M/S in a FastPrep-24, dilution plated onto mMRS medium, incubated under oxic and microoxic conditions, and visually inspected by colony morphology to confirm strain identity. If at least 200 CFU fly−1 of the expected bacterial strain were detected, the strain was deemed ‘present’ (see below for incorporation into statistical models). If at least 200 CFU fly−1 of an unexpected bacterial species were detected in 2 consecutive weeks, the vial was deemed contaminated. Differences between Acetobacter strains could usually not be determined by colony morphology, so Acetobacter contamination of other Acetobacter strains cannot be ruled out.
The lifespan analysis for flies bearing Escherichia coli mutants was conducted exactly as described above with one exception: because Escherichia coli persisted poorly in flies during the first lifespan experiment (see
Differences in fly lifespan with bacterial treatments were determined by a left- and right-censored Cox mixed-effects survival model in R (THERNEAU, '12, THERNEAU, '14) to account for differences in bacterial persistence in the flies. All flies entered the experiment at the time of egg transfer to sterile diet, and bacterial presence was indicated as a fixed effect with the value of “1”. If the F1 bacterial vials were uncontaminated and no bacteria were detected in two consecutive weeks, the bacteria-associated flies were marked to exit the experiment at the last day bacteria were detected in F1 vials (right-censored); at the same day, the same flies entered the experiment (left-censored) with a bacterial presence value of “0”. For F1 vials that were contaminated in 2 consecutive weeks, flies from the P vials were marked as leaving the experiment on the latest day that no contamination was detected. If the flies were contaminated from the first transfer onward, the entire vial was excluded from the analysis.
To predict bacterial genes that influenced lifespan, a meta-genome wide association (MGWA) approach was used, as in our previous work (CHASTON, '14). Amino acid sequences from each bacterial species used in the monoassociation experiments were obtained by whole-genome sequencing or from Genbank, and were clustered in orthologous groups (OGs) using a local installation of the OrthoMCL software with an inflation factor of 1.5. MGWA was performed using the MGWAR R package (SEXTON, '18). Differences in lifespan of the flies, relative to axenic flies, were determined by the right- and left-censored Cox mixed effects model described above. OGs were ranked according to p-value with an FDR-corrected p-value of ≤0.01 considered significant.
To identify functional categories that were enriched among the significantly-associated COGs a KEGG enrichment analysis was performed. KEGG categories were assigned to a representative sequence from each OG using BlastKOALA. The KEGG pathway assignments to each OG were retrieved in KEGG PATHWAY, and chi-square tests were performed to test for pathways that were enriched in the top 170 OGs relative to all 12980 clustered OGs. P-values were false-discovery rate (FDR) corrected for multiple tests. Chi-square tests and FDR correction were performed in R (CORE TEAM, '16).
The phylogenetic tree shown in
Expression of Klebsiella variicola CBS::CBL in A. fabarum DsW_054
Plasmid pAH1 was constructed by insertion of the 2.5 Kb CBS-CBL Klebsiella variicola operon [SEQ ID NO: 11] into expression vector pCM62. Genomic DNA was isolated from 1.5 ml of K variicola culture using the DNeasy Blood and Tissue Kit (Qiagen). The CBS-CGL fragment was amplified from with Pfx polymerase (Thermo Fisher Scientific) and the CBSCGL-xbaI-for (5′-NNNNtctagaATGTCACTGTTTCATTCC-3′ [SEQ ID NO: 12]; restriction enzymes in lowercase here and hereafter) and CGL-ecoRI-rev (5′-NNNNgaattcCGGAATAATCACTCCTCC-3′ [SEQ ID NO: 13]). The pCM62 plasmid was digested using Xbal and EcoRI (New England Biolabs) according to manufacturer's recommendations, and the CBS-CGL fragment was ligated into the plasmid using T4 DNA Ligase (New England Biolabs). The assembled pAH1 plasmids were then electroporated into Escherichia coli S17 λ-Pir, and selected on LB plates containing chlortetracycline at a concentration of 5 ng/mL. Successful cloning was initially screen by PCR across the polylinker, selecting for colonies with the 2.5 kB insert, and the sequence of the insert was verified in its entirety by Sanger sequencing at the BYU DNA Sequencing center using primers cbscgl-500 (5′-ACCACTTATTCAGCGAACC-3′ [SEQ ID NO: 14]), CBS_CGL-1000 (5′-GGAGGAACGATAATCGAAG-3′ [SEQ ID NO: 15]), CBS_CGL_1500 (5′-GATCCTGGCTGGTCGAAG-3′ [SEQ ID NO: 16]), CBS_CGL-2000 (5′-CCAACGCTTCTCCCTGCC-3′[ SEQ ID NO: 17]), CBS_CGL_2500 (5′-TGGATAAGGACAGTCACC-3′ [SEQ ID NO: 18]), and CBS_CGL_3000 (5′-GAAGTGGAGCGAGTCTGG-3′ [SEQ ID NO: 19]). This created plasmid pAH1 pAH1 was conjugated into Acetobacter fabarum DsW_054 as described previously, with minor changes (WHITE, '18). Briefly, S17 λ-Pir Escherichia coli containing pAH1 were grown overnight at 37° C. in LB-chlortetracycline, and A. fabarum DsW_054 was grown at 30° C. in potato medium. 500 μl of cells from each culture were centrifuged, the supernatants discarded, and each pellet was resuspended in 50 μl of potato medium. 50 μl of each cultured was mixed in microcentrifuge tube and incubated at 30° C. for 16 hours. The 100 μl mixture was then plated onto YPG medium (1.5% agar, 1% glycerol, 0.5% peptone, 0.5% yeast extract, 0.2% acetic acid and 20 mg/L chlortetracycline (CHASTON, '14). YPG plates containing transformed A. fabarum DsW_054 were incubated for 72 hours, after which colony PCR and plasmid isolation were performed to confirm the presence of the plasmid.
To study the effects that different bacterial species had on the lifespan of Drosophila melanogaster, we monoassociated the flies with different bacterial strains and measured fly lifespan. As controls we measured the lifespans of bacteria free flies and flies that were associated with a representative five-species bacterial community, as in our previous work (NEWELL, '14).
As shown in
Variation in lifespan effects of the different bacteria was specific for lifespan and was likely tied to specific bacterial functions. For example, the variation could not be attributed exclusively to developmental delays since lifespan varied over many days and microbial influence on development varies across less than 24 hours on the rich diet used in our experiments (see, e.g. (CHASTON, '14)). The effect of bacterial persistence was also independent of bacterial identity and, presumably, function. Frequent transfer of flies to fresh diets, as is necessary in lifespan experiments, can cause loss of the associated microbial communities in Drosophila (BLUM, '13). To test how bacteria persisted in the flies throughout our experiment and if it impacted fly lifespan, we measured the period over which bacteria were detected in Fl generation flies that hatched on spent media after P generation flies were transferred to fresh diet. There was wide variation in bacterial persistence in the flies with one Lactobacillus strain lost from flies after the first transfer, and numerous Acetobacter strains detectable in spent P generation vials for the entire experiment. When the period during which bacteria were transferred to spent P generation vials was included in a left- and right-censored survival model, the effect was significant. Additionally, there was a positive correlation between bacterial persistence and lifespan in Proteobacteria-associated flies, but not in flies bearing Firmicutes isolates or when the taxonomic groups were considered together, suggesting that the period during which bacteria were associated with the flies was associated with conspecific Acetobacter functions but was not a directly-influencing lifespan factor. Because the sampling of microbiota abundance is destructive, we were unable to determine if microbial load over time also contributed to the species-specific effects. Regardless, these findings together suggest that while the period during which bacteria are present can influence the lifespan effect, it is the identity of the persisting microbe to which the effect must be attributed. Given the varied influence of bacteria on Drosophila lifespan it may be possible that this finding will not be consistent in flies reared on other, possibly less nutritional, diets, where increases in bacterial load may be correlated with increased nutritional provisioning.
Identifying Bacterial Genes with Effects on Lifespan
To identify possible bacterial genes that influence Drosophila melanogaster lifespan, we performed a meta-genome wide association study. As shown in Table 2, 12 of the 12,980 OGs significantly reduced or extended lifespan. A hit was considered significant if the P value with the Bonferroni correction was less than or equal to 0.05. Within the top most significant OGs, genes involved in vitamin B2, vitamin B12, and methionine metabolism were detected. Visual inspection of other highly ranked genes revealed many genes with small p-values before correction for multiple tests that were associated with functions in vitamin B5, B6, and folate metabolism. To focus our study on classes of bacterial functions that were predicted to influence lifespan we performed a KEGG enrichment analysis, using all OGs with a false-discovery rate corrected p-value<0.01 (170 OGs total). As shown in Tables 2 and 3, two KEGG categories were significantly enriched among the top 170 OGs in the MGWA: glucagon signaling; and cysteine and methionine metabolism. Since glucagon signaling is an animal pathway and bacteria only bear homology to scattered genes in the pathway, we focused our remaining efforts on testing the hypothesis that microbial cysteine and methionine metabolism influences Drosophila melanogaster lifespan.
As a first test of the MGWA prediction that bacterial cysteine and methionine metabolism influence Drosophila melanogaster lifespan we performed a mutant analysis. We measured lifespan in Drosophila melanogaster that were monoassociated with Escherichia coli strains bearing mutations in methionine metabolism genes. The results from the measurements are shown in
On a nutritionally rich diet, associated microbes normally shorten Drosophila melanogaster lifespan (data not shown). To better understand the molecular basis for these effects we performed a screen to predict specific bacterial gene mutations that shorten or extend fruit fly lifespan, and validated predictions in fruit flies monoassociated with bacteria bearing mutations in the predicted genes. The mutant analysis identified mutations in the methionine cycle and transsulfuration that shorten (
To begin to understand why the pdxB and pdxK mutants shortened Drosophila lifespan, whereas the luxS mutants promoted longevity, we performed a global metabolomic analysis of flies or diets that had been monoassociated with the different mutants. When we compared the metabolomes of the flies bearing mutant versus wild-type Escherichia coli strains, we detected only 3 metabolites with significantly different abundant in the pdxB (methionine, ascorbate) and pcbcK (deoxyinosine) flies as shown in
Further, a transcriptomic analysis of flies bearing pdxK mutant or wild-type bacteria also revealed few differences (data not shown). Taken together, these findings suggest that the dramatic reduction in fly lifespan was not tied to generalized malaise in the flies; but to the small number of specific changes we detected. Similarly, a metabolomic analysis of diets on which flies were reared with the luxS mutant revealed few differences in the metabolite contents relative to diets with flies and wild-type bacteria (data not shown). As shown in
To test the idea that bacterial gene expression could be used to restrict dietary methionine in the flies, we monoassociated the flies with an A. fabarum strain that ectopically expressed transsulfuration genes CBS and CGL (+CBS:CGL). As shown in
Under normal circumstances as shown in
As shown in
As shown in
To create a strain of Lactobacillus that expresses CBS and CGL (ProL), we obtain and modify backbone integration vectors specific to Lactobacillus species. Since the current integration plasmids do not support constitutive expression, we modify the vectors to express the CBS and CGL genes behind the lactate dehydrogenase promoter (ldhL), a promoter that is constitutively active in Lactobacillus species (GEOFFROY, '00). We then clone the CBS and CGL genes into the newly constructed Lactobacillus integration vector and electroporate the vector into the Lactobacillus rhamnosus GG. We then perform Sanger sequencing to confirm the sequence and integration of the cloned genes. Together, these approaches create a recombinant strain of Lactobacillus rhamnosus GG that constitutively expresses the CBS and CGL genes stably from the chromosome. We also create a control strain to be used in all subsequent experiments by introducing the empty vector to Lactobacillus rhamnosus GG.
To test if ProL influences fruit fly lifespan we inoculate it to bacteria-free flies and measure their lifespan. To make mono-associated flies, Drosophila melanogaster eggs are surface-sterilized in bleach for 5 min., transferred to sterile diet in a sterile biosafety cabinet (˜30-50 eggs/vial), and are inoculated with the bacteria individually (NEWELL, '14, KOYLE, '16). Bacteria are cultured separately in mMRS liquid and solid media (NEWELL, '14), normalized to 0.1 OD600 and 50 μl are added to the sterile diet containing sterile eggs. To measure fruit fly lifespan, flies are transferred 2-3 times weekly to fresh diet. Diet transfers are frequent because the flies are actively mating and developing offspring affect diet quality. At each transfer the number and sex of dead flies is recorded. All work is done with sterile diets in a biosafety cabinet to ensure no contaminating microbes are introduced during transfer. Differences in fly lifespan are tested using a Cox mixed effects survival model (THERNEAU, '12, THERNEAU, '14) in R.
To determine whether ProL influences the Lactobacillus metabolome similarly to our recombinant Acetobacter strain, we measure the metabolomes of flies and their diets when inoculated with ProL versus control strains. At 7 and 30 days of age, 30 sex-separated flies are flash frozen on liquid nitrogen and undergo whole-metabolome analysis, together with samples from bacteria-free controls (48 samples total: 6 replicates for each of 2 ages, 2 sexes, 2 treatments). 30 mg of spent fly diet are flash-frozen and sent for analysis (24 samples total; 6 replicates, 2 ages, 2 treatments). ProL extends lifespan through methionine restriction since the methionine content of flies and diet is lower when inoculated with ProL versus the empty-vector control strain.
To determine the effect of a probiotic strain when inoculated to flies bearing an established microbial community, we inoculate flies with either the control or recombinant Acetobacter or Lactobacillus strains (4 treatments total: 2 test strains each with their own control), and measure their lifespan. At 5 and 30 days of age we collect diet and fly samples for metabolomic analysis as in our experiments above (144 samples total: 6 replicates for each of 2 ages, 2 sexes, 4 treatments+48 diet treatments). Further, we monitor persistence of ProL and the control throughout the experiment, and perform reinoculations on at least a weekly basis if either is lost from the flies over time. Any strain that acts as a probiotic extends the lifespan of bacteria-associated flies. Since there can be differences in how Acetobacter and Lactobacillus persist in the flies, we include the recombinant Acetobacter strain to control for differences in persistence on the probiotic effect.
Together, the approaches as described here create a candidate probiotic strain that has lifespan-extending effects in mammals. ProL functions similarly to the Acetobacter probiotic strain (
Bacteria can generally extend fly lifespan by methionine restriction and dietary methionine restrictions in mice extends mouse lifespans and alters the abundances of several serum biomarkers and other indicators of mouse health in 5-week old mice. Thus, bacteria extend mouse lifespans through methionine restriction which can be detected via methionine-restriction-like biomarkers in 8-week old mice fed the bacteria.
To presumptively assess if ProL administration mimics methionine restriction in mice, we feed ProL or the control strain (109 cfu) to each of 20 mice (5 mice per cage) in drinking water daily for 8 weeks while measuring methionine-restriction indicators on a weekly basis or at the end of the experiment as shown in Table 5 below. Table 5 shows Phenotypes to measure in probiotic-administered mice and controls. Over the indicated time periods these are methionine-restriction responsive phenotypes, as reported in (LEES, '14).
We use 3-week-old male C57BL/6J wild-type mice for these experiments. At the end of the experiment, blood samples are collected from each mouse, the mice are sacrificed, and liver and white adipose tissue are dissected from each animal within 1 hour of sacrifice. Serum, liver, and adipose biomarker levels are assessed in triplicate from each mouse sample as described previously (LEES, '14). Additionally, to determine gene expression in tissues that display distinctive methionine-restriction-like signatures, dissected tissues from the mice in each cage are pooled and gene expression in the sample analyzed by RNAseq at a depth of 40 million reads/sample (see also our published and current RNAseq work in Drosophila, e.g. (DOBSON, '16)), for a total of 5 replicates per treatment. Tissues analyzed are the liver and white adipose tissue. We collect fecal samples weekly to monitor the impact of the probiotic administration on the mouse microbiome on an individual mouse basis by a 16S rRNA marker gene survey as in our ongoing work (performed as described in (KOZICH, '13)).
ProL leads to methionine restriction in the mice since they eat more but weigh less, have greater glucose tolerance/insulin responsiveness, and have lower blood glucose, serum insulin, serum triglyceride, and liver triglyceride levels. Also, the bacteria persist and are abundant as indicated in 16S data. Taken together, these experiments reveal the probiotic potential of ProL.
While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention.
This application claims the benefit of U.S. Provisional Application No. 62/479,659 filed Mar. 31, 2017, the disclosure of which is incorporated in its entirety by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 62479659 | Mar 2017 | US |
