Patent Description:
The field of the invention is related to genetically modifying and selecting of gut microbes that have altered phenotypes and for use of these microbes in treating diseases.

Akkermansia muciniphilia are a bacteria found on the mucosal surface of the human intestinal track. This bacteria uses mucin as its single nutrient source. It accounts for <NUM>-<NUM>% of the intestinal bacteria in adults and is a species of bacteria that inhabits the large intestine. It is a gram-negative, obligate, anaerobic, non-motile, nonspore-forming elliptical eubacterium that is thought to be beneficial to the gut flora. However, Akkermansia has been found to be difficult to molecularly manipulate. The mechanisms by which Akkermansia physiologically influences the gut microbiome, mucosal and systemic immunity and glucose/lipid metabolism is not well understood. <NPL>) discloses parallel assessment of transposon delivery vectors in bacteria. <CIT> discloses systems and methods for genetic analysis of intractable microbes. <NPL>) discloses Akkermansia muciniphila gen. , a human intestinal mucin-degrading bacterium.

As such, there is a need for methods and systems for producing genetically altered Akkermansia strains to study its role in gut flora.

The present disclosure is based, in part, on the development of the inventors of a method to genetically alter Akkermansia bacteria using a transposon vector. Genetically altered Akkermansia bacteria and libraries of altered Akkermansia bacteria are also provided. Other aspects of the present disclosure are provided in all that is described and illustrated herein.

Specifically, in one aspect, the claimed invention provides a method of genetically altering Akkermansia bacteria, the method comprising: (a) introducing a exogenous transposon vector of SEQ ID NO:<NUM> into Akkermansia to produce a plurality of altered Akkermansia bacteria; and (b) culturing the plurality of altered Akkermansia to select for bacterium having incorporated the transposon of the vector into the genome to produce a plurality of genetically altered Akkermansia bacteria.

In another aspect, the claimed invention provides a genetically altered Akkermansia bacteria produced by the method described above. Herein the genetically altered Akkermansia bacteria genome contains the transposon (SEQ ID NO:<NUM>) of the transposon vector (SEQ ID NO:<NUM>).

In another aspect, the claimed invention provides a genetically altered Akkermansia bacteria produced by incorporating the transposon from vector of SEQ ID NO: <NUM> into an Akkermansia bacteria. In one example, the transposon is SEQ ID NO:<NUM> incorporated into the Akkermansia genome.

In another aspect, the claimed invention provides a library of altered Akkermansia bacteria, wherein the library is produced by the method described above.

Described, but not as such part of the claimed invention, is a method of selecting for an altered Akkermansia bacterium having an altered genetic regulator of a trait, the method comprising: (a) introducing an exogenous transposon vector of SEQ ID NO:<NUM> into a population of Akkermansia to randomly incorporate the transposon into the Akkermansia genome; (b) culturing the population Akkermansia to select for Akkermansia having the transposon integrated into their genome to produce a plurality of altered variants of Akkermansia; and (c) selecting for Akkermansia having the altered genetic regulator by culturing the Akkermansia under conditions in which the altered genetic trait is selected.

Described, but not as such part of the claimed invention, is a method of identifying novel genetic regulators of a trait in Akkermansia, the method comprising: (a) incorporating an exogenous transposon vector of SEQ ID NO:<NUM> into a population of Akkermansia to produce a population of altered Akkermansia incorporating the transposon into their genome; (b) culturing the Akkermansia in medium comprising chloramphenicol to select for Akkermansia having incorporated the exogenous transposon; (c) exposing the altered Akkermansia to two different conditions; and (d) analyzing by sequencing or PCR amplifying the genes disrupted by the transposon in the altered Akkermansia grown under the two different conditions to identify genes associate with the trait.

The foregoing and other aspects and advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there are shown, by way of illustration, preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

"About" is used to provide flexibility to a numerical range endpoint by providing that a given value may be "slightly above" or "slightly below" the endpoint without affecting the desired result. The term about as used herein refers to a range of +/- <NUM>% of the numerical value listed.

The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of and "consisting of" those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as <NUM>% to <NUM>%, it is intended that values such as <NUM>% to <NUM>%, <NUM>% to <NUM>%, or <NUM>% to <NUM>%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

As used herein, "treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. Optionally, the subject or patient is a human. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, mouse, rat, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. Optionally, the subject is a mouse, or a mouse model of a disease.

The present disclosure describes, in part, materials, systems and methods for the mutation and characterization of the gut microbe Akkermansia muciniphila and related species (including clinical strains) and uses thereof of the altered Akkermansia.

The present disclosure provides tools and methods to genetically manipulate the emerging pro-biotic Akkermansia muciniphila. The systems and methods allow for rapidly identifying factors related to a phenotypic trait, for example, factors mediating colonization of animals, including new enzymes mediating the breakdown of mucins and successful competition with other members of the microbiota. The systems allow for the generation of altered strains that are better suited as immunomodulators of chronic inflammation and with enhanced properties as protectors against diet-induced obesity and boosters of cancer immunotherapies.

The inventors have developed a method to genetically modify Akkermansia muciniphila that incorporates transposon mutagenesis (insertion of a transposon from a vector into the Akkermansia genome), phenotype analysis and PCR or sequenced-based mutational mapping to identify novel genetic regulators in Akkermansia found in the human microbiome in the intestine.

Akkermansia muciniphila is a Gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval-shaped bacterium found in the gut microbiome. Prior to this disclosure A. muciniphila was not known to be amenable to molecular genetic manipulation. They are known to process mucin, a glycosylated protein important in luminal protection of the gastrointestinal track.

The availability of carbohydrates in the gastrointestinal track plays a role in shaping the structure-function of the microbiota and determination of which microbes can grow and colonize the intestinal track. Utilization of microbes in promoting health is relies on the ability to colonize useful bacteria within the gut to maintain a healthy flora. There is still little known about the structural requirements for mucin degradation by gut bacteria and the limited functional characterization of enzymes that correlate with the strains able to degrade and utilize mucin and mucin glycans. Mucin is a large, highly glycosylated proteins. The present disclosure provides methods of making altered variants of Akkermansia, populations of altered Akkermansia, and use of the altered Akkermansia to colonize the colon and promote health in a subject.

The present invention provides a method of genetically altering Akkermansia bacteria, the method comprising: (a) introducing an exogenous transposon vector of SEQ ID NO:<NUM> into Akkermansia to produce a plurality of altered Akkermansia bacteria (comprising the transposon); and (b) culturing the plurality of altered Akkermansia to select for bacteria having incorporated the transposon into the genome to produce a plurality of genetically altered Akkermansia bacteria.

Methods of introducing an exogenous transposon vector of SEQ ID NO:<NUM> into Akkermansia to produce a plurality of altered Akkermansia bacteria are known in the art. These methods are generally referred to as transposon mutagenesis or transposition mutagenesis and allows for genes to be transferred to a host organism's chromosome, interrupting or modifying the function of the extant gene on the chromosome and causing mutation. This allows for the ability to induce single hit mutations within a genome, and the ability to identify the gene that has been mutagenized by being able to identify the adjacent sequences to the transposon. The transposon vector of SEQ ID NO:<NUM> has been specifically designed for use in Akkermansia as described in Example <NUM>. The vector of SEQ ID NO:<NUM> contains a modified mariner transposon, himar1C9, with a chloramphenicol resistance cassette (cat) and the transposase enzyme required to catalyze transposition. The vector further comprises lamba pir dependent origin of replication and is unable to replicate in strains such as Akkermansia that lack pir genes.

The transposon vector (SEQ ID NO:<NUM>) comprises the transposon (nucleic acids <NUM>-<NUM> of SEQ ID NO:<NUM> (e.g. SEQ ID NO:<NUM>)) and the transposase enzyme. The transposase enzyme is required for extracting and inserting the transposon into the Akkermansia genome. Once transposition occurs, the transposon (SEQ ID NO:<NUM> (nucleic acids <NUM>-<NUM> of SEQ ID NO:<NUM>)) is inserted into the genome of the altered Akkermansia strain. Thus, the altered Akkermansia strain/variants comprises the transposon of SEQ ID NO:<NUM>. In other words, the altered Akkermansia strain/variants comprises SEQ ID NO:<NUM> within its genome, but does not contain the rest of the transposon vector backbone.

The transposon vector contains the antibiotic resistance gene for chloramphenicol (cat) within the transposon, which was required for the use in Akkermansia, as prior vectors that used erythromycin as the antibiotic selection did not work in Akkermansia and resulted in spontaneous resistance.

One method of introducing the transposon vector into Akkermansia is by conjugation. Methods of conjugation are known in the art and for example, but not limited to, the method as described in Example <NUM>. Bacterial conjugation is the transfer of genetic material (e.g., the exogenous transposon vector of SEQ ID NO:<NUM>) between bacterial cells by direct cell-to-cell. In a preferred embodiment, the transposon vector is conjugated from an E. coli strain to the Akkermansia. The method of conjugating includes co-culturing the E. coli strain carrying the transposon vector (e.g., SEQ ID NO:<NUM>) with Akkermansia under aerobic conditions for about <NUM>-<NUM> hours at <NUM>.

Following conjugation, to counter select against E. coli and allow transposition to occur, the transconjugates were sub-cultured. Suitable methods of subculturing are known in the art. For example, as described in Example <NUM>, the transconjugates are subcultured multiple times, for example <NUM> times, under anaerobic conditions. This allowed for the selection for the altered (variant strains) of Akkermansia that have incorporated the transposon (e.g. SEQ ID NO:<NUM> corresponding to nucleic acids <NUM>-<NUM> of SEQ ID NO:<NUM>) into their genome from the other bacterial strains in the culture. This subculturing conditions are anaerobic conditions, and include a sub-culturing step in medium comprising chloramphenicol, the antibiotic resistance gene that is included in the transposon sequence.

Once subcultured, a population of altered Akkermansia comprising the transposon (including the antibiotic resistance gene) in their genome are produced. This population of altered Akkermansia can be grown and used as a library of altered Akkermansia for screening and treatment purposes. The library of altered (mutant variants of) Akkermansia can be used to screen for phenotypic traits. For example, in one embodiment, the library can be grown under conditions related to the phenotypic trait and screened to identify genes associated with the phenotypic trait.

The library of altered Akkermansia stains can also be used to characterize each altered Akkermansia strain by DNA sequencing or PCR analysis of the genomic sequence adjacent to the transposon inserted into the genome. This allows for determination of which gene has been altered by the insertion of the transposon.

In one embodiment, the method further comprises: culturing the plurality of genetically altered Akkermansia under conditions to select for a trait; and optionally identifying the gene associated with the trait by PCR or sequencing of the gene adjacent to the transposon within the Akkermansia genome.

Methods of DNA sequencing to identify the genes disrupted by the transposon are known in the art. For example, in one embodiment, the DNA sequencing method used can be INSeq/TnSeq as described in <NPL>), doi:<NUM>/nprot2011. Briefly, Insertion Sequencing (INSeq) is a method for determining the insertion site and abundance of transposon mutants in a mixed population using a modified mariner transposon containing MmeI sites at its ends, allowing for the cleavage at chromosomal sites <NUM>-<NUM> bp from the inserted transposon. See Goodman et al. Genomic regions that are adjacent to the transposons are amplified by linear PCR, and sequenced using a high-throughput instrument as described in Goodman.

The present disclosure contemplates a library of genetically altered Akkermansia, specifically, a library of genetically altered Akkermansia muciniphila. Further, libraries of genetically altered Akkermansia made from clinical strains of Akkermansia (e.g. strains isolated from a patient, for example, but not limited to, an obese patient, patient with chronic inflammation, among others). The library can be used for screening and culturing of altered Akkermansia that play a role in phenotypic traits.

In another embodiment, the disclosure provides a library of altered Akkermansia bacteria produced by the claimed method.

As used herein, the terms "altered Akkermansia," "altered Akkermansia strain", "genetically altered Akkermansia,", "variants of Akkermansia," "Tn mutant Akkermansia," "Tn mutants," and "mutant Akkermansia" are used interchangeably to refer to the genetically modified Akkermansia that have incorporated the transposon of the transposon vector of SEQ ID NO:<NUM> into their genome. Tn mutants refer to mutant strains made by insertion of a transposon (Tn) as noted in the art. The Akkermansia may be any known species of Akkermansia that falls within the genus, including, but not limited to, for example, Akkermansia muciniphila or clinical species isolated from patients.

For example, the library of altered Akkermansia strains can be used in method of screening for genes required for the utilization of mucin. In some embodiments, a library of genetically altered Akkermansia are cultured in medium with or without mucin. The library grown without mucin can be compared genetically to the library grown in medium containing mucin. Methods of genetically analyzing the genes in the altered Akkermansia grown in the presence of mucin and the genes in altered Akkermansia grown in the absence of mucin can be determined by sequence or PCR analysis, as detailed herein, and the genes from the two populations compared to identify genes that regulate mucin utilization. This is demonstrated in Example <NUM> herein.

In some embodiments, altered Akkermansia strain that has advantageous growth characteristics in the presence of mucin can be identified. These altered Akkermansia with advantageous growth characteristics can be used to colonize a subject's colon by administering the altered Akkermansia to the subject.

Suitable methods of administering the altered Akkermansia strains to a subject are known in the art, and include, administering the altered Akkermansia orally, rectally, or other routes that maintain the viability of the bacteria. In some embodiments, the altered stain can be administered orally, for example, but not limited to, in tablets, capsules, liquids, etc. that allow for delivery of the strains to the intestinal track. Suitably, the altered strains may be formulated into a composition that allows for the strain to maintain viability while being delivered to the intestinal track.

In another embodiment, the library of altered Akkermansia can be screened for phenotypic traits associated with stable colonization of the intestine. In some embodiments, the method comprises: introducing the plurality of genetically altered Akkermansia into a subject, and detecting the altered Akkermansia that have a growth advantage colonizing the intestine of the subject by genetically analyzing the genes in the altered bacteria growing within the colon of the subject. In some embodiments, the bacteria that are colonizing the intestine of the subject are obtained in a sample from the colon of the subject, culturing the sample under conditions suitable for growth of the altered Akkermansia (e.g. anaerobic conditions in the presence of chloramphenicol to select for Akkermansia with the transposon), and identifying the genes associated with the growth advantage by sequencing or PCR analysis of the altered Akkermansia strains grown. Suitably, DNA sequencing or PCR methods used to determine genes associated are specific to the transposon (e.g. use primers specific to the exogenous transposon) and thus allow for identification of the genetically altered Akkermansia strains that have the transposon as opposed to any wildtype bacteria that may be growing within the gut. In some embodiments, the subject is a mouse. In some embodiments, the subject is a mouse model of a disease (e.g., obesity mouse model, etc.).

The methods described herein can be used in methods of screening for other phenotypical traits. For example, the Tn/IN-seq to screen for variants with enhanced colonization under various diets/disease conditions. This can be done by feeding the subject a specific diet, and screening for the ability of the altered bacteria to colonize the colon under the specific diet conditions or under immune-status alteration. This can identify altered bacteria that have genes that specifically breakdown and utility the components of the diet.

In another embodiment, the methods described herein can be used to screen for genes involved in phenotypes that could affect colonization and interaction with host, for example biofilm formation, aggregation, capsule production, IgA binding, and resistance to antimicrobial peptides or bile salts. In some examples, the phenotype trait may be, for example, but not limited to, amino acid biosynthesis, carbohydrate metabolism, nutrient uptake, redox tolerance, adherence, invasion, growth, reproduction, and the like. A trait can include a genetically-determined characteristic that is important for the overall growth and survival of that bacteria, such as the ability to colonize the host intestine. For example, as demonstrated in the examples, some genetic regulators of Akkermansia growth include genes that are required for utilization of mucin, e.g., genes found in Table <NUM>. Other genes necessary for the growth and colonization of Akkermansia in the colon including the distal colon of a subject are found in Table <NUM>. The present disclosure is not limited to these genes as these are exemplary of what can be identified by the methods described herein.

In a further embodiment, the mutants can be screened for genes involved in activating host signaling pathways, for example the TLR2 signaling pathways that have been linked to intestinal health and prevention of obesity. For example, in one embodiment, the method involves screening for altered Akkermansia strains that have different levels of TLR2-mediated recognition by immune cells, or in another embodiment, administering the altered Akkermansia to a subject normal and obese subject (e.g., normal and obese mouse model) both being fed the same diet (e.g. normal or high fat), and screening for the genes associated with the bacteria within the obese subject compared to the non-obese subject.

Described, but not as such part of the claimed invention, is a method of selecting for an altered Akkermansia bacterium having an altered genetic regulator of a trait, the method comprising: (a) introducing an exogenous transposon vector of SEQ ID NO:<NUM> into a population of Akkermansia to randomly incorporate the transposon into the Akkermansia genome; (b) culturing the population Akkermansia to select for Akkermansia having the transposon integrated into their genome to produce a plurality of altered variants of Akkermansia; and (c) selecting for Akkermansia having the altered genetic regulator by culturing the Akkermansia under conditions in which the altered genetic trait is selected. In some aspects, step (a) comprises conjugation of the Akkermansia with E. coli containing the transposon vector of SEQ ID NO:<NUM>. In further aspects, the methods of step (b) comprises subculturing the bacteria under anaerobic conditions to select for the altered variants of Akkermansia in the presence of chloramphenicol. Methods of determining the altered genetic regulator can be done by methods known in the art, including, but not limited to, sequencing (e.g., but not limited tom INSeq/TnSeq described in Goodman et al. <NUM>), or PCR analysis of the genome adjacent to the transposon element.

Described, but not as such part of the claimed invention, is a system that comprises a discovery platform using a transposon vector for genetically manipulating Akkermansia bacteria, including the probiotic Akkermansia muciniphila for the treatment of chronic inflammation in a subject.

Described, but not as such part of the claimed invention, is genetically manipulating the probiotic Akkermansia muciniphila for the treatment of treatment of diet-induced obesity in a subject.

The genetically altered Akkermansia can be used to boost immune checkpoint inhibitors in cancer immunotherapies. This can be done by administering an effective amount of the altered Akkermansia (e.g., altered Akkermansia muciniphila) in a subject undergoing checkpoint inhibitor therapy to enhance the anti-cancer properties of the checkpoint inhibitor (See, e.g., <NPL>)).

Methods of enhancing checkpoint inhibitor therapy are contemplated (but do not form part of the claimed invention). The methods comprising administering an effective amount of the altered Akkermansia (e.g., altered Akkermansia muciniphila) in a subject undergoing checkpoint inhibitor therapy to enhance the anti-cancer properties of the checkpoint inhibitor. For example, the altered Akkermansia (e.g., altered Akkermansia muciniphila) can be used to increase the efficacy of PD-<NUM> based immunotherapies (e.g., PD-<NUM> antibody (i.e., pembrolizumab, nivolumab, cemiplimab, etc. which are commercially available, for example, pembrolizumab, and anti-PD-<NUM> antibody, available from Merck and Co and described in <CIT>, <CIT>, <CIT> and <CIT>; nivolumab, an anti-PD-<NUM> antibody, available from Bristol-Myers Squibb Co and described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and<CIT>).

Methods of treating diet-induced obesity are contemplated (but do not form part of the claimed invention). The methods comprise administering an effective amount of an altered Akkermansia strain to the subject in order to treat the diet-induced obesity. The altered Akkermansia strain can have an altered gene selected from Table <NUM>.

Methods of treating an inflammatory disorder are contemplated (but do not form part of the claimed invention), the method comprising administering an effective amount of an altered Akkermansia strain to treat the inflammatory disorder.

Described, but not as such part of the claimed invention, is using the systems and methods described herein for genetically manipulating the emerging pro-biotic Akkermansia muciniphila to generate strains that are better suited as immunomodulators of chronic inflammation and with enhanced properties as protectors against diet-induced obesity.

The present disclosure also contemplates a genetically altered Akkermansia bacteria containing the transposon from SEQ ID NO: <NUM>.

In another aspect, the present disclosure contemplates a genetically altered Akkermansia bacteria which has a disruption of any one of the genes listed in Table <NUM>. These genetically altered Akkermansia bacteria have genes altered in the ability to utilize mucin.

In another embodiment, the present disclosure contemplates a genetically altered Akkermansia bacteria has disruption of any one of the genes listed in Table <NUM>. These genetically altered Akkermansia bacteria have genes required for the utilization of mucin. In another embodiment, the present disclosure contemplates a genetically altered Akkermansia bacteria has disruption of any one of the genes listed in Table <NUM>. These genetically altered Akkermansia bacteria have genes that provide a growth advantage in colonizing the colon of a subject. Methods of using any of the contemplated genetically altered Akkermansia bacteria having one or more of the genes listed in the table disrupted is contemplated for use in administering to a subject (but do not form part of the claimed invention).

Described, but not as such part of the claimed invention, are kits for carrying out the methods described herein are provided. The kits may contain the necessary components with which to carry out one or more of the above-noted methods.

The kit can comprise the vector of SEQ ID NO:<NUM> and instructions for transposition within a bacteria. Optionally, the kit comprises instructions on how to isolate and alter a strain of Akkermansia, including, but not limited to, Akkermansia muciniphila or a related species, including clinical strains.

The kit can comprise an altered Akkermansia muciniphila strain comprising the transposon of SEQ ID NO:<NUM>.

It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the scope of the appended claims. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. The term "consisting essentially of" and "consisting of" should be interpreted in line with the MPEP and relevant Federal Circuit interpretation. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. "Consisting of" is a closed term that excludes any element, step or ingredient not specified in the claim. For example, with regard to sequences "consisting of" refers to the sequence listed in the SEQ ID NO. and does refer to larger sequences that may contain the SEQ ID as a portion thereof.

The systems and methods provided herein have many commercially important biological from microbial communities associated with humans, livestock and industrial settings.

The invention will be more fully understood upon consideration of the following non-limiting examples.

To be able to do genetic screening for genes and altered bacteria with altered phenotypes, a tool for mutating the bacteria was necessary. A modified version of a previously described vector was generated for use in Akkermansia. The original vector, pSAM_Bt<NUM>, was designed for use in Bacteroides thetaiotaomicron. The vector encodes both a modified mariner transposon, himar1C9, with an erythromycin resistance gene and the transposase enzyme required to catalyze transposition. The plasmid uses a lamba pir dependent origin of replication and is unable to replicate in strains such as Akkermansia that lack pir genes.

To make pSAM_Bt compatible for use in Akkermansia, the original erythromycin resistance marker on the transposon (ermG) was replaced with a chloramphenicol resistance cassette (cat). Initial attempts to use erythromycin as a selection marker in Akkermansia were unsuccessful and growth with erythromycin repeatedly resulted in spontaneous resistance. The transposase enzyme was then codon optimized for expression in Akkermansia. We generated an Akkermansia codon table by concatenating a series of housekeeping genes to make a <NUM><NUM> bp sequence to use as an input for codon analysis. Rare codons in the himar1C9 sequence were replaced codons used preferentially in Akkermansia. The resulting plasmid was named pSAM_Akk (<FIG>, SEQ ID NO:<NUM>). We found that these alterations to the vector were essential for mutagenesis in Akkermansia, and alteration of the resistance marker or the transposase alone was insufficient for transposition to occur. Similarly, we have not had success using Akkermansia promoters to drive the expression of himar1C9. As such, pSAM-Akk vector has been specifically made to allow for mutagenesis of Akkermansia muciniphila.

The transposon vector (SEQ ID NO:<NUM>) was introduced into Akkermansia by conjugation with an E. coli donor strain. Akkermansia starter cultures sub-cultured <NUM>:<NUM> into <NUM> synthetic medium<NUM> and grown to OD600 = <NUM> - <NUM>. The cells were then harvested by centrifugation in <NUM> tubes at <NUM><NUM> xg, <NUM>, <NUM>. In parallel, E. coli S17 pSAM_Akk cultures were grown aerobically in LB + <NUM> ug/ml ampicillin, <NUM> ug/ml chloramphenicol at <NUM> <NUM> rpm to an optical density (OD) OD600 = <NUM> - <NUM>. To avoid shearing conjugation pili, E. coli was centrifuged at <NUM><NUM> xg, <NUM>, and washed once with sterile PBS. coli and Akkermansia pellets were combined in a total volume of <NUM> in synthetic medium and the suspension was used to make <NUM>µl puddles on pre-reduced synthetic medium plates. The plates were incubated aerobically at <NUM> for <NUM> - <NUM> depending on the Akkermansia strain. Aerobic incubation is critical for successful conjugation.

Following conjugation, the plates were transferred to an anaerobic chamber and the cells were scraped into <NUM> of a <NUM>:<NUM> mix of PBS and <NUM>% glycerol (glycerol is optional but required to store cultures at -<NUM>). To counter select against E. coli and allow transposition to occur, the transconjugants were sub-cultured three times. A <NUM>µl aliquot of the cell suspension was used to inoculate <NUM> synthetic medium with <NUM>µg/ml kanamycin and <NUM>µg/ml gentamicin and incubated at <NUM>, anaerobic, <NUM>. The culture was then sub cultured two more times as described above at <NUM> intervals. These sub-culturing steps are required to cure the plasmid and obtain transposon mutants (<FIG>). After the third round of sub-culturing, <NUM> - <NUM>µl of culture was spread on synthetic medium agar plates supplemented with <NUM>µg/ml gentamicin, <NUM>µg/ml kanamycin, and <NUM>µg/ml chloramphenicol, and incubated anaerobically at <NUM> for <NUM> days. This medium is required to inhibit the growth of residual E. Once transconjugants have grown, single colonies were picked with a pipette tip and arrayed into <NUM>-well plates containing with <NUM>µl per well synthetic medium with <NUM>µg/ml gentamicin, <NUM>µg/ml kanamycin, and <NUM>µg/ml chloramphenicol and incubated anaerobically at <NUM> for <NUM> days.

To confirm that the transposon had inserted into the genome, PCR for the β-lactamase gene was used to test for the absence of the plasmid backbone (bla) and for the presence of the transposon (cat). Finally, a Southern blot was performed on a subset of mutants to confirm the Tn insertion had occurred as aa single insert and at multiple locations in the genome (<FIG>).

To screen for genes required for mucin utilization, arrayed Tn mutants were used to inoculate duplicate <NUM>-well plates containing either mucin medium<NUM> or synthetic medium. The plates were incubated anaerobically at <NUM> for <NUM> days. Following growth, the OD600 was measured using a plate reader. Mutants that grew in synthetic medium, but not in mucin, were selected for additional characterization. To confirm the initial screen, mutants of interest were tested for mucin growth defects by running growth curve assays in a plate reader, taking measurements every <NUM> for <NUM> (<FIG>). Arbitrary PCR was used to locate the transposon insert sites and to identify the genes required for growth on mucin. The screen led to the identification of genes specifically required for growth on mucin, but not on monosaccharides (Table <NUM>).

A second approach used to screen the Tn mutants was to create a large pooled library for use in for transposon insertion sequencing (Tn/IN-seq)<NUM>. This method identifies conditionally essential genes by passaging large pools of mutants though various conditions and subsequently using next-generation sequencing to test the abundance of each mutant in the input and output pools. Genes required for survival in the specific conditions will be depleted from the input pool. We used Tn/IN-seq to identify genes required for colonization of the mouse intestinal tract.

To create the pooled library, equal volumes of the arrayed Tn mutants were pooled into a single suspension. The cell suspension was diluted <NUM>:<NUM> into synthetic media and incubated anaerobically at <NUM> for <NUM> (this growth step is optional). The cultures were then washed once with sterile PBS and concentrated <NUM>-fold, for a final concentration of approximately <NUM><NUM> CFU/ml. The cell suspension was used to gavage germ free C57BL/<NUM> mice with ~ <NUM><NUM> CFU. After one week of colonization, the mice were euthanized and the cecal contents were collected for DNA isolation. The DNA was then used to prepare sequencing libraries following the protocol described by Goodman et al. , with a modified primer set to allow for sequencing on Illumina's Hiseq <NUM> platform.

Analysis of the Tn/IN-seq data identified genes required for stable colonization of the intestinal tract (<FIG>). Genes required for colonization included putative components of the type II secretion system, type IV pili proteins, and glycosyl hydrolases among others (Table <NUM>). Conversely, inactivation of certain genes led to an increased abundance, suggesting that the library could potentially be screen for hypercolonizing variants (Table <NUM>). In addition, mutants unable to grow on mucin were dramatically depleted from the population, confirming that growth on mucin is occurring in vivo and that it is important for Akkermansia colonization.

All sequences associated with NCBI protein and gene accession numbers found in the tables can be found at www. gov/ [ncbi. The genomic sequence of Akkermansia can be found under accession number: NC_010655.

Intestinal samples from mice as described in example <NUM> were taken, sectioned and stained with antibodies against mucin and Akkermansia. As shown in <FIG>, Akkermansia is very closely associated with the mucin layer within the intestinal tract.

Further, the ability of wild-type Akkermansia or mutant Amuc_0544 was also examined in mice. Sections of the proximal and distal colon were obtained, sectioned and stained for mucin or Akkermansia. As demonstrated in <FIG>, while both wt and mutant Akkermansia were able to colonize the proximal colon, the gene Amuc-<NUM> was required for the colonization of the distal colon in mice.

Claim 1:
A method of genetically altering Akkermansia bacteria, the method comprising:
(a) introducing a exogenous transposon vector of SEQ ID NO:<NUM> into Akkermansia to produce a plurality of altered Akkermansia bacteria; and
(b) culturing the plurality of altered Akkermansia to select for bacterium having incorporated the transposon of the vector into the genome to produce a plurality of genetically altered Akkermansia bacteria.