Spore representation method

Abstract

The present invention provides artificial bacterial spores comprising a cargo protein bound to a spore coat protein. Also provided are methods for producing the bacteria and the spores, and methods for delivering a cargo protein to a subject using the spores.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Application No. 63 / 351,606, filed on June 13, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] [Sequence Listing] This application includes an XML - formatted sequence listing entitled "166118.01328_ST26.xml". This is 34,921 bytes and was created on June 13, 2023. The sequence listing was electronically submitted with this application via the Patent Center and is hereby incorporated by reference in its entirety.

Background Art

[0003] Spores are a very stable phenotype of spore - forming microorganisms such as Bacillus subtilis. Spores have strong resistance to heat, pH, proteases, and solvent challenges and can be used as protein carriers in many potential applications such as vaccines, probiotic delivery, bioremediation, enzyme immobilization, and bioprocesses. Spore display is a technique for loading a protein of interest into or onto the surface of a spore. This is achieved by binding the protein of interest to a spore coat protein and fixing it to the spore. There are at least 44 Bacillus subtilis spore coat proteins that may function as anchor proteins for spore display, but only 12 of these proteins have been used for enzyme display. Therefore, to improve the modularity of the Bacillus subtilis spore display method, it is necessary to identify additional anchor proteins and display strategies.

Summary of the Invention

[0004] In a first aspect, the present disclosure provides a bacterial spore comprising a cargo protein bound to a spore coat protein.

[0005] In a second aspect, the present disclosure provides a bacterium that produces the spores described herein.

[0006] In a third aspect, the present disclosure provides a method for producing the spores described herein. In some embodiments, the method includes culturing a bacterium that expresses a fusion protein comprising a spore coat protein and a cargo protein in a sporulation medium.

[0007] In other embodiments, the method includes culturing a bacterium that expresses both (i) a spore coat protein fused to a first protein tag and (ii) a cargo protein fused to a second protein tag that specifically binds to the first protein tag in a sporulation medium.

[0008] In further embodiments, the method includes (a) culturing a first bacterium that expresses a spore coat protein fused to a first protein tag in a sporulation medium, (b) culturing a second bacterium that expresses a cargo protein fused to a second protein tag that specifically binds to the first protein tag, (c) isolating the cargo protein produced by the second bacterium in step b, and (d) combining the spores produced by the first bacterium in step a with the cargo protein separated from the second bacterium in step c.

[0009] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the modified spores described herein.

[0010] In a fifth aspect, the present disclosure provides a method for delivering a cargo protein to a subject in need thereof. The method includes administering the pharmaceutical composition described herein to the subject.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] [Detailed Description] The present invention provides artificial bacterial spores comprising a cargo protein bound to a spore coat protein. Also provided are methods for producing bacteria and spores, and methods for delivering a cargo protein to a subject using the spores.

[0013] Bacterial spores can be used as protein carriers in various applications because they have strong resistance to heat, pH, proteases, and other stability challenges. The Bacillus subtilis spore coat is composed of more than 44 known spore coat proteins, all of which may function as anchor proteins for spore display. However, only 12 of these spore coat proteins have been used for enzyme display. Therefore, to identify novel anchor proteins, the inventors tested whether 44 known Bacillus subtilis spore coat proteins could be used for the display of the enzyme beta-glucuronidase (GusA). From this screening, in addition to the 12 previously used ones, 13 novel spore coat proteins that can be used as anchor proteins for enzymes were identified.

[0014] The newly identified anchor proteins may each offer different advantages for specific applications. For example, some of these anchor proteins are excellent at stabilizing enzymes and may thus be suitable for promoting catalysis, while other anchor proteins may be suitable for use as vaccines because they increase the availability of antigen surfaces. Thus, by increasing the number of known Bacillus subtilis anchor proteins, the inventors expanded the protein toolbox available for spore display and increased the likelihood that anchor proteins suitable for specific applications would be available. The inventors tested the ability of a subset of the identified Bacillus subtilis anchor proteins to stabilize conjugated enzymes against harsh conditions (such as high temperature and extreme pH). Their data suggest that the spore coat protein SscA is an excellent display anchor equal to or better than commonly used anchor proteins (such as CotB, CotC, CotG, CotX, CotY, and CotZ).

[0015] Conventional spore display methods rely on directly fusing cargo proteins to spore coat proteins. For these methods to function, the resulting fusion protein needs to be appropriately expressed, folded, and loaded into spores during sporulation. However, fusion proteins are often misfolded, and even properly folded fusion proteins can interfere with spore assembly, prevent cargo protein loading, or weaken the spore structure. These problems become more severe as the size of the protein increases. Therefore, these methods may not function when the cargo protein is a large protein such as a multimeric enzyme or a large antibody.

[0016] To avoid this problem, the inventors designed a spore display system that does not rely on direct fusion of cargo proteins to anchor proteins. Instead, their system uses pairs of protein tags (e.g., SpyCatcher / SpyTag) that spontaneously form covalent bonds, allowing the cargo protein to fold independently of the anchor protein before being loaded into the spore. Their cargo loading system is advantageous because (1) it can functionally load more proteins into spores and (2) it can shorten the length of the design, construction, and test cycle of spore display.

[0017] B. subtilis spore display is useful for various applications, such as a means of delivering therapeutic proteins and enzyme immobilization strategies for use in synthetic biochemistry platforms. These applications have conventionally relied on the use of recombinant production organisms (such as E. coli). However, in some applications, there is a risk of recombinant genetic material leaking into the environment. Recombinant genetic material can potentially provide new functions to organisms and lead to unintended and potentially tragic consequences (such as the spread of antibiotic resistance genes). Proteins are essentially "non-hereditary" in that organisms do not have the genetic material necessary to create copies of the proteins. Therefore, for safety reasons, proteins can be used after being purified from the genetic material that encodes them (such as in cell-free systems). However, such methods pose challenges in both the storage and delivery of proteins due to protein instability. Spore display is known to confer stability to covalently attached proteins, extend shelf life, and enhance robustness against stability challenges (such as extreme pH, temperature fluctuations, solvents, detergents). Spore display simplifies the protein purification process because spores can be easily separated and concentrated by centrifugation and / or filtration. Furthermore, gene-free spores (i.e., spores that deliver cargo proteins without the genetic material encoding those proteins) can be created by translocating cargo proteins generated by another cell to the spore surface, eliminating the risks associated with the leakage of potentially harmful recombinant genetic material.

[0018] (Spore:) In a first aspect, the invention provides a bacterial spore comprising a cargo protein bound to a spore coat protein.

[0019] "Bacterial spores" or "spores" are metabolically inactive, stripped-down forms that certain bacteria in the phylum Bacillota can reduce themselves to. When spores are formed inside cells, they may be called "endospores". Spores are highly resistant to harsh conditions (such as high temperature, radiation, dryness, chemicals, etc.), so bacteria can remain dormant for long periods, sometimes for centuries, by means of spores. Spore formation is usually triggered by nutrient deprivation. When a bacterium begins the process of forming a spore, the spore divides asymmetrically to form a larger mother cell and a smaller forespore. The forespore is engulfed by the mother cell and then undergoes dehydration and maturation to become a spore. Finally, the mother cell is destroyed by programmed cell death, and the spore is released into the environment. When the environment becomes more favorable, the spore can reactivate into a vegetative state.

[0020] The artificial spores of the present invention are designed for use in spore display. "Spore display" is a technique for loading a protein of interest into or onto the surface of a spore. Spore display can be used to stabilize and / or deliver a cargo protein. As used herein, the term "cargo protein" refers to a protein that is bound to a spore. In some embodiments, the cargo protein is an enzyme, a pharmaceutical, an antibody, a small molecule, or another genetically encoded molecule. As used herein, an "enzyme" is a protein that functions as a catalyst (i.e., increases the rate of a specific biochemical reaction). Examples of enzymes suitable for use as cargo proteins include cellulase (e.g., cellulase from Clostridium cellulovorans), dehydrogenase (e.g., xylose reductase from Neurospora crassa, phosphite dehydrogenase from Pseudomonas stutzeri, formate dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, acetaldehyde dehydrogenase, aldehyde dehydrogenase), agarase (e.g., agarase from Pseudomonas vesicularis, agarase from Zobellia galactanivorans), lyase (e.g., phenylalanine ammonia lyase from Anabaena variabilis), isomerase (e.g., L - arabinose isomerase from Lactobcillus sakei), antibiotic degrading enzyme (e.g., beta - lactamase from Escherichia coli, broad - spectrum beta - lactamase, macrolide esterase, N - acetyltransferase, O - adenyltransferase, chloramphenicol acetyltransferase, AAC(6’)-Ib - cr acetyltransferase), phosphorylase (e.g., trehalose phosphorylase from Thermoanaerobacter brockii, sucrose phosphorylase from Bifidobacterium adolescentis), decarboxylase (e.g., malonate decarboxylase and its subunits from Geobacillus stearothermophilus), and plastic - degrading enzyme (e.g., PETase), among others, but not limited to these. Other suitable enzymes are also contemplated.

[0021] Cargo proteins attach to the spore coat of spores through binding to coat proteins. The "spore coat" is a proteinaceous shell that encloses the genomic material of bacterial spores and plays a major role in their survival. As used herein, the term "coat protein" refers to any protein capable of forming part of the spore coat. As described in Example 1, the inventors tested 44 known B. subtilis coat proteins for their ability to function as anchor proteins for spore display. As used herein, an "anchor protein" or "coat anchor protein" is a coat protein that can bind cargo proteins without inhibiting the ability to be loaded onto spores. The inventors identified 26 functional anchor proteins from the screening, 13 of which were novel (i.e., had not previously been shown to function as anchor proteins). Thus, in some embodiments, the coat protein is selected from the list of 26 functional anchor proteins shown in Table 2. In a preferred embodiment, the coat protein is selected from the list of 13 novel functional anchor proteins shown in Table 3. In certain embodiments, the coat protein is SscA.

[0022] The cargo can be bound to the spore coat protein by any means known in the art. As described in Example 3, the cargo protein can bind directly or indirectly to the spore coat protein. For example, in some embodiments, the spore coat protein and the cargo protein are directly bound by expressing these proteins as a fusion protein. A "fusion protein" is a protein that contains at least two domains, each of which is encoded by a separate gene, and these genes are joined and transcribed and translated as a single unit to produce a single polypeptide. The spore coat protein and the cargo protein can be fused in any order. That is, the C-terminus of the spore coat protein can be fused to the N-terminus of the cargo protein, or the N-terminus of the spore coat protein can be fused to the C-terminus of the cargo protein. The inventors have determined that in the case of the enzyme GusA, it is preferred to fuse the N-terminus of the enzyme to the C-terminus of the spore coat protein (i.e., to best maintain enzyme function). However, for other enzymes, a different orientation may be preferred.

[0023] Unfortunately, fusion proteins are often misfolded, and even properly folded fusion proteins may interfere with spore assembly. Thus, in other embodiments, the spore coat protein and the cargo protein are indirectly linked by fusing each component to a different member of a protein tag pair. A "protein tag" is a short amino acid sequence that is added to another protein. Many protein tags are known in the art and include, for example, myc tag, FLAG tag, hemagglutinin tag, polyhistidine tag, and strep tag. Since these tags are small in size (usually 5-15 amino acids), adding a protein tag usually does not affect the function of the protein to which the tag is attached. As used herein, a "protein tag pair" refers to two protein tags that specifically bind to each other. The term "specific" refers to the ability of a protein to bind to one molecule over other molecules. Under appropriate conditions, a protein that specifically binds to a target molecule will bind to that target molecule without binding to other molecules present in large amounts in the sample. Specific binding means binding to the target molecule with an affinity that is at least 25%, at least 50%, at least 100% (2-fold), at least 10-fold, at least 20-fold, or at least 100-fold greater than its affinity for other molecules.

[0024] One major problem with most protein tags commonly used in the art is the instability of their interaction with non-covalent partners. A stable covalent bond between the spore coat protein and the cargo protein is preferred over a non-covalent bond because it can continue to properly display the cargo protein on the spore surface when the spore encounters harsh environments. Covalent bonds are also preferred for applications where functionalized spores are used as long-term enzyme storage devices, therapeutic delivery vehicles, or catalytic microparticles. Thus, in a preferred embodiment, the protein tag pair forms a covalent bond. A "covalent bond" is a strong chemical bond that results from the sharing of a pair of electrons between two atoms.

[0025] In some embodiments, the covalent bond is an isopeptide bond. An "isopeptide bond" is an amide bond formed between the carboxyl / carboxamide group of one amino acid and the amino group of another amino acid, with at least one of these groups being outside the protein backbone. Isopeptide bonds are stable under conditions where non-covalent interaction rapidly dissociates, for example, they are stable under long-term (e.g., several weeks), high temperature (>95°C), high force, or harsh chemical treatment (e.g., pH 2 - 11, organic solvents, detergents, denaturants). Isopeptide bonds are irreversible under biological conditions and are resistant to most proteases. Examples of protein tag pairs that form isopeptide bonds include, but are not limited to, SpyTag / SpyCatcher, SpyTag002 / SpyCatcher002, SpyTag003 / SpyCatcher003, SnoopTag / SnoopCatcher, DogTag / DogCatcher, and SdyTag / SdyCatcher. Isopeptide bonds can also be formed by sortase-mediated ligation, butelase-mediated ligation, disulfide bond formation, and the use of non-standard amino acids.

[0026] In Example 3, the inventors describe two ways to bind a cargo protein to a spore coat protein using the SpyCatcher-SpyTag system (see Figures 5 and 6). Specifically, the inventors designed a method of expressing the spore coat protein as a fusion protein with SpyCatcher and expressing the cargo protein as a fusion protein with SpyTag. When these protein tags spontaneously form an isopeptide bond, they indirectly bind the cargo protein to the spore coat protein. Thus, in some embodiments, the first protein tag is SpyCatcher and the second protein tag is SpyTag. In other embodiments, the first protein tag is SpyTag and the second protein tag is SpyCatcher, the spore coat protein is fused to SpyTag, and the cargo protein is fused to SpyCatcher.

[0027] The SpyCatcher-SpyTag system is described in detail in U.S. Patent No. 9,547,003, the entire disclosure of which is incorporated herein by reference. In this system, the peptide SpyTag (SEQ ID NO: 27, 13 amino acids) spontaneously reacts with the protein SpyCatcher (SEQ ID NO: 28, 12.3 kDa) to form an isopeptide bond. SpyTag and SpyCatcher were formed by splitting and engineering the CnaB2 domain of the protein FbaB from Streptococcus pyogenes, which naturally forms an intramolecular isopeptide bond. Specifically, SpyTag was formed from the C-terminal beta strand of CnaB2 containing the reactive aspartic acid D556, and SpyCatcher was formed from the remaining beta strands containing the reactive lysine K470 and catalytic glutamic acid E516. The DNA sequences encoding SpyTag and SpyCatcher can be recombinantly introduced into the DNA sequences encoding the two target proteins to form two fusion proteins (i.e., one containing SpyTag and the other containing SpyCatcher). Subsequently, the two fusion proteins can be covalently bonded simply by mixing in the reaction. The conditions suitable for this reaction are known in the art. For example, this reaction can be carried out in phosphate-buffered saline (PBS) for in vitro tests and in the cytoplasm of cells for in vivo tests.

[0028] In subsequent generations of the SpyCatcher-SpyTag system, the binding is faster than the original. For example, in the second-generation SpyTag002 (SEQ ID NO: 29) / SpyCatcher002 (SEQ ID NO: 30) created by phage display, the protein tag pair reacts up to 12 times faster than the original pair (rate constant: 2.0 ± 0.2 × 10 4 M -1 s -1)。The third-generation SpyTag003 (SEQ ID NO:31) / SpyCatcher003 (SEQ ID NO:32) created by rational design allows the protein tag pair to react up to 400 times faster than the original pair (rate constant: 5.5 ± 0.6 × 10 5 M -1 s -1 ). Thus, in some embodiments, the protein tag pair used to attach the cargo protein to the spore coat protein is SpyTag002 / SpyCatcher002 or SpyTag003 / SpyCatcher003.

[0029] (Bacteria:) In a second aspect, the present invention provides a genetically modified bacterium that produces spores loaded with the cargo described herein.

[0030] As described in Example 3, the spores loaded with the cargo of the present invention can be produced using either direct protein fusion or indirect protein binding. Thus, the bacteria of the present invention can be genetically modified to (1) express a single fusion protein comprising a cargo protein fused to a spore coat protein, or (2) express the spore coat protein and the cargo protein as separate proteins (i.e., proteins that are transcribed and translated separately). In embodiments where the bacterium expresses the spore coat protein and the cargo protein as separate proteins, a protein tag pair can be used to indirectly attach the cargo protein to the spore coat protein. Specifically, in some embodiments, the spore coat protein is fused to a first protein tag and the cargo protein is fused to a second protein tag that forms an isopeptide bond with the first protein tag. As described above, the inventors designed a binding method in which the spore coat protein is expressed as a fusion protein with the peptide SpyCatcher and the cargo protein is expressed as a fusion protein with the peptide SpyTag. Thus, in a preferred embodiment, the first protein tag is SpyCatcher and the second protein tag is SpyTag.

[0031] Any bacterium that forms spores can be used in the method of the present invention. Examples of such bacteria include, but are not limited to, Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus megaterium, Clostridium botulinum, Clostridium tetani, etc. However, the anchor protein identified by the inventors in Example 1 is derived from Bacillus subtilis. Therefore, in a preferred embodiment, the bacterium is derived from the genus Bacillus. In a specific embodiment, the bacterium is Bacillus subtilis. Advantageously, B. subtilis is generally regarded as safe (GRAS) by the US Food and Drug Administration (FDA). In some embodiments, the bacterium is modified to cause germination failure or a decrease in germination efficiency by inactivation of specific genes including, but not limited to, cotH, cotG, cotB, cotE, cotT, cwlD, cwlJ, gerAA, gerAB, gerAC, gerD, gerBA, gerBC, gerBB, gerKA, gerKB, gerKC, gerKD, gerE, gerM, gerQ, gerT, pdaA, pdaB, sleB, spoVAC, spoVAD, spoVAE, sscA, ypeB, and combinations thereof.

[0032] (Method for producing spores:) In a third aspect, the present invention provides three methods capable of generating spores loaded with the cargo of the present invention.

[0033] The first method of generating spores (shown in Figure 4) involves (a) culturing bacteria that express a fusion protein containing a spore coat protein and a cargo protein in a sporulation medium. As used herein, the term "culturing" refers to the process of growing cells in an artificial environment. In this method, the bacteria are cultured in a sporulation medium. As used herein, the term "sporulation medium" refers to any non-nutrient medium that induces spore formation. Examples of sporulation media include the 2xSG medium defined by Nicholson (Journal of bacteriology 172.1 (1990):7-14), which is hereby incorporated by reference in its entirety.

[0034] In this method, the bacteria should be cultured for a time sufficient for the bacteria to form spores in the sporulation medium. Suitably, the bacteria are cultured for 24 to 96 hours.

[0035] In some embodiments, this first method further comprises introducing into the bacteria a construct encoding the fusion protein prior to step (a). As used herein, the term "construct" refers to a recombinant polynucleotide, i.e., a polynucleotide formed by combining at least two polynucleotide components from different natural or synthetic sources. For example, a construct can include (1) a coding region of one gene operably linked to another gene found within the same genome, (2) derived from the genomes of different species, or (3) a synthetic promoter. Constructs can be generated using conventional recombinant DNA methods. In addition to the sequence encoding the fusion protein, the construct may further include regulatory elements that facilitate the transcription and translation of the fusion protein. The construct may be a vector such as a plasmid.

[0036] As used herein, "introducing" refers to the process of introducing an exogenous polynucleotide into a recipient cell. Suitable introduction methods include, but are not limited to, bacteriophage or virus infection, natural transformation, electroporation, heat shock, lipofection, microinjection, and particle bombardment. In some embodiments, the construct is introduced into the bacteria using a carrier. Suitable carriers include, but are not limited to, lipid carriers (e.g., Lipofectamine) and polymeric nanocarriers.

[0037] A second method of generating spores (shown in Figure 5) involves culturing, in a sporulation medium, bacteria that express (i) an exosporium protein fused to a first protein tag and (ii) a cargo protein fused to a second protein tag that specifically binds to the first protein tag. In this method, the tagged exosporium protein and the tagged cargo protein are expressed independently within the same spore-forming mother cell.

[0038] In some embodiments, this second method further involves introducing into the bacteria, prior to step (a), (i) a first construct encoding an exosporium protein fused to a first protein tag and (ii) a second construct encoding a cargo protein fused to a second protein tag.

[0039] In contrast, in the third method, the tagged exosporium protein and the tagged cargo protein are expressed in separate bacterial cells. Specifically, a third method of generating spores (shown in Figure 6) involves (a) culturing a first bacterium that expresses an exosporium protein fused to a first protein tag in a sporulation medium, (b) culturing a second bacterium that expresses a cargo protein fused to a second protein tag that specifically binds to the first protein tag in a sporulation medium, (c) isolating the cargo protein produced by the second bacterium in step (b), and (d) combining the spores produced by the first bacterium in step (a) with the cargo protein isolated from the second bacterium in step (c). In this third method, the first bacterium that expresses the tagged exosporium protein can be any spore-forming bacterium, and the second bacterium that expresses the tagged cargo protein can be any bacterium suitable for the production of recombinant proteins (e.g., E. coli, Bacillus subtilis).

[0040] In step (c), the cargo protein produced by the second bacterium can be isolated using any protein purification method known in the art (e.g., size exclusion chromatography, separation based on charge or hydrophobicity, affinity chromatography, high performance liquid chromatography, etc.). At a minimum, the second bacterium needs to secrete the cargo protein or be lysed to release the cargo protein. As used herein, the term "lysis" is used to represent the disruption of the cell membrane. Lysis allows the contents to flow out of the cell and become accessible. Examples of lysis methods include, but are not limited to, chemical lysis, heat lysis, mechanical lysis, and osmotic lysis. In some embodiments, the crude lysate of the second bacterium is combined with the culture of the first bacterium or the spores isolated therefrom in step (d).

[0041] In some embodiments, this third method further comprises (i) introducing into the first bacterium, prior to step (a), a first construct encoding an exosporium protein fused to a first protein tag, and / or (ii) introducing into the second bacterium, prior to step (b), a second construct encoding a cargo protein fused to a second protein tag.

[0042] In some embodiments of the second and third methods, the first protein tag forms an isopeptide bond with the second protein tag. In certain embodiments, the first protein tag is SpyCatcher and the second protein tag is SpyTag.

[0043] Any of these three methods for generating spores may further include isolating spores produced by sporogenic bacteria from the supernatant of the culture after step (a). In the third method, this additional separation step can be performed before or after the spores tagged in step (d) are bound to the tagged cargo protein. That is, the spores may be separated from the supernatant of the culture of the first bacterium before step (d) so that the separated spores are bound to the cargo protein separated in step (d), or alternatively, the separated cargo protein may be added to the culture of the first bacterium in step (d) and then the spores (filled with the cargo protein) may be separated.

[0044] After sporulation, when the mother cell undergoes programmed cell death, the spores are released into the environment. Therefore, the spores can be easily separated from the supernatant of the culture by centrifugation. "Centrifugation" is a method of separating molecules of different densities by rotating them at high speed around an axis in a solution. Examples of suitable centrifugation conditions for separating bacterial spores from cell cultures include 21,000 g for 2 minutes, or 3,000 g for 5 minutes.

[0045] Any of these methods can be used to generate the spores described herein, although the second and third methods may be preferred. This is because when the cargo protein and the spore coat protein are fused to a short protein tag (e.g., the 14 - amino - acid SpyTag), the likelihood of these proteins being misfolded is significantly reduced compared to when they are expressed as a fusion protein of a single cargo protein and spore coat protein.

[0046] (Pharmaceutical composition:) In a fourth aspect, the present invention provides a pharmaceutical composition comprising the spores described herein and a pharmaceutically acceptable carrier.

[0047] Pharmaceutically acceptable carriers are known in the art and include, but are not limited to, diluents (e.g., tris hydrochloride, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizers (e.g., glycerol, polyethylene glycol), emulsifiers, liposomes, nanoparticles, and adjuvants. The pharmaceutically acceptable carrier can be an aqueous or non-aqueous solution, suspension, or emulsion. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Aqueous carriers include isotonic solutions, alcohol / aqueous solutions, emulsions, and suspensions including physiological saline and buffered media.

[0048] The pharmaceutical composition of the present invention may further include additives such as albumin and gelatin for preventing absorption on the surface, detergents (e.g., Tween20, Tween80, Pluronic F68, bile salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking agents or tonicity modifiers (e.g., lactose, mannitol).

[0049] (Method for delivering a cargo protein to a subject:) In a fifth aspect, the present invention provides a method for delivering a cargo protein to a subject in need thereof. The method includes administering to the subject the spore or pharmaceutical composition described herein.

[0050] As used herein, the term "administering" refers to introducing a substance into the body of a subject. Administration methods are well known in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, intratympanic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including injections such as intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. In certain embodiments, the composition is administered orally.

[0051] The "subject" to which the method is applied can be a non - mammalian animal such as a mammal or a bird. Suitable mammals include, but are not limited to, humans, cows, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, the method can be practiced on laboratory animals (such as mice and rats) for research purposes. In other embodiments, the method is used to treat commercially important livestock (such as cows, horses, pigs, rabbits, goats, sheep, chickens, etc.) or pets (such as cats and dogs). In a preferred embodiment, the subject is a human.

[0052] In some embodiments, the cargo protein is delivered to a specific cell type or tissue within the subject. For example, in some embodiments, the cargo protein is delivered to the intestinal tract of the subject.

[0053] Antibiotics are prescribed approximately 275 million times per year in the United States. However, some antibiotics are non-specific, meaning that while they kill pathogenic microorganisms that cause infection, they also commonly kill many beneficial bacteria that inhabit the gut. The death of these beneficial bacteria can cause serious problems. For example, the disruption of the natural microbiota by antibiotics creates an opportunity for pathogenic bacteria such as Clostridioides difficile to colonize the gut. In the United States alone, approximately 450,000 infections and 30,000 deaths are caused by Clostridioides difficile each year. Clostridioides infections are the most common healthcare-associated infections and are estimated to have accounted for more than 2% of all hospital costs in the United States in 2009. Additionally, antibiotic-induced gut microbiota imbalance (i.e., dysbiosis) has been associated with adverse health effects such as depression, certain cancers, colitis, and other chronic diseases. Accordingly, in some embodiments, the methods of the present invention are used to deliver an enzyme that degrades antibiotics to the gut to prevent disruption of the microbiota by antibiotics. In these embodiments, the spore composition needs to be formulated to be able to reach the gut of the subject. For example, when a spore-displaying enzyme is orally administered, it passes through the stomach and is delivered to the intestine where it can degrade antibiotics, and thus the composition may be formulated as an orally administered liquid or tablet.

[0054] Proteins are fragile and are destabilized and degraded by low pH, various salt concentrations, and proteases in the stomach and intestines, making it difficult to efficiently deliver functional proteins to the gut. For this reason, enzymes are often delivered to the gut within the cells in which they are produced (e.g., SYNB1618 from Synlogic). However, such products contain genetic material encoding the enzyme and are not gene-free. Spores can stabilize proteins and make them gene-free by translocating, and thus can be used to deliver antibiotic-degrading enzymes without incurring the risk of spreading antibiotic resistance genes. Accordingly, the gene-free spores of the present invention provide a safer means for delivering enzymes to the gut. Delivery of antibiotic-degrading enzymes is one of many potential uses of the artificial spores described herein.

[0055] It will be apparent to those skilled in the art that many additional modifications beyond those already described are possible without departing from the inventive concept. In interpreting this disclosure, all terms should be interpreted as broadly as possible within the context. Variations of the term "comprising" should be interpreted as a non-exclusive reference to an element, component, or step, such that the referenced element, component, or step can be combined with other elements, components, or steps not expressly referenced. An embodiment referred to as "comprising" certain elements is also contemplated as "essentially comprising" and "consisting of" those elements. The terms "essentially comprising" and "consisting of" should be interpreted consistent with the interpretations of the MPEP and the relevant Federal Circuit Court. The transitional phrase "essentially comprising" limits the scope of a claim to certain materials or steps that "do not materially affect the basic and novel characteristics" of the claimed invention. "Consisting of" is a limiting term that excludes elements, steps, or ingredients not explicitly recited in the claim. For example, with respect to a sequence, "consisting of" refers to the sequence recited in SEQ ID NO. and to a larger sequence that may include SEQ ID NO. as a part of itself.

[0056] While the present invention has been described in terms of one or more preferred embodiments, it should be understood that many equivalents, alternatives, variations, and modifications, aside from those expressly described, are possible and fall within the scope of the invention. [Example]

[0057] The present invention will be more fully understood in light of the following non-limiting examples.

[0058] [example] (Example 1: Identification of a novel Bacillus subtilis spore membrane anchor protein) To identify novel Bacillus subtilis spore membrane anchor proteins, we first conducted an exhaustive literature search to create a list of known Bacillus subtilis spore membrane proteins. Some of these have been shown to be already functional enzyme anchor proteins, while others have been shown to be non-functional. We created a final list of 44 exosporium proteins and modified the B. subtilis genome to design 88 constructs encoding fusion proteins in which the enzyme beta-glucosidase (GusA) was fused to the N-terminus or C-terminus of each exosporium protein (SCP). Of the 88 designed constructs, 52 could be synthesized.

[0059] To form each of these constructs, we used a co-transformation method that enables scarless editing of the SCP locus (Figure 1). In this method, the wild-type B. subtilis strain 168 is co-transformed with two DNA molecules. One is a molecule carrying the kanamycin resistance (kanR) gene, which is integrated into the non-essential ganA locus, and the other is a molecule carrying the sequence encoding the SCP-GusA fusion protein, which is integrated into the SCP locus. The co-transformed cells are plated on agar plates containing 2xSG sporulation medium, kanamycin, and the chromogenic substrate X-gluc. As a result, only cells that have taken up the kanR gene can grow on the plate. A part of the population (~5%) has taken up both DNA molecules. When the sequence encoding the SCP-GusA fusion protein is also integrated into the genome, the SCP-GusA fusion protein is expressed when the cells form spores. When the integration, expression, and folding of the SCP-GusA fusion protein are successful, the spores produced by the bacteria are loaded with functional GusA enzyme. Functional GusA reacts with X-Gluc in the substrate and turns the colonies blue. Therefore, the formation of blue colonies in this assay indicates that the exosporium protein forms a functional structure that can be loaded onto the spore surface. Colony PCR was used to confirm the genomic integration of the SCP-GusA construct at each native locus.

[0060] Based on this screening, functional fusion proteins were obtained from 34 out of the 52 synthesized constructs (Table 1). The 34 successful constructs represent 26 unique anchor proteins (Table 2), 13 of which are new enzyme anchor proteins (Table 3).

[0061] [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] (Example 2: Evaluation of the Activity and Accessibility of Cargo Proteins Displayed on Spores) The activity of the SCP-GusA fusion protein identified in Example 1 was tested. Specifically, the Gus activity assay was used to test the activity of the 34 constructs listed in Table 1. These constructs encode the enzyme GusA with its N-terminus or C-terminus fused to the 26 SCP anchors listed in Table 2. It was found that the activity of GusA displayed on spores varied depending on the spore coat anchor protein, and the highest GusA activity was observed with the anchor protein SscA-C (Figure 2).

[0065] Furthermore, the surface availability of GusA expressed from 34 different constructs was tested by labeling the recombinant spores with anti-GusA antibody. The purified spores were incubated with a primary anti-GusA polyclonal antibody and a secondary fluorescent antibody and analyzed using flow cytometry. It was found that the surface accessibility of GusA displayed on spores varied greatly depending on the spore coat anchor protein (Figure 3).

[0066] (Example 3: Assembly of Cargo-Loaded Spores) In the following example, three methods for generating spores containing cargo proteins fused to spore coat anchor proteins are described.

[0067] (1) Cis-Loading by Direct Fusion The assembly of cis-loading spores by directly fusing a cargo protein to a spore coat anchor protein is shown in Figure 4. Briefly, a B. subtilis strain containing DNA encoding the desired SCP-cargo fusion protein is cultured in a starvation medium that induces sporulation. After 48 - 60 hours, most of the cell population forms spores. Once sporulation is complete, the functionalized spores can be easily purified by centrifugation.

[0068] (2) Cis-Loading Using the SpyCatcher-SpyTag System The assembly of cis-loading spores using the SpyCatcher-SpyTag system is shown in Figure 5. SpyCatcher is fused to the desired spore coat protein. The cargo protein is expressed in the same mother cell fused to SpyTag. The mother cell is cultured in a starvation sporulation medium. When spores are formed in the mother cell, SpyCatcher and SpyTag spontaneously form a covalent isopeptide bond, binding the spore coat protein to the cargo protein. Once sporulation is complete, the functionalized spores can be easily purified by centrifugation.

[0069] (3) Trans-Loading Using the SpyCatcher-SpyTag System The assembly of translocating spores using the SpyCatcher-SpyTag system is shown in Figure 6. SpyCatcher is fused to the target spore coat protein. Once sporulation is complete, the SpyCatcher-functionalized spores can be purified by centrifugation. The fusion protein of the cargo protein and SpyTag is produced in a second production host. After expression, the fusion protein of the cargo protein and SpyTag is released from the production host by lysis and purified if necessary. The purified spores and cargo protein are mixed in a buffer (e.g., PBS, pH = 7.0) to load the cargo protein onto the surface of the spores. After assembly, the functionalized spores can be easily purified by centrifugation.

[0070] (Example 4: Evaluation of the Stability of Cargo Proteins) Enzymes are biological catalysts used in chemical manufacturing, agriculture, bioremediation, therapy, and other industries. Protein spore display often enhances the stability of the displayed protein, so this technology has the potential to be used in all these industries. It is predicted that due to the stabilization brought about by spore display, enzyme cargo proteins can maintain their activity even in the face of various stability challenges such as time, temperature, pH, proteases, solvents, and detergents. Therefore, the B. subtilis anchor protein identified in Example 1 is being tested for its ability to stabilize the binding enzyme against all these stability challenges. Preliminary data are shown below.

[0071] [Temperature Challenge] Heat helps to speed up chemical reactions, and many enzymes can catalyze reactions faster at high temperatures. However, if the temperature is too high, the enzyme becomes destabilized and / or denatured and can no longer catalyze the reaction. Therefore, stabilizing the enzyme against temperature challenges allows the reaction to proceed faster and longer at high temperatures. Many techniques use immobilization to enhance the stability of enzymes.

[0072] When the enzyme GusA was fused to the various B. subtilis anchor proteins identified in Example 1, it was tested whether it would be stabilized against temperature challenges. Specifically, GusA was fused to the spore coat proteins CgeA-C, CotG-C, CotX-C, CotY-C, and SscA-C, and the spore-displayed GusA was incubated at 25, 37, 50, and 60 °C to test its stability. The loss of free GusA enzyme activity begins at 37 °C. All of the tested spore-displayed enzymes maintained or acquired activity up to 50 °C, but the magnitude of the effect varied depending on the anchor protein (Figure 7). For example, while the free GusA enzyme completely loses activity when incubated at 60 °C, GusA fused to CgeA-C or SscA-C maintains more than 50% activity at this temperature.

[0073] [pH Challenge] Spore display can also be used to enhance the stability of enzymes against extreme pH. When the anchor proteins CotX-C (Figure 8B) and SscA-C (Figure 8C) were tested for their ability to confer stability against extreme pH compared to the free enzyme (Figure 8A), it was found that binding the enzyme GusA to these spore coat proteins resulted in higher relative activity than the free enzyme at pH > 8.

[0074] (Example 5: Evaluation of Different Cargo Proteins) In addition, the activity of the second enzyme, DuraPETase, was also tested. This is a modified form of an esterase derived from Ideonella sakaiensis (ACS Catalysis 11(3):1340-1350, 2021). DuraPETase was fused to SCP anchors CgeA-C, CotG-C, CotX-C, CotY-C, and SscA-C using a p-nitrophenyl acetate esterase activity assay (Sci Total Environ 709:136138, 2020). Similar to GusA, it was found that the activity of DuraPETase displayed on the spores differed depending on the spore coat anchor protein, and the highest GusA activity was confirmed with the anchor protein SscA (Figure 9). Indeed, the esterase activity with SscA was higher than that of any SCP tested so far.

Claims

1. A bacterial spore containing a cargo protein bound to a spore coat protein, A bacterial spore in which the spore coat protein is selected from the proteins listed in Table 3 or Table 2.

2. The bacterial spore according to claim 1, wherein the spore coat protein is fused to a first protein tag, and the cargo protein is fused to a second protein tag that specifically binds to the first protein tag.

3. The bacterial spore according to claim 2, wherein the second protein tag forms an isopeptide bond with the first protein tag.

4. A bacterium that produces bacterial spores according to any one of claims 1 to 3.

5. The bacterium according to claim 4, wherein the bacterium expresses the spore coat protein and the cargo protein as a fusion protein.

6. The bacterium according to claim 4, wherein the bacterium expresses the spore coat protein and the cargo protein as separate proteins.

7. The bacterium according to claim 6, wherein the spore coat protein is fused to a first protein tag, and the cargo protein is fused to a second protein tag that specifically binds to the first protein tag.

8. The bacterium according to claim 7, wherein the second protein tag forms an isopeptide bond with the first protein tag.

9. The bacterium according to claim 4, wherein the bacterium is a bacterium of the genus Bacillus.

10. The bacterium according to claim 9, wherein the bacterium is Bacillus subtilis.

11. a) A step of culturing bacteria in a spore-forming medium, wherein the bacteria express a fusion protein comprising the spore coat protein and the cargo protein, A method for producing bacterial spores according to any one of claims 1 to 3, including the following:

12. a) A step of culturing bacteria in a spore-forming medium, wherein the bacteria express (i) a spore coat protein fused to a first protein tag and (ii) a cargo protein fused to a second protein tag that specifically binds to the first protein tag. A method for producing bacterial spores according to any one of claims 1 to 3, including the following:

13. b) The method according to claim 11, further comprising the step of isolating bacterial spores produced by the bacteria from the supernatant of a culture.

14. The method according to claim 13, wherein the bacterial spores are isolated by centrifugation.

15. a) A step of culturing a first bacterium in a spore-forming medium, wherein the first bacterium expresses a spore coat protein fused to a first protein tag, b) A step of culturing a second bacterium in a spore-forming medium, wherein the second bacterium expresses a cargo protein fused to the second protein tag that specifically binds to the first protein tag, c) A step of isolating the cargo protein produced by the second bacterium in step (b), d) A step of combining the spore coat protein produced by the first bacterium in step (a) with the cargo protein isolated from the second bacterium in step (c), A method for producing bacterial spores according to any one of claims 1 to 3, including the following:

16. The method according to claim 15, further comprising the step of separating the spores produced by the first bacterium in step (a) from the supernatant of the culture before step (d).

17. The method according to claim 16, wherein the spores are separated by centrifugation.

18. The method according to claim 12, wherein the second protein tag forms an isopeptide bond with the first protein tag.

19. The bacterial spore according to any one of claims 1 to 3, wherein the cargo protein is an enzyme.

20. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a bacterial spore according to any one of claims 1 to 3.

21. A bacterial spore according to any one of claims 1 to 3, for use in delivering cargo proteins to a subject.

22. The bacterial spore for use according to claim 21, wherein the cargo protein is an enzyme.

23. The bacterial spore for use according to claim 22, wherein the enzyme degrades the antibiotic.

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