Anti-pathogen phages produced from microbes and methods of use

Abstract

The present disclosure provides genetically modified microbes. A genetically modified microbe includes a lysogenic P22 phage and at least one exogenous coding region. The exogenous coding region can encode a Salmonella modification system methylase, a C2 protein, a C2-inhibiting protein, rfb gene cluster coding regions and a rfc coding region, or a combination thereof. Also provided are compositions that include the genetically modified microbe and methods of using the genetically modified microbe.

Description

ANTI-PATHOGEN PHAGES PRODUCED FROM MICROBES AND METHODS OFUSE

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 508,322, filed June 15, 2023, which is incorporated by reference herein in its entirety.

[0003] SEQUENCE LISTING

[0004] This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an XML file entitled "0703000001W001.xml" having a size of 83,879 bytes and created on June 13, 2024. The information contained in the Sequence Listing is incorporated by reference herein.

[0005] GOVERNMENT FUNDING

[0006] This invention was made with government support under 1R35GM147484 awarded by the National Institute of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] The human gastrointestinal tract is densely colonized with myriad microorganisms important to human health, including bacteria and viruses (Nature 2012, 486 (7402), 207- 214. doi.org / 10.1038 / naturel 1234), which play essential roles in metabolic processes and immunological responses. (Brestoff et al., Nat Immunol 2013, 14 (7), 676-684. doi.org / 10.1038 / ni.2640. Commensal.) Key to the continued symbiotic relationship between mammalian host and microbe is the preservation of microbial diversity in the gut microbiome and its resilience against environmental perturbations that may lead to dysbiosis-related diseases in the short term (Brestoff et al., Nat Immunol 2013, 14 (7), 676- 684. doi.org / 10.1038 / ni.2640. Commensal.), but possibly to chronic disease in the long term (Sommer et al., Nat Rev Microbiol 2017, 15 (10), 630-638. doi.org / 10.1038 / nrmicro.2017.58.). Despite the growing understanding of the symbioticrelationship between host and microbe and their prophylaxis for pathogen infection, gastrointestinal diseases are prevalent worldwide.

[0009] Salmonella is a clinically relevant enteric pathogen that often emerges in food-poisoning outbreaks, representing one of the four major global causes of diarrheal diseases. (Coburn et al., Immunology and cell biology 2007, 85 (2), 112-118. doi.org / 10.1038 / sj.icb.7100007; WHO. Salmonella (non-typhoidal). who.int / news-room / fact-sheets / detail / salmonella-(non- typhoidal)) In the United States, widespread food-poison agents that causes ~1.3 million cases and more than $300 million of direct medical costs per year. S. Enteritis and S. Typhimurium are the two most common nontyphoidal serovars (NTS) that cause diarrhea, with a reported death rate reaching up to 24 % in developing countries, especially among infants. (Chimalizeni et al., Advances in Experimental Medicine and Biology 2010, 659, 33-46. doi.org / 10.1007 / 978- 1-4419-0981-7_3; Feasey et al., Lancet (London, England) 2012, 379 (9835), 2489-2499. doi.org / 10.1016 / S0140-6736(l 1)61752-2.) Mild symptoms of NTS infections require only treatment with fluids, while severely ill patients require antimicrobials, including ampicillin, quinolones, and cephalosporins. (Fabrega et al., Clinical microbiology reviews 2013, 26 (2), 308-341. doi.org / 10.1128 / CMR.00066-12.) Despite the efficacy, the broad use of antibiotics for Salmonella treatment has led to the spread of multi drug-resistant S. Typhimurium, a major global threat. (Meakins et al., Microbial drug resistance (Larchmont, N.Y.) 2008, 14 (1), 31-35. doi.org / 10.1089 / mdr.2008.0777; Stevenson et al., Antimicrobial agents and chemotherapy 2007, 51 (1), 195-197. doi.org / 10.1128 / AAC.00222-06; Borges et al., Revista Brasileira de Ciencia Avi cola 2019, 21 (1). doi.org / 10.1590 / 1806-9061-2018-0827.)

[0010] Bacteriophages (or phages) are the natural predators of bacteria and are a potential alternative antimicrobial strategy, one capable of precisely targeting bacterial pathogens in their native environment with minimal disruption to the gut microbiota. (Sommer et al., Nat Rev Microbiol 2017, 15 (10), 630-638. doi.org / 10.1038 / nrmicro.2017.58.) A major challenge for phage therapy, especially for the treatment of enteric infections, is having sufficient high quantities throughout the gastrointestinal tract to eradicate the bacterial pathogen before it can establish an infection.

[0011] SUMMARY OF THE APPLICATION

[0012] The present disclosure provides a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase. The P22 phage can be present in the genome of the microbe and can include an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein. In some embodiments, the microbe is not Salmonella enterica serovar Typhimurium. The microbe can further include a coding region encoding a C2 protein, and the coding region encoding the C2 protein can be operably linked to a constitutive promoter. The microbe can further include a coding region encoding a C2-inhibiting protein, such as a P22 anti-repressor protein or a mutant C2 protein. In some embodiments, the coding region encoding a c2-inhibiting protein is operably linked to an inducible promoter, such as an anhydrotetracycline inducible pTet-promoter. The microbe can further include rfb gene cluster coding regions and a rfc coding region. The genetically modified microbe can be a commensal microbe, such as a Gram-negative microbe. An example of a Gram-negative microbe is E. coli.

[0013] Also provided by the present disclosure are methods for increasing the presence of P22 phage in the gastrointestinal tract of a subject. In one embodiment, the method includes administering to a subject a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase. The P22 phage can be present in the genome of the microbe and include an inactivated coding region encoding a C2 protein, or encode an inactive C2 protein. In some embodiments, the subject has or is at risk of infection by S. enterica serovar Typhimurium.

[0014] In one embodiment, the method includes treating a subject having a S. enterica serovar Typhimurium infection, or for treating a subject having diarrhea. The method can include administering to a subject a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase. The P22 phage can be present in the genome of the microbe and include an inactivated coding region encoding a C2 protein, or encode an inactive C2 protein. The administering can include oral administration of at least IxlO2microbes, and theadministering can include administration of the microbe to the upper gastrointestinal tract or to the lower gastrointestinal tract. In some embodiments, the microbe is not Salmonella enterica serovar Typhimurium. The microbe can further include a coding region encoding a C2 protein, and the coding region encoding the C2 protein can be operably linked to a constitutive promoter. The microbe can further include a coding region encoding a C2- inhibiting protein, such as a P22 anti-repressor protein or a mutant C2 protein. In some embodiments, the coding region encoding a C2-inhibiting protein is operably linked to an inducible promoter, such as an anhydrotetracycline inducible pTet-promoter. The microbe can further include rfh gene cluster coding regions and a rfc coding region. The genetically modified microbe can be a commensal microbe, such as a Gram-negative microbe. An example of a Gram-negative microbe is E. coll.

[0015] The present disclosure also provides compositions. In one embodiment, a composition includes a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase and a pharmaceutically acceptable carrier. The P22 phage can be present in the genome of the microbe and can include an inactivated coding region encoding a C2 protein, or encoding an inactive C2 protein. In some embodiments, the microbe is not Salmonella enterica serovar Typhimurium.

[0016] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0017] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and singlestranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.

[0018] While the polynucleotide sequences described herein are listed as DNA sequences, it is understood that the complements, reverse sequences, and reverse complements of the DNA sequences can be easily determined by the skilled person. It is also understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uridine nucleotide.

[0019] A "coding region" is a polynucleotide sequence that encodes an RNA molecule. The boundaries of a coding region are generally determined by a transcription initiation site at its 5' end and a transcription terminator at its 3' end. A coding region typically includes at least one polynucleotide sequence that encodes a protein. A polynucleotide sequence encoding a protein, also referred to as an open reading frame (ORF), has boundaries that are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A coding region can encode an RNA molecule that includes one or more open reading frames. An RNA molecule that includes one open reading frame can be referred to as a "monocistronic message." An RNA molecule that includes two or more open reading frames can be referred to as a "polycistronic message."

[0020] A regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

[0021] As used herein, the term "protein" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "protein" also includes molecules which contain more than one protein joined by disulfide bonds, ionic bonds, or hydrophobic interactions, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (e g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and polypeptide are all included within the definition of protein and these terms are usedinterchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

[0022] As used herein, "genetically modified microbe" refers to a microbe into which has been introduced an exogenous polynucleotide and has been altered from its natural state. For example, a microbe is a genetically modified microbe by virtue of introduction into a suitable microbe of an exogenous polynucleotide, such as a P22 phage or a C2 protein.

[0023] As used herein, an "exogenous polynucleotide" refers to a polynucleotide that is not normally or naturally found in a microbe. An exogenous polynucleotide includes a coding region that is not normally found in a microbe, and a coding region that is normally found in a microbe but is operably linked to a regulatory region to which it is not normally linked. An "endogenous polynucleotide" is also referred to as a "native polynucleotide."

[0024] Polynucleotide and / or polypeptides or protein sequences may include one or more forms of typographical emphasis (e.g., underlined text, bolded text, italicized text). It is understood that the typographical emphasis is non-limiting. Sequences stated with typographical emphasis include the stated sequence without the typographical emphasis. Additionally, polynucleotide sequences may be displayed in capital letters, lower case letters, or a combination thereof. The case of the letters in the polynucleotide sequences is nonlimiting. Unless otherwise stated, lower case and upper-case letters simply indicate the identity of the nucleobase.

[0025] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0026] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0027] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0028] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0029] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of and / or "consisting essentially of are also provided. The term "consisting of means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0030] Conditions that are "suitable" for an event to occur, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event.

[0031] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations,compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0033] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0034] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0035] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0038] FIG. 1 shows a conceptual framework for the engineering of a microbe that carries a lytic prophage P22 for target Salmonella enterica serovar Typhimurium (STm). (FIG. 1A) Instead of administering oral phage (post-infection), basal levels of lytic phage that can provide prophylactic protection against future infection will be first established and, second, the phage levels can be inducible for higher levels. (FIG. IB) Genetic engineering of bacteria to encode the lytic prophage and all circuitry to produce a phage that targets a pathogenic microbe, including the prophage, the phage repressor, the modification gene of a restriction modification system, and a phage induction mechanism. Whereas basal phage levels can act as a prophylactic measure, chemically triggered phage induction may lead to higher phage levels desirable during more severe infections. TCGAATAATCGC (SEQ ID NO: 1).

[0039] FIG. 2 shows a P22 lysogen of E. coli produces an active P22 phage. (FIG. 2A-C) Bacterial growth is not significantly affected by the presence of the lysogen in E. coli MG1655, mouse commensal E. coli MP1 and STm. Lines represent the average of five biological replicates, and error bars represent the standard error of the mean (SEM). (FIG. 2D) Levels of spontaneous phage production over time for P22 lysogens of E. coli and STm. Phage levels from (FIG. 2E) E. coli MG1655 and (FIG. 2F) E. coli MP1 lysogens after 12-16 h growth in liquid culture. The AA to AE mutations correspond to the deletions in the phage genome described by Baaziz and coworkers (Baaziz et al., iScience, 2024, 27(4): 109595), having the kanamycin resistance gene (KanR) replacing each one of the regions indicated. Mutants having the KanRdeleted later were indicated as AKanR.

[0040] FIG. 3 shows lysogens produce an active P22 phage and can overcome STm restriction system. The efficiency of plating (EOP) represents the ratio of phage infection in the tested host and host from the phage source (phage infection calculated in pfu / mL). (FIG. 3A) EOP of P22 phage from STm or E. coli when tested against wild-type STm or R-M mutants. (FIG. 3B) EOP of P22 phage from STm, E. coli or E. coli expressing the modification gene from StyLTI when tested against different strains of STm. (FIG. 3C)The EOP for P22 produced form E. coli (P22E coh) increases with the promoter strength, leading to infectivity of STm wild-type to similar levels of P22 produced from STm.

[0041] FIG. 4 shows chemically inducible promoters trigger higher levels of phage from E. coli P22. (FIG. 4A) Bacterial growth and (FIG. 4B) GFP fluorescence signal for E. coli lysogens carrying the aTc-inducible trigger construct in a high copy number plasmid. (FIG. 4C) The increase in Phage / bacteria for the aTc-treated condition illustrates the efficacy of the phage-triggered mechanism.

[0042] FIG. 5 shows lytic P22 induced from E. coli can kill Salmonella. (FIG. 5A) Lytic and the lysogenic P22 phages produced from E. coli can be differentiated by clear and turbid plaque phenotypes. (FIG. 5B-C) Co-culture experiments for STm missing the StyLTI and StySA restriction system and E. coli mutants. (FIG. 5B) Phage titer was determined by post-assay method with the P22-susceptible E. coli with phage lysates from each coculture. (FIG. 5C) After 8 h, co-culture aliquots were spotted in LB-agar plates supplemented with different antibiotics to determine STm (chloramphenicol resistant) and E. coli (kanamycin resistant). (FIG. 5D) The same co-culture experiment was conducted with wild-type STm and E. coli mutants carrying the StyLTI-Mod gene under different promoters. In this experiment, STm growth was determined by platting the co-cultures in LB-plates supplemented with tetracycline, and E. coli lysogens in kanamycin.

[0043] FIG. 6 shows mouse groups treated with E.col21showed a better survival rate when challenged with wild-type STm. E. coli non-lysogenic and lysogenic groups were infected with STm dosages of (FIG. 6A-B) 102CFU and (FIG. 6C-D) 106CFU four days after E. coli colonization. Mice weight and health status were monitored for 14 days post-infection. Mice were sacrificed when loss exceeded 20% of initial weight or poor body condition.

[0044] FIG. 7 shows mice groups treated with the lysogenic E. coli present stable levels of P22 in the gut. Levels of (FIG. 7A) E. coli (lysogenic and non-lysogenic), (FIG. 7B) STm, and (FIG. 7C) P22 in mouse stool.

[0045] DETAILED DESCRIPTION

[0046] Diarrheal diseases are commonly associated with infectious, water- and food-borne pathogens that colonize the gut. Both the access and the microbiota complexity pose a challenge to the elimination of the pathogen without causing dysbiosis. Conventional applications of phages as antimicrobials ("phage therapy") use virulent-free phages to kill an established infection. While there has been some success in this approach, there are a number of ways by which bacteria can overcome this. The inventors hypothesized that greater efficacy could be achieved by targeting an infecting pathogen at its most vulnerable point, during the initial stages of infection when the number of pathogens is low and before it can stably engraft. As described herein, a non-pathogenic gram-negative was engineered to harbor a virulent mutant of a prophage, a phage integrated into the microbe’s DNA, that normally targets Salmonella enterica serovar Typhimurium (also referred to as Salmonella enterica subsp. enterica serovar Typhimurium). The non-pathogenic microbe can colonize a patient’s gut and produce phage. Phage production can occur at a low level or be induced to result in high levels of phage in the gut.

[0047] Accordingly, the present disclosure provides genetically modified microbes, compositions that include the microbes, and methods for using the microbes. A microbe, also referred to herein as a bacterium, can be a commensal microbe, such as a Gram-negative microbe. Examples of gram-negative microbes include, but are not limited to, commensal gut microbes such as E. coli, Klebsiella, Bacteroides, Prevotella, and Akkermansia, In one embodiment, a microbe useful herein is not Salmonella enterica serovar Typhimurium

[0048] In some embodiments, a microbe of the present disclosure can be administered to a subject to treat certain conditions of the gastrointestinal tract, thus a microbe useful herein is non- pathogenic in the subject to which it will be administered. As described in greater detail herein, a subject can be, but is not limited to, a human. Whether a microbe is pathogenic can be determined using routine animal models, including murine animal models.

[0049] A microbe of the present disclosure includes one or more P22 bacteriophage (also referred to herein as P22 phage, P22, and phage) (Byl and Kropinski, Journal of Bacteriology, 2000, 182(22). doi.org / 10.1128 / jb.182.22.6472-6481.2000; Susskind and Botstein, 1978,Microbiol Rev 42. doi.org / 10.1 128 / mr.42.2.385-413). A microbe of the present disclosure that includes a lysogenic P22 can be referred to as a "host microbe." P22 is a member of the Podoviridae family that infects Salmonella enterica serovar Typhimurium and has limited, and in some embodiments, undetectable ability to infect any other microbe. P22 is commercially available from the American Type Culture Collection (ATCC, Virginia), and can be obtained from strains of Salmonella enterica serovar Typhimurium containing a lysogenic P22.

[0050] The P22 present in a host microbe is integrated into the DNA of the microbe, such as the microbe’s circular chromosome or a plasmid present in the microbe. In this integrated state the phage is in a dormant lysogenic state and replicates with the DNA of the microbe. The integrated lysogenic phage is also referred to herein as a prophage. An integrated P22 can cease to be lysogenic and enter a lytic cycle, which results in accelerated replication of the phage genome, the release of new phage, and death of the host microbe by lytic release of the new phage. Integration of P22 into the Gram-negative E. coli had no identifiable negative impact on the growth of the E. coli. The combination of the host microbe and P22 useful herein is engineered to have a controllable lysogenic stage in the host microbe. P22 useful herein is also engineered to be incapable of lysogeny, e.g., always initiate the lytic stage, when it enters the target microbe Salmonella enterica serovar Typhimurium. These two activities can be accomplished by modification of a P22 C2 protein coding region, and modification of the host microbe to include a C2 protein coding region and a coding region encoding a protein that inhibits the activity of a C2 protein.

[0051] The P22 C2 protein is encoded in the genome of a wild-type P22 and inhibits the integrated phage from entering the lytic cycle. A P22 present in a host microbe includes one or more mutations that cause a reduced, in one embodiment undetectable, amount of C2 protein to be expressed by the phage. Thus, a P22 useful herein can be referred to as an engineered P22. The host microbe is engineered to express the c2 coding region, thereby complementing the inactivated prophage copy of the c2 coding region and maintaining the lysogeny of the phage in the host microbe.

[0052] A P22 can be modified to result in a reduced or undetectable amount of C2 protein to be expressed by the phage. Modifications can include mutations to inactivate a coding region encoding a C2 protein, e.g., a c2 coding region is not expressed, or encode an inactive C2 protein, e.g., a C2 protein is expressed but it cannot inhibit the integrated phage from entering the lytic cycle. Examples of mutations include, but are not limited to, altering the nucleotides of the c2 promoter so that it is no longer active and driving expression of the c2 coding region, engineering the c2 coding region to include one or more stop codons so a truncated and inactive C2 protein is produced or no C2 protein is produced, or engineering mutations of the c2 coding region so an altered and inactive C2 protein is produced. The genetic structure of P22 is known (Byl and Kropinski, J. Bacteriol., 2000, 182(22):6472- 6481) and the nucleotide sequence of P22 is readily available. One example of a nucleotide sequence encoding a C2 protein is present in Table 4A, nucleotides 1753-2401 (SEQ ID NO:71) of "c2 phage repressor," see also GenBank accession number AF217253. One example of an amino acid sequence of a C2 protein is encoded by nucleotides 1753- 2401 of Table 4A is:MNTQLMGERIRARRKKLKIRQAALGKMVGVSNVAISQWERSETEPNGENLLALSKA LQCSPDYLLKGDLSQTNVAYHSRHEPRGSYPLISWVSAGQWMEAVEPYHKRAIENW HDTTVDCSEDSFWLDVQGDSMTAPAGLSIPEGMI ILVDPEVEPRNGKLWAKLEGE NEATFKKLVMDAGRKFLKPLNPQYPMIEINGNCKI IGWVDAKLANLP (SEQ ID NO:2).One example of an amino acid sequence of a C2 protein is GenBank accession number AAF75024 1 . Other examples of C2 proteins are those having structural similarity to SEQ ID NO:2. In addition, the structure and function of the C2 protein is known (De Anda et al., I. Bacteriol., 1983, 258(17): 10536-10542; Biochemistry, 1981, 20(12):3591-3598). The skilled person can design mutations of a P22 c2 coding region to reduce expression and / or express a C2 protein with reduced or undetectable activity. In one embodiment, a c2 protein coding region of a P22 is modified to include one or more stop codons after the start codon.

[0053] A P22 can be modified to include one or more deletions of regions that are non-essential to function. Specific regions that can be deleted include one or more of the following: (i) the region having the gtrC to gtrA coding regions, which encode proteins that alter the bacterialO-antigen to inhibit adsorption by superinfecting phage (referred to as AA); (ii) the region with the sieA coding region, which encodes an inner membrane protein that acts to exclude superinfecting phage (referred to as AB); (iii) the region having the ninB to ninH coding regions, which encode proteins involved in N-independent transcriptional termination (referred to as AC); (iv) the region having the orf25 to orf80 coding regions, whose products facilitate a pseudo-lysogenic state after phage infection (referred to as AD); and (iv) the region having the mnt to ant coding regions, which act as a secondary immunity region (referred to as AE)(Baaziz et al., iScience, 2024, 27(4): 109595).

[0054] The host microbe includes a c2 coding region that maintains the lysogenic state of the integrated P22. A coding region encoding a functional C2 protein can be present in any location of the host microbe’s genome, including inserted in the circular chromosome or in a plasmid present in the microbe. The skilled person will understand that an inserted exogenous coding region should not overlap or separate coding regions that are part of the host metabolism, and the effect the promoter of the inserted exogenous coding region has on host coding regions should be minimized. The c2 coding region can be operably linked to a promoter. In one embodiment, a promoter operably linked to a c2 coding region is constitutive. Constitutive promoters that function in gram-negative microbes are known to the skilled person (Shimada et al., 2014, PLoS ONE 9(3): e90447. doi: 10.1371 / joumal. pone.0090447).

[0055] The P22 present in a host microbe is in a lysogenic state, e.g., it is integrated and replicates with the replication of the microbe’s genome; however, the P22 and host microbe are designed to cause the P22 to enter the lytic stage under certain conditions, release newly produced P22 that does not express a C2 protein, and be lytic when present in Salmonella enterica serovar Typhimurium. In one embodiment, a host microbe is further engineered to include a coding region encoding a C2-inhibiting protein that interferes with the ability of C2 protein to maintain the lysogenic state of P22. The presence of the C2-inhibiting protein results in the integrated P22 entering the lytic cycle, accelerating replication of the phage genome, releasing of new phage, and death of the host microbe by lysis during release of the new phage.

[0056] Examples of C2-inhibiting proteins include, but are not limited to, proteins that act to reduce expression of a C2 coding region or reduce the activity of a C2 protein. An example of a protein that reduces the activity of a C2 protein is a P22 C2 anti-repressor protein (Ant). Without intending to be bound by theory, the Ant protein inactivates the C2 protein through the non-covalent binding between the C2 and Ant2, which prevents the DNA binding activity of C2. An example of a C2 anti-repressor protein is available at GenBank accession number AAF75059.1, and an example of a coding region encoding a c2 anti-repressor protein is available at GenBank accession number NC_002371.2.

[0057] Another example of a protein that reduces the activity of a C2 protein is a mutant C2 protein. A C2 protein has two domains, a N-terminal DNA binding domain and a C- terminal dimerization domain. A mutant C2 protein that lacks a DNA binding activity and does have a dimerization activity acts as a dominant-negative variant of the C2 protein, e.g., it inhibits the ability of a C2 protein to maintain lysogeny of an integrated P22. An example of suitable mutant C2 proteins include those with one or more mutations that reduce or block the DNA binding activity. Thus, in one embodiment, a full length mutant C2 protein is expressed but only has the dimerization activity. Another example of suitable mutant C2 proteins include those with a deletion of some or all of the DNA binding domain. Thus, in another embodiment, a truncated mutant C2 protein is expressed. An example of a mutant c2 coding region that encodes a truncated mutant C2 protein with dimerization activity is present in Table 4B, nucleotides 2384-2846 (SEQ ID NO:3) of "C2DN::cro::gfp defective phage repressor", which encode a protein having the amino acid sequenceMAIENWHDTTVDCSEDSFWLDVQGDSMTAPAGLSIPEGMI ILVDPEVEPRNGKLW AKLEGENEATFKKLVMDAGRKFLKPLNPQYPMIEINGNCKI IGWVDAKLANLP (SEQ ID NO:4).Other examples of mutant C2 proteins are those having structural similarity to SEQ ID NO:4. The skilled person can design mutations of a P22 C2 coding region so it expresses a mutated C2 protein that does not have DNA binding activity.

[0058] A coding region encoding a C2-inhibiting protein can be present in any location of the host microbe’s genome, including inserted in the circular chromosome or in a plasmid present inthe microbe. The coding region encoding a C2-inhibiting protein can be operably linked to a promoter. In one embodiment, a promoter operably linked to a c2 coding region is inducible. Inducible promoters that function in gram-negative microbes are known to the skilled person (Rincon & Farney, Microbial Biotechnology, 2023, 16:961-976. doi:https: / / doi.org / 10.1101 / 2022.04.23.489285). In one embodiment, a suitable inducible promoter is an anhydrotetracycline (aTc) inducible pTet-promoter (Kotula et. al., 2014, 11 l(13):4838-4843).

[0059] In one embodiment, a host microbe can be modified to include a cro coding region that encodes a Cro protein. Cro binds to the operator driving c2 expression, and is a transcriptional repressor of c2. An example of the use of a mutant c2 coding region and a cro coding region is shown in Table 4B. In addition to the mutant C2 coding region that encodes a truncated mutant C2 protein with dimerization activity (Table 4B, nucleotides 2384-2846 (SEQ ID NO:3) of "c2DN::cro::gfp defective phage repressor"), nucleotides 2760-2846 (SEQ ID NO:70) are an example of a cro coding region that encodes a Cro protein. The inclusion of both coding region encoding a truncated mutant C2 protein and a coding region that encodes a Cro protein provide a redundancy in mechanisms to induce phage lysis in the host microbe. For instance, in an embodiment where the coding region encoding a truncated mutant C2 protein and coding region that encodes a Cro protein are both under the control of an inducible promoter, induction will trigger higher levels of phage induction, resulting in better ability to eradicate high pathogen loads. For instance, in the presence of an agent such as anhydrotetracycline (aTc) that induces expression from an inducible promoter, both the dominant negative c2 gene, missing the DNA binding domain, and the cro gene are expressed under the same promoter. While the product of the c2 gene C2DNdimerizes with the functional C2 monomer, making the phage repressor nonfunctional, the Cro protein represses the expression of the c2 phage repressor gene. Both responses contribute to switch the phage life cycle and induce phage lysis.

[0060] The microbial receptor of P22 is present on Salmonella enterica serovar Typhimurium, but is not present on most other commensal microbes, such as gram-negative microbes. The P22 receptor is the repeating polysaccharide of O antigen that can be synthesized by the products of the rfb gene cluster and the rfc gene. In some embodiments, a host microbe ofthe present disclosure optionally includes rfb gene cluster coding regions and the rfc coding region. The nucleotide sequences of the rfb gene cluster coding regions and the rfc coding region are known (X56793.1 and AE006468.2) and have been cloned onto a single plasmid, pPR1347 (Neal et al., 1993, J. Bacteriol., 175(21):7115-7118). The skilled person can transform a suitable commensal microbe, including a Gram-negative, such as an E. coli, with pPR1347 to engineer a host microbe that can be exposed to, and infected by, P22. Alternatively, the skilled person could use standard molecular biology techniques to add each of the rfb gene cluster coding regions and the rfc coding region to a suitable commensal microbe, including a Gram-negative, such as an E. coli, to obtain a host cell that could be infected by P22.

[0061] E. coli includes atiP22, a specific site where P22 integrates into the genome. In one embodiment, a commensal microbe, such as a Gram-negative microbe, can be engineered to include the integration / attachment site for P22 in E.coli attP 22) Lindsey et al., 1992, J. Bacteriol., 174(11): 3834383), thereby resulting in a modified microbe that can be infected by P22.

[0062] As discussed herein, P22 present in a host microbe can enter the lytic cycle and produce P22 that go on to infect a target Salmonella enterica serov ar Typhimurium. Salmonella enterica serovar Typhimurium typically include a restriction-modification (R-M) system that can act as a type of immune system that reduces the likelihood introduced DNA, such as P22, will survive within the Salmonella enterica serovar Typhimurium. R-M systems typically include two coding regions, one encoding a restriction enzyme that recognizes and cleaves a specific site, and a methylase that methylates at least one nucleotide of the specific site and thereby prevents the restriction enzyme from cleaving the methylated site (Beaulaurier et al., Nat Common 6, 7438 (2015). doi.org / 10.1038 / ncomms8438). In order to have P22 produced by a host microbe be capable of successfully infecting and lysing a target Salmonella enterica serovar Typhimurium, a host microbe can be modified to include a coding region that encodes a R-M system methylase. There are at least three R- M systems in Salmonella enterica serovar Typhimurium, the StyLTI, StySA, and StySB systems, with the StyLTI being the most common (De Backer et al., J. Bacteriol., 1991, 173(3): 1321-1327). Thus, a host microbe can be modified to express a methylase from theStyLTI R-M system, the StySA R-M system, the StySB R-M system, or a combination thereof. These R-M system coding regions that encode a methylase are known and can be used by the skilled person to modify a host microbe of the present disclosure. One example of a nucleotide sequence encoding a methylase of the StyLTI system is present in Table 4C, nucleotides 267-3974 (SEQ ID NO:5) of " StyLTI R'M+system", which encode a protein having the amino acid sequenceMLKDNQKHNESVAPNSAFLSELQRALPEFFTADRYNEQGELIAKGGFDLARFERAL KARNIDELTSGYQIDFIGKDYAKKQAGEKSVTVIVPDVEHNTLAENKNSHNLFLTG DNLDVLRHLQNNYADTVDMI YIDPPYNTGSDGFVYPDHFEYSDRALQDMFGLNDTE LARLKS I QGKSTHSAWLS FMYPRLFLARKLLKDTGFI FI S I DDNEYANLKLMMDE I FGEGGFVTNVMWKRKKEISNDSDNVS IQGEYILVYAKTGQGALRLEPLSKEYIQKS YKEPTEQFPEGKWRPVPLTVSKGLSGGGYTYKITTPNGTVHERLWAYPEASYQKLV ADNLVYFGKDNGGIPQRVMYAHHSKGQPTTNYWDNVASNKEGKKEILDLFGDNVFD TPKPTALLKKI IKLAIDKDGWLDFFAGSGTTAHAVMALNEEDGGQRTFILCTIDQ ALSNNT IAKKAGYNT IDE I SRERI TRVAAKIRANNPATNSDLGFKHYRFATPTQQT LDDLDSFDIATGHFINTSGQLAAFTESGFTDMINPFSARGLGVPGGASGEETLLTT WLVADGYKMDIDVQTVDFSGYCARYVDNTRLYLIDERWGTEQTRDLLNHIGTHQLP VQT I VI YGYS FDLES IRELE I GLKQLDQKVNLVKRY(SEQ ID NO:6).Other examples of methylases of the StyLTI system are those having structural similarity to SEQ ID NO:6.

[0063] The expression of a coding region encoding a methylase can be tuned control the level of phage methylation, which can modify the infectivity of the phage on the target pathogen S. entericci serovar Typhimurium. Non-limiting examples of promoters are shown in Table 5.

[0064] A host microbe that includes a P22 described herein can be produced using standard methods of microbial genetics and molecular biology. For instance, lysogenic host microbes, such as E. coli, can be produced using generalized transduction methods and tested using a virulent P22 such as P22 H5 (see "Engineering E. coliP22lysogens" in Example 1). Individual coding regions encoding proteins such as C2, C2-inhibiting protein, and methylase protein can be cloned in genetic constructs that facilitate transfer of the desired coding regions into a host microbe (see "Molecular cloning" in Example 1).The genome of a lysogenic host microbe, such as E. coli, can be modified to include one or more of the desirable coding regions and / or modify the genome of a lysogenic P22 using transformation and generalized transduction methods (see "Bacterial engineering" inExample 1). Without intending to be limiting, in one embodiment a method of making a host microbe includes performing the following steps in order: prepare P22 lysogens in the host microbe, add a c2 coding region the host microbe, modify the P22 coding region in the lysogenic P22, and then add R-M system by adding a methylase coding region to the host microbe. Of note, P22 lysogens may be obtained by at least two alternative methods: (1) by transforming the plasmid pPR1347 that harbors the rfb gene cluster coding regions and the rfc coding region to make the host microbe susceptible to infection by P22, followed by phage infection and streaking individual plaques for colony purification; or (2) generalized Pl transduction of the P22 prophage into the recipient host microbe. On both cases, the lysogen selection would be carried out in LB-supplemented plates based on the antibiotic marker present in the prophage genome.

[0065] A C2 protein, C2-inhibiting protein such as C2 anti-repressor protein or a mutant C2 protein, or a methylase of the StyLTI system that is useful herein can have an amino acid sequence that is structurally similar to a reference protein disclosed herein. As used herein, a protein is "structurally similar" to a reference protein if the amino acid sequence of the C2 protein possesses a specified amount of sequence similarity and / or sequence identity compared to the reference protein . An example of a C2 protein reference protein is SEQ ID NO:2, an example of a C2 anti-repressor protein reference protein is GenBank accession number AAF75059.1, an example of a mutant C2 protein reference protein is SEQ ID NO:4, and an example of a reference protein for a methylase of the StyLTI system is SEQ ID N0:6.

[0066] Structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (for example, a candidate protein and a reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate protein is the protein being compared to the reference protein.

[0067] A pair-wise comparison analysis of amino acid sequences or nucleotide sequences can be conducted, for instance, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented through 2004).

[0068] In one embodiment, the algorithm used to determine structural similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[0069] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Na l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0070] In the comparison of two amino acid sequences, structural similarity may be referred to by percent "identity" or may be referred to by percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, non-polar amino acids include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Polar amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine. The uncharged amino acids include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine, among others.

[0071] Thus, as used herein, reference to a protein as described herein, such as reference to an amino acid sequence described herein, can include a protein with at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference protein.

[0072] Alternatively, as used herein, reference to a protein as described herein, such as reference to an amino acid sequence described herein, can include a protein with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference protein.

[0073] The present disclosure also provides compositions that include a host microbe. In general, a composition can be formulated to be compatible with its intended route of enteral (e.g., oral) administration. In one embodiment, a formulation may be a liquid composition. Liquid compositions include, but are not limited to, , suspensions, dispersions, and the like. In one embodiment, a formulation may be a solid composition. Solid compositions include, freeze-dried preparations of a host microbe. A solid composition can be, for instance, a pill or capsule that includes the host microbe as solid. Those formulations may include a pharmaceutically acceptable carrier to render the composition appropriate for administration to a subject. As used herein "pharmaceutically acceptable carrier" includes pharmacologically inactive compounds compatible with pharmaceutical administration. A pharmaceutically acceptable carrier can be, and typically is, United States Pharmacopeia (USP) grade. Examples of pharmaceutically acceptable carriers include gut-transit protectants. A gut-transit protectant is a compound or composition that can protect a host microbe during gut transit, e.g., transit through the high acidity and digestive enzymes present in the stomach and / or intestine. Examples of gut-transit protectants include, but are not limited to, a dairy product such yogurt, a milk, or a reconstituted powdered milk.

[0074] A composition of the present disclosure may be administered by any method suitable for depositing in the gastrointestinal tract, such as the mouth, stomach, colon, or rectum, of a subject. Examples of routes of administration include rectal administration (e.g., bysuppository, enema, upper endoscopy, upper push enteroscopy, flexible sigmoidoscopy, or colonoscopy), intubation through the nose or the mouth (e.g., by nasogastric tube, nasoenteric tube, or nasal jejunal tube), or oral administration (e.g., by a solid such as a pill or capsule, or by liquid). In one embodiment, a composition is formulated for oral administration as a liquid that is swallowed by the subject.

[0075] The present disclosure is further directed to methods of using the host microbes and compositions described herein. One method includes administering to a subject in need thereof an effective amount of a composition described herein. The subject can be anyone in need. A subject can be an infant, child, adolescent, or adult. The administering is done under conditions suitable for deposition of the composition such that the host microbe in the composition colonizes one or more region of the gastrointestinal tract. For instance, administration may be into one or more areas of the upper gastrointestinal tract, e g., stomach and small intestine (duodenumjejunum, and ileum), or into the lower gastrointestinal tract, e.g., the terminal ileum, cecum, colonic areas, and rectum, or a combination thereof. When orally ingested, STm reaches the gastrointestinal tract and replicates predominately in the large intestine. The pathogen then uses both its flagella and its chemotactic system to find intestinal epithelium cells and deliver virulence factors using the STm secretion system that ultimately will trigger the inflammation process in the host intestine (Galan, Nat Rev Microbiol 19, 716-725 (2021). doi. org / 10.1038 / s41579-021- 00561-4). In addition, systemic invasion can occur in the small intestine. As used herein, an "effective amount" relates to a sufficient amount of a composition described herein, to provide the desired effect. For instance, in one embodiment an "effective amount" is an amount effective to alleviate one or more symptoms and / or signs of a disease or a condition as described herein. In some embodiments, an effective amount is an amount that is sufficient to affect a reduction in a symptom and / or sign associated with a disease as described herein. A reduction in a symptom and / or a sign is, for instance, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, or at least 100 % in a measured sign as compared to a control, a nontreated subject, or the subject prior to administration of the composition. A dosage can be at least 1 x 102host microbes, at least 5* 102host microbes, at least 1 x 103host microbes, at least 5* 103host microbes, at least 1 x 104host microbes, at least 5x 104host microbes, atleast 1 x ] 05host microbes, at least 5* 1O5host microbes, at least IxlO6host microbes, at least 5* 106host microbes, at least IxlO7host microbes, at least 5* 107host microbes, at least I x lO8host microbes, at least 5x 108host microbes, at least I x lO9host microbes, at least 5x 109host microbes, at least I x lO10host microbes, at least 5x IO10host microbes, at least I x lO11host microbes, at least 5x 1011host microbes, at least IxlO12host microbes, or at least 5x 1012host microbes. In one embodiment, a dosage is no greater than 5x 1012host microbes. Examples of ranges include at least I xlO2host microbes to no greater than 5x 1012host microbes, at least 1x103host microbes to no greater than 5x 1012host microbes, at least I x lO4host microbes to no greater than 5x 1012host microbes, or at least I x lO5host microbes, at least I x lO6host microbes to no greater than 5xl012host microbes, at least I x lO7host microbes to no greater than 5x 1012host microbes, at least I x lO8host microbes to no greater than 5x 1012host microbes, at least I x lO9host microbes to no greater than 5x 1012host microbes, at least Ix lO10host microbes to no greater than 5x 1012host microbes, or at least I x lO11host microbes to no greater than 5x 1012host microbes. It will be understood, however, that in some embodiments the total dosage of the compositions as disclosed herein will be decided by the attending professional, e.g., physician, within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type and extent of disease or condition being treated.

[0076] In one embodiment, a method of the present disclosure includes treating certain diseases in a subject in need of treatment. The subject may be a mammal, such as a human. In some embodiments animal models may be used, such as a mammal, including a rat, a mouse, a hamster, a gerbil, or a primate. As used herein, the term "disease" refers to any deviation from or interruption of the normal structure or function of a part, organ, or system, or combination thereof, of a subject that is manifested by a characteristic symptom or clinical sign. Diseases include those resulting from intestinal infection by S. enterica serovar Typhimurium. Such diseases include, but are not limited to, diarrhea, abdominal cramps, nausea, and vomiting.

[0077] As used herein, the term "symptom" refers to subjective evidence of disease or condition experienced by the subject and caused by disease. As used herein, the term "clinical sign, or simply "sign," refers to objective evidence of a disease present in a subject. Symptomsand / or signs associated with diseases referred to herein and the evaluation of such signs are routine and known in the art. Typically, whether a subject has a disease, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the disease. Symptoms and signs of intestinal infection by . enterica serovar Typhimurium include, but are not limited to, loose stool, stomach cramps, nausea, vomiting, fever, and dehydration.

[0078] Treatment of a disease can be prophylactic or, alternatively, can be initiated after the development of a disease. Treatment that is prophylactic, for instance, initiated before a subject manifests signs of a disease, is referred to herein as treatment of a subject that is "at risk" of developing a disease. An example of a subject that is at risk of developing a disease is a person having a risk factor. An example of a risk factor for diarrhea from S. enterica serovar Typhimurium is exposure, or anticipated exposure, to conditions that include poor public hygiene, such as contaminated food or water. Treatment can be performed before, during, or after the occurrence of a disease described herein. Treatment initiated after the development of a disease may result in decreasing the severity of the signs of the disease, or completely removing the signs. The lysogenic P22 of host microbes described herein will have spontaneous low levels of phage production, and this spontaneous production of phage in subject's gastrointestinal tract is expected to protect the subject from the onset of infection. In those embodiments where infection in the subject by S. enterica serovar Typhimurium is severe, expression of the C2-inhibiting protein results in a substantial increase in the production of phage and increased destruction of the pathogen.Accordingly, a method of treatment can include, in addition to administering a composition that includes a host microbe described herein, administering a compound that induces expression of the coding region encoding a C2-inhibiting protein. Administration of the inducing compound results in increased numbers of host microbes to enter the lytic cycle, produce P22 phage that is virulent in the target pathogen S. enterica serovar Typhimurium, and reduce the number of viable pathogens in the subject.

[0079] The invention is defined in the claims. However, below there is provided a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspectsmay be combined with any one or more features of another example, embodiment, or aspect described herein.

[0080] Exemplary Aspects

[0081] Aspect 1 is a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and includes an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

[0082] Aspect 2 is the genetically modified microbe of any of Aspects 1 or 3-29, wherein the microbe further includes a coding region encoding a C2 protein.

[0083] Aspect 3 is the genetically modified microbe of any of Aspects 1-2 or 4-29, wherein the coding region encoding the C2 protein is operably linked to a constitutive promoter.

[0084] Aspect 4 is the genetically modified microbe of any of Aspects 1-3 or 5-29, wherein the microbe further includes a coding region encoding a C2-inhibiting protein.

[0085] Aspect 5 is the genetically modified microbe of any of Aspects 1-4 or 6-29, wherein the C2-inhibiting protein includes a P22 anti-repressor protein or a mutant C2 protein.

[0086] Aspect 6 is the genetically modified microbe of any of Aspects 1-5 or 7-29, wherein the coding region encoding a C2-inhibiting protein is operably linked to an inducible promoter.

[0087] Aspect 7 is the genetically modified microbe of any of Aspects 1-6 or 8-29, wherein the inducible promoter includes an anhydrotetracycline inducible pTet-promoter.

[0088] Aspect 8 is the genetically modified microbe of any of Aspects 1-7 or 9-29, wherein the microbe includes rfb gene cluster coding regions and a rfc coding region.

[0089] Aspect 9 is the genetically modified microbe of any of Aspects 1-8 or 10-29, wherein the microbe is a gram-negative microbe.

[0090] Aspect 10 is the genetically modified microbe of any of Aspects 1-9 or 1 1-29, wherein the gram-negative is E. coli.

[0091] Aspect 11 is a method for increasing the presence of P22 phage in the gastrointestinal tract of a subject, including: administering to a subject a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and includes an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

[0092] Aspect 12 is the method of any of Aspects 1-11 or 13-29, wherein the subject has or is at risk of infection by S. enterica serovar Typhimurium.

[0093] Aspect 13 is a method for treating a subject having a S. enterica serovar Typhimurium infection, including: administering to a subject a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and includes an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

[0094] Aspect 14 is a method for treating a subject having a diarrhea, including: administering to a subject a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and includes an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

[0095] Aspect 15 is the method of any of Aspects 1-14 or 16-29, wherein the subject has a S. enterica serovar Typhimurium infection.

[0096] Aspect 16 is the method of any of Aspects 1-15 or 17-29, wherein the administering includes oral administration of at least IxlO2microbes.

[0097] Aspect 17 is the method of any of Aspects 1-16 or 18-29, wherein the administering includes administration of the microbe to the upper gastrointestinal tract.

[0098] Aspect 18 is the method of any of Aspects 1-17 or 19-29, wherein the administering includes administration of the microbe to the lower gastrointestinal tract.

[0099] Aspect 19 is the method of any of Aspects 1-18 or 20-29, wherein the microbe further includes a coding region encoding a C2 protein.

[0100] Aspect 20 is the method of any of Aspects 1-19 or 21-29, wherein the coding region encoding the C2 protein is operably linked to a constitutive promoter.

[0101] Aspect 21 is the method of any of Aspects 1-20 or 22-29, wherein the microbe further includes a coding region encoding a C2-inhibiting protein.

[0102] Aspect 22 is the method of any of Aspects 1-21 or 23-29, wherein the C2-inhibiting protein includes a P22 anti-repressor protein or a mutant C2 protein.

[0103] Aspect 23 is the method of any of Aspects 1-22 or 24-29, wherein the coding region encoding a C2-inhibiting protein is operably linked to an inducible promoter.

[0104] Aspect 24 is the method of any of Aspects 1-23 or 25-29, wherein the inducible promoter includes an anhydrotetracycline inducible pTet-promoter.

[0105] Aspect 25 is the method of any of Aspects 1-24 or 26-29, wherein the microbe includes rfb gene cluster coding regions and a rfc coding region.

[0106] Aspect 26 is the method of any of Aspects 1-25 or 27-29, wherein the microbe is a gramnegative microbe.

[0107] Aspect 27 is the method of any of Aspects 1-26 or 28-29, wherein the gram-negative is E. coli.

[0108] Aspect 28 is the method of any of Aspects 1-27 or 29, the method further including administering to the subject a compound that induces expression of the coding region operably linked to the inducible promoter.

[0109] Aspect 29 is a composition including a genetically modified microbe including a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase and a pharmaceutically acceptable carrier, wherein the P22 phage is present in the genome of the microbe and includes an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

[0110] EXAMPLES

[0111] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

[0112] Example 1

[0113] Virulent phage production from prophage sequences in bacteria

[0114] The genetic engineering of an E. coli bacteria that carries a lytic prophage P22 for Salmonella sv. Typhimurium inactivation that can be administered as a prophylaxis measure to protect the patient at the onset of the infection (FIG. 1). We first demonstrate the impact of the heterologous phage on bacterial growth and phage production levels and describe the design of lysogens that carry phage mutants that lead to the highest phage production levels. The defense mechanism of Salmonella was then investigated against phages coming from this bacteria sentinel and the genetic engineering modifications required to overcome Salmonella's restriction-modification (R-M) system that typically protects against phage infection. To ensure that the P22 phage produced from lysogens of E. coli is capable of solely killing Salmonella, we engineer the P22 prophage to remain lysogenic when integrated into the E. coli chromosome but to produce a virulent P22 phage when induced. The engineered bacteria also carry an inducible promoter that can trigger high levels of phage production in the presence of anhydrotetracycline (aTc). Finally, it is demonstrated that virulent P22 is effective against Salmonella in a co-culture experiment and enhances the protection against STm infection in a mouse model. Overall, the presentdisclosure describes a new therapeutic strategy to ameliorate diseases caused by enteric pathogens by precisely targeting the causative microbial factors in the gastrointestinal tract.

[0115] Results

[0116] Engineering E. coli strains carrying the heterologous P22 phage for STm inactivation.

[0117] E. coli that is lysogenized with P22 can produce high levels of P22 phage. E. coli is not a natural host for P22 phage, but if the appropriate phage receptor is expressed, it can sustain its lytic propagation and prophage integration. Previous work has shown that the plasmid pPR1347 expressing rfb and rfc genes of STm leads to the expression of the necessary O- antigen modifications to the E. coli lipopolysaccharide so that it is susceptible to P22 phage (Neal et al., 1993, J. Bacteriol., 175(21):7115-7118). It was found that the growth of E. coli strains (FIG 2A-B) and STm (FIG. 2C) were not substantially impacted by lysogeny with P22 phage. Furthermore, P22 lysogens of E. coli MG1655 and STm could produce high levels of free P22 phage within ~ 4 h of culture (FIG. 2D), whereas the mouse commensal E. coli MP1 strain showed lower phage levels. E. coli lysogens harboring phage mutants having regions of the genome replaced by a kanamycin resistance gene were also tested for phage production after growth overnight in liquid culture, demonstrating predominantly higher levels of phage mutants missing either the A or the B regions (Baaziz et al ., iScience, 2024, 27(4): 109595). The further deletion of the kanamycin cassette (AKanR) through the recombination of the FRT did not show a major impact for most of the mutants but led to high phage levels for E. coli MP1 mutants missing the D region, one of the key lysogens to establish a stable bacterial growth in the mammalian gut.

[0118] Overcoming STm restriction mechanism

[0119] Expression of modification genes from STm R-M systems can protect E. coli-Aevwe P22 phage from inactivation when infecting STm. R-M systems are frequently occurring mechanisms by which bacteria protect themselves from foreign DNA. Modification enzymes produced by STm modify the bacterial DNA in a manner that protects it from recognition by restriction enzymes. STm encodes multiple R-M systems, of which StySA and StyLTI are the most prevalent. As shown in FIG. 3 A, P22 phage produced from E. coli(P22E coh) has a substantially higher efficiency of plating (EOP) than P22 phage produced from STm (P22STm) when tested against a wildtype STm host. If one deletes the StyLTI restriction enzyme, the EOP of P22Ecrecovers nearly 3-log. Deletion of the second restriction enzyme related to the StySA system fully recovers the EOP of P22Ec. Together, this data indicates that P22E co11lacks the necessary modification to protect itself from restriction STm R-M systems and that the StyLTI R-M system has the greatest impact on P22E CO11efficacy against STm. To determine if this P22E cohefficacy against STm could be recovered, we first expressed the StyLTI modification gene from a high copy number plasmid in source E. colt MG1655. The P22 phage produced (P22E coli::pGGAsel::StyLTI) showed a near complete recovery in EOP to levels similar to P22STmwhen tested against wildtype STm (FIG. 3A). When integrated into the bacterial genome, one single copy of the StyLTI-modifi cation gene does not impact bacterial growth, regardless of the strengths of the promoter that controls the StyTLI gene expression (FIG. 3B) (Yan and Fong, J Biol Eng, 11, 33, 2017). The EOP for P22E co11increases with the promoter strength, leading to infection values for P22STmfor the phage from E. coli with StyLTLMod under the strongest promoter (FIG. 3C).

[0120] Phage levels are enhanced by chemically inducible promoter

[0121] Spontaneous phage induction is essential for the sentinel bacteria to confer protection at the onset of the infection while the pathogen levels are minimal. However, higher phage levels might be needed in in extreme cases of ingestion of excessive amounts of pathogen. The E. col22engineered herein harbors a chemically triggered genetic construct (Kotula et. al., 2014, 111(13):4838-4843) that, in the presence of anhydrotetracycline (aTc), induces the expression of a dominant-negative variant of the phage repressor gene c2 ' the cro gene and the gfp reporter. The gene product from the c2DNpresents the protein dimerization domain but lacks the DNA binding domain; therefore, either expression of a C2DNand / or Cro can induce the lytic lifecycle of the P22 phage. Although E. co / zP22carrying this construct in a plasmid presents a late growth in the presence of the chemical trigger, all bacteria presented appreciated growth levels (FIG. 4A), with a substantial fluorescence signal from GFP (green fluorescence protein), demonstrating effective induction for lysogens treated with aTc (FIG. 4B). The increase in the fluorescence signal with thebacteria growth demonstrates the consistent aTc induction with the cell growth. The 100- fold increase in phage concentration, normalized by cell growth, for lysogens treated with aTc demonstrates the potential of this approach to induce high phage levels to treat severe pathogen infections (FIG. 4C).

[0122] Generating a virulent P22 phage with stable lysogeny of E. coli

[0123] Virulent P22E eo11eliminates STm. To ensure that the primary outcome of P22 infection is bacterial lysis, it is necessary to eliminate the possibility of lysogeny. Lambdoid phages, like P22 phage, maintain lysogeny by expressing a repressor protein that functions as a genetic switch. For P22 phage, lysogeny is maintained by expression of the C2 repressor protein. The production of phage can be induced by degradation of C2, which leads to the de-repression of lytic genes encoded by P22. For P22 phage to act as an antibacterial, it cannot be capable of lysogeny once infecting STm. Therefore, to maintain a lysogenic P22 prophage in E. coli that will produce a virulent P22 phage upon induction, c2 from the E. coli chromosome in a region distal from the P22 prophage was constitutively expressed, and then the c2 gene endogenous to P22 phage was disrupted. As a result, a virulent P22 (P22vzr) phage that is stably maintained in E. coli lysogens was provided (see phenotype difference in FIG. 5A). Co-culture experiments of E.coliP22and STm lack the R-M systems demonstrate the P22LJHC P22TemPerate reach similar levels in culture after 8 h (FIG. 5B), but only the lysogens carrying the lytic version of P22 can totally eradicate STm in the conditions tested, evidencing that either the free P22tytic or the E. coli lysogens carrying the temperate phage are unable to eradicate STm.

[0124] STm inactivation in bacterial co-culture

[0125] To demonstrate that a virulent P22 phage is capable of killing wildtype STm, we cultured STm with various formulations of P22 phage. As shown in FIG. 5D, STm cultured overnight is capable of readily surviving when growing with a free lytic P22 phage (P22Lytic) or wildtype E. coli (MG1655). This also evidences that nutritional competition is not sufficient for inhibiting pathogen growth. Co-cultures with the E. coli lysogens carrying the P22 ytic demonstrate a major drop in the levels of the pathogen, with undetectable levels of STm in the presence of lysogens with the StyLTI under the two strongest promoters.

[0126] E. coli lysogens confer protection against STm in a mouse model

[0127] The efficacy of the engineered E. coli to prevent STm propagation was tested in the C57BL / 6 mouse model. The control group infected with the lowest STm dosage (102CFU) showed significant weight loss, leading to the death of all mice after 9 days (FIG. 6A-B). This contrasted with the group treated with the E. coif22, which presented a more cadenced weight loss and a significantly higher survival rate. The E. coli lysogens also demonstrated a better performance than the non-lysogenic E. coli (control) when challenged with higher STm dosage (106CFU) (FIG. 6C-D). The dramatic weight loss for the groups treated with the high STm dosage illustrates the limitation of this mouse model for studying intra- intestinal STm infections that is, even though the treatment with E.coli carrying P22 is effective, all the mice died because, in this mouse model, STm tend to escape and became systemic (Walker et al., Current Protocol, 2023, e824, Volume 3. doi: 10.1002 / cpz 1.824). Regardless, the paced weight loss and the improvement in the survival rate for the lysogenic treated groups demonstrate the efficacy of the E. colE'22for protection against STm.

[0128] These experiments also provided evidence on the bacteria and phage levels in the mammalian gut, taking their concentration in the stool as a proxy of their prevalence in the intestine. Our findings demonstrated that the E. coli strains stably colonize the mammalian gut before the STm infection (FIG. 7A). The consistent drop in the E. coli levels is due to the increase in the STm concentration, with a lower level of STm observed for the E. co IE22treated groups compared to their respective controls (FIG. 7B). Regardless of the STm dosage and the A". coli concentration over time, stable levels of P22 seem to be established in the mouse gut, which may protect against further pathogen infection even after the clearance of the prophylactic bacterial lysogens.

[0129] Conclusions

[0130] Traditional applications of phage therapy require the use of phages as treatments of disease, added after infection has started. Herein, the disclosure demonstrates the engineering of a non-pathogenic bacteria to produce phages capable of targeting bacterial species. We show that the STm phage, P22, can lysogenize E. coli and be engineered to produce virulent P22phages that eliminate STm in coculture. Using this strategy, the disclosure shows an application for these lysogens to continually produce virulent phages as a prophylactic protection against future infection.

[0131] Materials and Methods

[0132] Engineering E. coliP22lysogens.

[0133] Bacteria strains used in the study are presented in Table 1. E. coli MG 1655 strain was used to create a P22-sensitive strain by electroporation transformation using the plasmid pPR1347 carrying the rfc gene and the rfb gene cluster that for the expression the lipopolysaccharide phage receptors (Neal et al., 1993, J. Bacteriol., 175(21 ):7115-7118). This strain was lysogenized with P22 derived from Salmonella Typhimurium using the spot assay method for generating the lysogens. Molten agar was prepared by mixing 3 mL of top agar (modified LB medium with 0.5 % of agar, 10 mM calcium chloride, 10 mM magnesium sulfate, ~ 45 °C) and 100 pL of E. coli log-phage culture (ODeoo ~ 0.6), poured in a LB plate and let it harden. The top agar overlay was spot with diluted phage produced from the supernatant of Salmonella lysogen overnight culture in Luria Broth medium (LB, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) with phage stock store at 4 °C prior to use. After 12-16 h of incubation at 37 °C, plaques in the top agar overlay were streaked in LB plates supplemented with both kanamycin (50 pg / mL) and virulent p22 H5 phage (100 pL of 108pfu / mL phage suspension spread on top of the plate) and incubated overnight at 37 °C. Phage resistant lysogens were streaked again in LB plates supplemented only kanamycin (50 pg / mL) and incubated overnight at 37 °C to generate the purified E. coliP22lysogens.

[0134] Table 1. Bacteria strains and phage used in this study.Bacteria and phage strains DescriptionE. coll Strai n MG 1655E.Strain MG1655 transformed with plasmid pPR1347 from E. coli 2442E. coliP22Strain MG1655 lysogenized by P22 phage derived from Salmonella enterica sv. Ty phi murium Salmonella™ Strain Salmonella enterica sv. Typhimurium lysogenized with P22::AD::kanRprophageE. coli TB10 Strain MG1655, nadA::TnlO cI857 A(cro-bioA)(Johnson et al., J. Bacteriol., 2004 , 186(8):2418-2429)E. coli'. :pKD46 E. coli MG1655 carrying the pKD46 plasmidP22 P22AD phage originally obtained from Salmonella enterica sv. Typhimurium and modified as described elsewhereP22-Lytic P22AD phage mutant with two stop codons added at the beginning of the c2 geneP22-H5 P22 lytic phagePl Pl lytic phage for transduction -lytic Lambda lytic phage for E. coli lysis

[0135] Molecular cloning.

[0136] Gene constructs were prepared using Golden Gate Assembly using Q5 High-Fidelity polymerase (NEB) to amplify all the fragments with the Bsal recognition site and complementary overhangs. For phage repressor engineering (c2 phage repressor gene), the homology arms were PCR amplified, ampicillin resistance cassette flanked by FRT sites, proC promoter, and the c2 phage gene. The defective phage repressor was prepared by PCR amplifying the homology arms, the kanamycin resistance cassette (Tn5 neomycin phosphotransferase) flanked by FRT sites, TetR repressor, TetA promoter, and only part of the phage repressor that encodes the C-terminal of C2 repressor protein (c2DN, dominant-negative phage repressor). A repressor version that presents the cro gene, and the gfp reporter downstream to the cPN, separated by the insulator and the RBS sequences (Levin- Karp et al., ACS Synth. Biol., 2013, 2, 6, 327-336) were tested. For the restrictionmodification system, the homology arms were amplified, the gene that encodes DNA methylase with the kanamycin cassette (aph gene) replacing the gene restriction (StyLTI R’ M+). The source for each fragment amplification, primers and oligos, plasmids, and promoter are detailed in Tables 2, 3, 4, 5, respectively. Golden Gate reactions were conducted with NEB ridge Golden Gate Enzyme Mix (Bsal-HF v2), T4 DNA Ligase Reaction Buffer, and the pGGAselect DNA cloning vector. Ligation reactions were transformed by heat-shock in NEB 5-alpha competent E. coll (New England Biolabs), and selected using the antibiotic cassette in the gene construct. Selected strains were used as a template to amplify the entire genome construct and had their genome confirmed by Sanger sequencing. The constructs nomenclature, sequence and amplicon are described in Tables 2-5.

[0137] Table 2. Source for fragment amplification.Gene construct Amplicon Source c2 phage Upstream homology arm to araB E. coli MG1655 repressor regionFRT-ampicillin cassette-FRT pKD46 plasmid c2 repressor geneDownstream homology arm to araC E. coli MG1655 region c:PN::cro::gfp Upstream homology arm to lacZ E. coli MG1655 defective phage region repressorFRT-kanamycin cassette-FRT pKD13TetR::TetA promoter E. coli MG1655::tetP:: cro::araCC-terminal of p22 phage repressor P22 cro gene P22Super-folded gfp gene pSLQ1211 (Stirling et al., 2017, Molecular Cell 68:686- 697)Downstream homology arm to lacZ E. coli MG1655 regionStyLTI R M+Upstream homology arm E. coll MG1655 system StyLTI modification gene STm 14028s:: StyLTI: :Res’ (Porwollik et al., 2014, PLoS ONE 9(7): e99820. doi: 10.1371 / journal. pone.0099820)FRT-kanamycin cassette-FRT pkD4Upstream homology arm E. coli MG1655

[0138] Table 3. Primers and oligos used in this study.

[0139] Table 4. DNA for the genetic engineered constructs. Homology arms sequence are underlined and the functional gene sequence is marked in bold.

[0140] Table s. Nucleotide sequences for engineered promoters that control the StyLT-Res‘ gene.Sequences in underline and italics correspond to the homology region in E. coli (rbsA gene) and the homology region in the StyLTl Res' gene. The nucleotide sequence in upper case letters corresponds to the chloramphenicol resistance gene and its promoter (both from the commercially available pGGAselect plasmid). The UP sequence (upstream element) is indicated in bold, the promoter sequence is underlined, and the RBS sequence is in italic (Yan and Fong, J Biol Eng 11, 33 (2017). doi.org / 10.1186 / sl3036-017-0075-2).

[0141] Bacterial engineering.

[0142] For genome construct integration into the E. coliP22lysogens, phage repressor, trigger, and DNA methylation gene constructs were firstly recombined into E. coli genome via lambda red homologous recombination (Datsenko & Wanner, 2000, PNAS, 97(12):6640-6645. 10.1073 / pnas.120163297), followed by Pl transduction of each individual construct and antibiotic selection using the process described elsewhere (Thomason et al., Curr Protoc Mol Biol., 2007 JukChapter 1 : 1.17.1-1.17.8. doi: 10.1002 / 0471142727.mb0117s79). Briefly, phage repressor gene construct was integrated into E. coli TB10 strain using the genes integrated into the bacterial genome (Johnson et al., J. Bacteriol., 2004 , 186(8):2418-2429) and Pl transduced to A. coliP22lysogens followed by antibiotic selection with LB-agar plates supplemented with ampicillin (E. coliP22c2). After recombination, both kanamycin and ampicillin antibiotic cassettes were eliminated by expressing the FLP recombinase gene from the pCP20 helper plasmid that was later cured by growing the cells at 37 °C. Oligo recombination was then conducted into the P22 prophage (Cenens et al., 2013, PLoS Genet 9(2): el003269. doi: 10.1371 / journal.pgen.1003269) mutant with two stop codons at the beginning of the c2 gene of the prophage that goes directly to the lytic cycle when infecting Salmonella. Lysogens carrying the lytic prophage were screened by generating phage supernatant for each candidate and assessing the plaque phenotype for infection in E. coli susceptible to P22. Candidates that generated phage that led to clear plaques were Sanger sequenced to confirm the mutation into the c2 repressor gene into the prophage. Streptomycin resistant mutants were generated by spontaneous mutation by platting overnight culture of E. coliP22' Lytlc: c2 into LB-agar plates supplemented with streptomycin, followed by Sager sequencing to confirm the mutation into the rpsL gene that confers streptomycin resistant. E. coliP22'L rtwc2::StrRreceived by Pl transduction the StyLTI gene from the E. coli MG1655, and selected by LB-agar plates supplemented with kanamycin, followed by a new round of antibiotic cassette deletion using the FLP recombinase expression from pCP20. Lastly, the E. coliP22'yilcc2: :StrR: : StyLTI obtained by Pl transduction the construct with the defective repressor, under the pTet triggered promoter, from the E. coli TB10, followed by selection with LB-agar plates supplemented with kanamycin. The final E. coli strain carrying all the gene constructs, E. coliP22~Lytlc::SttJi::c2:: StyLTI: :c2DN:: Kat iRwas used to conduct both the in-vitro competition experiments and the mouse experiments for Salmonella killing.

[0143] Bacterial Growth.[00144J Salmonella and E. coli strains harboring the prophage or the different constructs were grown in LB broth, supplement with different antibiotic depending on the construct, at 37 °C and 200 rpm of orbital shaking for 12-16 h. For bacteria growth experiment, overnight growth cultures were back diluted in LB-broth to a ODeoo -1.25 and 100-fold diluted into 96-multiwell plates to investigate bacteria growth in the plate reader at 37 °C for 12 h based on the cell culture optical density.

[0145] Phage Production.

[0146] High phage titer stocks were prepared using the plaque assay method. Log-phase cultures (100 pL, ODeoo -0.6) of P22-susceptible E. coli or Salmonella in LB broth supplemented with the kanamycin (50 pg / mL) was first mixed with P22 phage from Salmonella (100 pL, -106pfu / mL) and let it sit on the bench for 30 min. This mixture was used to inoculate 3 mL of molten top agar (LB with 0.5 % agar, supplemented with 10 mM CaCh and 10 mM MgSCL at 45 °C) and immediately poured into LB agar plates to harden. After 12-16 h incubation at 37 °C, the top agar from the highest density of plaques was resuspended in 3 mL sterile phage buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCh, and 0.01% gelatin at pH 7.5), and incubated static at 4 °C for 1-2 h. The mixture was pelleted by centrifuging at 3,700 rpm for 10 min at 4 °C, and the supernatant was collected and filtered through a 0.45 pm syringe filter. This phage lysate for different strains was stored at 4 °C before using. To investigate phage production over a time, E. coliP22or Salmonellcf22cultures in LB broth supplemented with the kanamycin (50 pg / mL) were prepared by overnight incubation (12- 16 h) at 37 °C and 200 rpm orbital shaking. Cells were pelleted (same condition as above) and resuspended in LB broth with kanamycin followed by pelleting. This washing cycle was repeated once more and the cells were resuspended in LB broth with kanamycin, back diluted to a ODeoo of 1.25, and used to inoculate 5 mL of LB broth with kanamycin. After incubation (37 °C, 200 rpm orbital shaking) for 1 to 16 h, the cell culture was treated with3-4 drops of chloroform, pelleted, and the supernatant was filtered through a 0.45 pm syringe filter for storage at 4 °C.

[0147] Quantification of Phage Infection and Efficiency of Plating.[00148J Phage titer was always verified using top agar overlay method against P22-susceptible E. coli. Log-phage culture for P22-susceptible E. coli (100 pL, ODeoo ~0.6, prepared in LB broth supplemented with kanamycin) was used to inoculate 3 mL of molten top agar and poured in immediately poured into LB agar plates to harden. After 30 min, 2 pL of 10-fold serial diluted phage pipetted in the top agar layer, let it dry under the flame and incubated at 37 C overnight. Spots presented individual plaques were exanimated to calculate the plaque forming units per milli-liter of phage stock (PFU / mL). For the efficiency of platting (EOP), the number of plaques forming units was determined based on the plaque assay method with P22-susceptible E. coli or Salmonella mutants in the top agar overlay (100 pL, ODeoo -0.6), and 100 pL of phage stock from Salmonella or different E. coli strains. After 37 °C overnight incubation, the number of plaques for each Salmonella mutant plate was divided by the plaques in E. coli plate to determine the EOP.

[0149] Integration and testing of different promoter strengths on StyLTI Res'

[0150] E. coli lysogens were modified with different promoters upstream of the StyLTI Mod gene to control the levels of DNA phage methylation and, ultimately, the EOP in wild-type STm. Lysogens were first transformed with pKD46, made competent, and transformed with SOO- SOO ng of the linear amplicons harboring the chloramphenicol-resistant gene and one of the different promoters. The amplicons with different promoters were obtained by amplification (with primers RB127-RB128) of the ordered G-Blocks (Table 3). Recombinants were selected in LB plates supplemented with chloramphenicol and streak purified for colony purification (Johnson et al., J. Bacteriol., 2004 , 186(8):2418-2429). Purified lysogens were used to produce phage in overnight liquid culture, and the purified phage was tested for EOP against wild-type STm.

[0151] Chemically induced phage production.

[0152] E. coli lysogen cultures were prepared by inoculating LB broth and incubating at 37 °C and 220 RPM for 12-16 h. Cell cultures harboring the chemical inducible genetic construct pTet::c2DN::cro::gfp, either in a plasmid or integrated into the bacterial genome, were incubated in LB broth supplemented with kanamycin. The cell cultures we pellet by centrifugation (6,000 g, 2 min at 25 °C) and resuspended in LB-broth or LB-kanamycin. This step was repeated for three times, and the cells had their ODeoo first adjusted to 0.125. This culture was back-diluted 100-fold in the same media and incubated in a plate reader for growth under shaking at 37 °C. After 2 h, the culture was induced with anhydrotetracycline to a final concentration of 100 ng / mL and incubated for 16 h to measure the cell growth based on the optical density and the promoter induction based on the GFP fluorescence signal (ex 485 nm / em 528 nm) recorded each 5 min. A similar experimental setup was carried out to measure phage production 8 h after aTc induction. The cell culture was treated with 2-3 drops of chloroform, centrifuged (6,000 g, 10 min at 4 °C) and the supernatant was sterile filtered (0.22 pm) and stored at 4 °C. Phage titer was determined by the spot assay method for the STM: AStyLTIASTySA as cell host in the top agar.

[0153] Coculture for Salmonella Inactivation in-vitro.

[0154] For this experiment, Salmonella and different E. coli strains were prepared overnight at 37 °C in LB broth supplemented with chloramphenicol (25 pg / mL) or kanamycin (50 pg / mL), respectively. All strains were washed twice with LB-broth and resuspended in LB-only to a ODeoo -1.25. LB-broth (5 mL) was inoculated with E. coli (50 pL), Salmonella (0.5 pL) or both. When required, P22 ytic from E. coli (50 pL, 106PFU / mL) or X-lytic from E. coli (50 pL, 106PFU / mL) were also added to the co-culture. The following scenarios were used for testing Salmonella inactivation: (i) Salmonella-orAy , (ii) Salmonella and P22i.vuc , (iii) Salmonella and E. coli, (iv) Salmonella and E. coif22, (v) Salmonella and E. colf22Ayiw, (vi) Salmonella, E. coli, and L-lytic, and (vii) Salmonella, E. coli, and P22i.vtic. After incubation for 8 h at 37 °C and 200 rpm, bacterial culture was 10-fold serial diluted and plated in LB- agar plates supplemented with chloramphenicol for Salmonella detection, or kanamycin for E. coli detection. Plates were examined after overnight incubation at 37 °C to determine the number of colony-forming units per milliliter of overnight culture (CFU / mL). Phage titerfor the phage stock harvested from each co-culture was determined using the spot assay using the top agar overlay supplemented with P22-susceptible E. coll.

[0155] Animal Experiment.[00156J Animal work was conducted according to the VT Biological Sciences Department Protocol using the department's animal facilities (IACUC protocol #23-260). Male C57BL / 6 mice (Jackson Labs), 12-14 years old, were acclimated in the vivarium, having food and drinking water ad libitum. Drinking water was replaced with a streptomycin sulfate USP grade solution (5 g / L) 24 h before E. coli oral gavaging. The gavaged bacteria was prepared by inoculating LB-broth supplemented with 50 pg / mL streptomycin and 25 pg / mL chloramphenicol with one single E. coli, followed by incubation for 12-16 h at 37 °C. The cell culture was pelleted by centrifugation (3,000 g, 10 min at 4 °C) and resuspended in PBS. This procedure was repeated twice, and the bacteria solution had the ODeoo adjusted to yield 109CFU / mL. Two mice groups (6 mice each) were orally gavaged with 100 pL of non-lysogenic E. coli (control groups), and the other two were gavaged with the engineered E. coli*22.

[0157] The groups were infected with STm four days later. STm cultures were prepared similarly to E. coli, using LB broth supplemented with 50 pg / mL streptomycin and 25 pg / mL tetracycline. After PBS washes, cultures were adjusted to 103and 107CFU / mL, and 100 pL were orally gavaged to the mice groups after 4 h of fasting. One of the non-lysogenic E. coli and one of the E. coli lysogenic treated groups received the low STm dosage; the same applied to the high STm dosage.

[0158] Mice weight loss and health indicators were recorded daily. Stool samples were collected each day to quantify the bacteria and phage in the gut. Phage was quantified by immediately resuspending the collected stool samples in phage buffer using sterile wooden sticks (50 mg stool / mL) and keeping them on ice. Samples were treated with 2-3 drops of chloroform, vortexed, and centrifuged (6,000 g, 10 min at 4 °C). The supernatant was serially diluted, and the phage titer was determined using the spot assay method in a top agar overlay with STm::AStyLTIASTySA as the bacteria host. Both E. coli and STm were quantified by resuspending the fresh stool pellets in PBS using sterile wooden sticks (50mg stool / mL) and keeping them on ice. The resuspended samples were serially 10-fold diluted and spiral-platted in MacConkey agar plates supplemented with chloramphenicol for E. coli quantification and tetracycline for STm quantification.

[0159] Mice were euthanized (CO2 and cervical dislocation) before the end of the experiment in case the weight loss was below 20% or if they looked excessively lethargic. After 14 days of STm infection, all the remaining mice were euthanized. Data were analyzed in Excel and GraphPad Prism.

[0160] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e g., GenBank and RefSeq, and amino acid sequence submissions in, e g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0161] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0162] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0163] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

CLAIMS1. A genetically modified microbe comprising a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and comprises an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

2. The genetically modified microbe of claim 1, wherein the microbe further comprises a coding region encoding a C2 protein.

3. The genetically modified microbe of claim 2, wherein the coding region encoding the C2 protein is operably linked to a constitutive promoter.

4. The genetically modified microbe of claim 2, wherein the microbe further comprises a coding region encoding a C2-inhibiting protein.

5. The genetically modified microbe of claim 4, wherein the C2-inhibiting protein comprises a P22 anti-repressor protein or a mutant C2 protein.

6. The genetically modified microbe of claim 4, wherein the coding region encoding a C2-inhibiting protein is operably linked to an inducible promoter.

7. The genetically modified microbe of claim 6, wherein the inducible promoter comprises an anhydrotetracycline inducible pTet-promoter.

8. The genetically modified microbe of claim 1, wherein the microbe comprises rfb gene cluster coding regions and a rfc coding region.

9. The genetically modified microbe of claim 1, wherein the microbe is a gramnegative microbe.

10. The genetically modified microbe of claim 9, wherein the gram-negative is E. coli.

11. A method for increasing the presence of P22 phage in the gastrointestinal tract of a subject, comprising: administering to a subject a composition comprising a genetically modified microbe comprising a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and comprises an inactivated coding region encoding a c2 protein, or encodes an inactive c2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

12. The method of claim 11, wherein the subject has or is at risk of infection by S. enterica serovar Typhimurium.

13. A method for treating a subject having a 5. enterica serovar Typhimurium infection, comprising: administering to a subject a composition comprising a genetically modified microbe comprising a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and comprises an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

14. A method for treating a subject having a diarrhea, comprising:administering to a subject a composition comprising a genetically modified microbe comprising a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase, wherein the P22 phage is present in the genome of the microbe and comprises an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

15. The method of claim 14, wherein the subject has a S. enterica serovar Typhimurium infection.

16. The method of any one of claims claim 11, 13, or 14, wherein the administering comprises oral administration of at least IxlO2microbes.

17. The method of any one of claims claim 11, 13, or 14, wherein the administering comprises administration of the microbe to the upper gastrointestinal tract.

18. The method of any one of claims 11, 13, or 14, wherein the administering comprises administration of the microbe to the lower gastrointestinal tract.

19. The method of any one of claims claim 11, 13, or 14, wherein the microbe further comprises a coding region encoding a C2 protein.

20. The method of claim 19, wherein the coding region encoding the C2 protein is operably linked to a constitutive promoter.

21. The method of claim 19, wherein the microbe further comprises a coding region encoding a C2-inhibiting protein.

22. The method of claim 21, wherein the C2-inhibiting protein comprises a P22 antirepressor protein or a mutant C2 protein.

23. The method of claim 21, wherein the coding region encoding a C2-inhibiting protein is operably linked to an inducible promoter.

24. The method of claim 23, wherein the inducible promoter comprises an anhydrotetracycline inducible pTet-promoter.

25. The method of any one of claims 11, 13, or 14, wherein the microbe comprises rfb gene cluster coding regions and a rfc coding region.

26. The method of any one of claims 20, 22, or 23, wherein the microbe is a gramnegative microbe.

27. The method of claim 26, wherein the gram-negative is E. coli.

28. The method of claim 23, the method further comprising administering to the subject a compound that induces expression of the coding region operably linked to the inducible promoter.

29. A composition comprising a genetically modified microbe comprising a P22 phage and at least one exogenous coding region encoding a Salmonella modification system methylase and a pharmaceutically acceptable carrier, wherein the P22 phage is present in the genome of the microbe and comprises an inactivated coding region encoding a C2 protein, or encodes an inactive C2 protein, and wherein the microbe is not Salmonella enterica serovar Typhimurium.

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