Modified Escherichia coli strain NISSSLE and treatment of gastrointestinal disorders

Abstract

The present invention relates to the field of modified Escherichia coli strain Nissle 1917 (EcN) and its use in the treatment of gastrointestinal diseases. The present invention is based on the study of the mechanism of probiotic properties of Escherichia coli strain Nissle 1917 (EcN). This study demonstrates that the EcN ClbP protein (an enzyme that activates the genotoxin colicin) is also required for the siderophore-microcin activity of the probiotic EcN, but interestingly, it is not its enzyme domain that cleaves the colicin precursor to form active colicin. Therefore, this study opens the way for the safe use of EcN, and therefore the present invention provides an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding a ClbP protein that inactivates the peptidase domain, and its use as a medicament, in particular for the treatment of gastrointestinal diseases.

Description

Technical Field

[0001] The present invention generally relates to the field of modified Escherichia coli strain Nissle 1917 (EcN) and its use for treating gastrointestinal disorders. Background Art

[0002] The probiotic Escherichia coli strain Nissle 1917 (EcN) was isolated by Alfred Nissle from soldiers fighting a severe diarrheal outbreak during World War I (1, 2). EcN was initially studied for its ability to combat bacterial gastrointestinal infections. It has been demonstrated to prevent intestinal colonization with enterohemorrhagic serovars of S. Typhimurium (3, 4) and to exhibit antibacterial activity against enterohemorrhagic E. coli strains (5). EcN is an excellent colonizer of the human intestine and has shown beneficial effects on a variety of intestinal disorders, such as acute diarrhea in infants and young children (6), chronic constipation (7), and abdominal pain in patients with irritable bowel syndrome (8). It has been widely used to treat inflammatory bowel disease (1) and has been shown to be as effective as the gold standard mesalamine for maintaining remission in children and adults with ulcerative colitis (9).

[0003] The probiotic activity of EcN is thought to be based on a variety of specific properties and fitness determinants, including antimicrobial activity against other bacteria (10). EcN reduces S. Typhimurium intestinal colonization by competing for iron, thanks to a large number of siderophores (enterobactin, sammycin, yersinia, and aerobactin) and multiple siderophore receptors and iron transport systems (3). Enterobactin, sammycin, and yersinia are nonribosomal peptides (NRPs) or polyketide (PK)-NRP hybrids that are synthesized by NRP synthases and PK synthases (NRPSs and PKSs) activated by cognate phosphopantetheine transferases (PPTases). In addition to this competition for limiting nutrients, EcN also exhibits direct antimicrobial activity associated with the production of two microcins (Mcc), H47 (MccH47) and M (MccM) (4, 11-13). Mcc is a secreted low-molecular-weight peptide that is synthesized by the ribosome and undergoes post-translational modifications, and it exhibits potent bactericidal activity against phylogenetically related bacteria (14,15). MccH47 and MccM are referred to as “siderophore-Mcc” because they are post-translationally modified by the attachment of catechol siderophores (13,16). The C-terminus of the Mcc peptide is covalently linked to a linearized and glycosylated derivative of enterobactin (13,16,17). This siderophore moiety is recognized by the catechol siderophore of the target bacteria (12,16). Therefore, siderophore-Mcc can enter and kill sensitive bacteria through a “Trojan horse” strategy by mimicking the iron-siderophore complex.

[0004] Comparative genomic analysis showed that EcN is closely related to pathogenic E. coli strains such as the uropathogenic strain CFT073 (18-20). EcN and CFT073 share eight genomic islands, including the pks / clb island that encodes the NRPS-PKS assembly line that synthesizes the genomic toxin colibactin (21, 22). Colibactin is produced as a prodrug moiety and exported in the periplasm by the efflux pump ClbM (23), where it is then hydrolyzed by the periplasmic membrane-bound ClbP protein with peptidase activity to release active colibactin (24, 25). Colibactin is not only a bona fide virulence factor (26, 27) but also a putative carcinogenic compound. Colibactin alkylates host cell DNA, leading to DNA cross-links, double-strand breaks, chromosomal aberrations, and genetic mutations in vitro and in vivo (21, 22, 28-30). Colibactin-producing E. coli are overrepresented in biopsies from patients with colorectal cancer (31,32) and have been shown to promote colorectal cancer in mouse models (31,33).

[0005] The contradiction between the pathogenicity and probiotic potential of EcN was revealed when the inventors showed that certain enzymes of the pks / clb island are able to synthesize analgesic lipopeptides (34) and that the probiotic properties of EcN are associated with the presence of the pathogenicity island (35). In the study of Olier et al. (2012), the inventors inactivated the gene encoding the phosphopantetheine transferase (PPTase) ClbA, which was considered specific for the synthesis of colicin. However, recent studies have shown that ClbA has pleiotropic effects and also regulates the synthesis of siderophores as well as the synthesis of analgesic lipopeptides (34, 36). As expected, the use of probiotic strains that produce genotoxins is a public health problem. Therefore, the inventors attempted to clearly separate genotoxicity from probiotic activity. In this study, the inventors used their knowledge of the biosynthetic pathways of colicin and other secondary metabolites produced directly or indirectly by the pks / clb island to specifically eliminate the genotoxic activity of colicin. The inventors examined the mutants' ability to inhibit pathogen growth while still producing beneficial secondary metabolites. The inventors successfully separated the probiotic activity from the genotoxic activity, opening a path to optimizing EcN. However, the inventors were surprised to observe that the pks / clb island was more closely linked to the probiotic activity of EcN than we expected, and that pathogenicity and probiotic properties in bacteria co-evolved. EcN is in some ways like the "magic drug" aspirin. Although, like aspirin, this bacterial strain has been used successfully for more than a century, it is critical to understand the method of action and consider safety and potential side effects. Summary of the invention

[0006] The present invention is based on the study of the mechanisms involved in the probiotic properties of the Escherichia coli strain Nissle 1917 (EcN). In addition to producing two siderophores-microcins (Mcc) responsible for its direct antimicrobial action, EcN also synthesizes the genotoxin colicin encoded by the pks island. Colicin is a virulence factor and a recognized carcinogenic compound. Therefore, this study allowed the inventors to separate the probiotic activity of EcN from its genotoxic activity. They demonstrated that the pks-encoded ClbP, the enzyme that activates the genotoxin colicin, is required for the siderophore-microcin activity of the probiotic EcN, but interestingly, not its enzyme domain that cleaves the colicin precursor into active colicin. This study allowed the inventors to separate the siderophore-microcin from the genotoxic activity by specifically targeting the peptidase domain of ClbP, which opens the way for the safe use of EcN. In fact, the ClbP peptidase activity used for colicin production has no role in the production of siderophore-microcins, which provides a method for constructing a non-genotoxic strain that retains its antibacterial activity. In addition, the inventors demonstrated in an in vivo animal model infected with an enteric bacterial pathogen (S. Typhimurium) that the administration of an EcN modified strain with a clbP gene encoding a ClbP protein with an inactivated peptidase domain (i.e., ClbP mutated at position S95) was non-genotoxic (no colicin production) but maintained bacterial antagonist activity and reduced pathogen colonization and virulence by maintaining the production of siderophore-microcins (Figure 8).

[0007] Therefore, in a first aspect, the present invention provides an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding a ClbP protein with an inactivated peptidase domain, wherein the peptidase domain of the ClbP protein having the amino acid sequence of SEQ ID NO: 3 is involved in the activation of the genotoxin colicin.

[0008] A second object of the present invention relates to the Escherichia coli strain Nissle 1917 (EcN) bacterium as defined above, for use as a medicament.

[0009] A third object of the present invention relates to the Escherichia coli strain Nissle 1917 (EcN) bacterium as defined above, for use in the treatment of gastrointestinal diseases. DETAILED DESCRIPTION OF THE INVENTION

[0011] Modified Escherichia coli strain Nissle 1917

[0012] In a first aspect, the present invention provides an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding a ClbP protein with an inactivated peptidase domain,

[0013] The peptidase domain of the ClbP protein having the amino acid sequence of SEQ ID NO: 3 is involved in the activation of the genotoxin colicin.

[0014] The inventors have shown that the native (wild-type) ClbP protein having the amino acid sequence of SEQ ID NO: 1, i.e. the enzyme that activates the genotoxin colicin, is required for the siderophore-microcin activity of the probiotic EcN, but interestingly, not its enzymatic domain that cleaves the colicin precursor into the active colicin. In particular, the inventors have shown that mutant EcN bacteria according to the invention encoding a gene inactivated only for the peptidase domain of the ClbP protein (i.e. mutants S95A, S95R, K98T or ClbP-3H), these mutants retain their antibacterial activity in vitro and in vivo (by siderophore-MCC production), but not mutant EcN bacteria with an inactivated gene encoding a complete ClbP protein (i.e. see the Examples section). Figure 4 ). These results demonstrate that the ClbP peptidase activity for colicin production has no role in the production of siderophores-microcins, which provides a method for constructing non-genotoxic ecobiotic strains that retain their antibacterial activity. Therefore, the mutant EcN bacteria according to the invention elicit a safer and protective response and therefore constitute very promising new probiotics, as demonstrated by the in vivo data using the modified EcN strains according to the invention (see Figure 8).

[0015] The term “Escherichia coli strain Nissle 1917” (also known as EcN or DSM6601) refers to the isolate of E. coli by Alfred Nissle from soldiers fighting a severe diarrheal outbreak during World War I (1,2). EcN was initially studied for its ability to combat bacterial gastrointestinal infections. Enteric serotypes of Salmonella typhimurium have been shown to prevent intestinal colonization (3,4) and to exhibit antibacterial activity against enterohemorrhagic E. coli strains (5). EcN is an excellent colonizer of the human intestine and exhibits beneficial effects on a variety of intestinal disorders, such as acute diarrhea in infants and young children (6), chronic constipation (7), and abdominal pain in patients with irritable bowel syndrome (8). It has been widely used to treat inflammatory bowel disease (1) and has been shown to be as effective as the gold standard, mesalamine, for maintaining remission in children and adults with ulcerative colitis (9).

[0016] Escherichia coli Nissle 1917 (EcN) is (Ardeypharm GmbH, Herdecke, Germany), It is a probiotic drug licensed in several countries for the treatment of a variety of intestinal diseases (10). ECN is known to have a genomic island called pks, which carries a gene cluster capable of synthesizing hybrid peptidoglycans, especially a genotoxin called colicin (21). Colicin is a structurally uncharacterized PK-NRP that is thought to be produced by a prodrug called colicin precursor, which has not been fully elucidated structurally (Li, ZR et al. Nat Chem Biol 12, 773-5 (2016); Bode, HB Angew Chem Int Ed Engl 54, 10408-11 (2015)). This toxin is produced by a complex biosynthetic mechanism involving the sequential action of proteins ClbA to ClbS (Taieb, F., et al EcoSal Plus 7 (2016)). The core machinery consists of three polyketide synthetases, three non-ribosomal peptide synthetases and two hybrid PKS-NRPSs. This mechanism also employs additional maturation proteins and efflux pumps.

[0017] Colicin is produced as a prodrug moiety that is exported into the periplasm via the efflux pump ClbM (23) and then hydrolyzed by the periplasmic membrane-bound ClbP protein with peptidase activity, releasing active colicin (24, 25). Colicin is not only a bona fide virulence factor (26, 27) but also a putative carcinogenic compound.

[0018] The term "ClbP protein" refers to a peptidase encoded by the pks genomic island of Escherichia coli (and other Enterobacteriaceae). The ClbP protein contains an N-terminal signal sequence that targets the protein to the inner membrane, a periplasmic peptidase domain containing a protease active site (AA residues 39-337 forming the peptidase), and three C-terminal transmembrane helices (AA residues 390-412, 433-455, and 465-485 forming the three transmembrane helices). The ClbP crystal structure and mutagenesis experiments revealed a serine active site and primary structural features associated with peptidase activity (24). ClbP allows maturation of the colicin precursor in genotoxic colicin by the ClbP peptidase activity that removes the N-acyl-D-asparagine prodrug scaffold from the colicin precursor (24, 25). S95 K98 and Y186 are key residues for ClbP peptidase activity, and mutants of these residues are unable to cleave the colicin precursor to release the mature active genotoxin (24, 25).

[0019] The ClbP sequence is shown in Table 1 below:

[0020]

[0021]

[0022]

[0023] According to the present invention, the term "gene encoding a ClbP protein inactivated for the peptidase domain" refers to a gene with a mutation that encodes a non-functional peptidase domain (such as ClbP S95, K98, Y186 mutants) or no peptidase domain at all (such as ClbP-3H mutant). According to the present invention, the inactivation of a specific domain of a gene can be carried out by various methods known to the skilled person. Examples of methods for inactivating a gene are in particular directed mutagenesis or homologous recombination, as described in Conde-Alvarez R. et al., Cell Microbiol 2006 Aug; 8 (8): 1322-35. Those skilled in the art also know how to design specific "nucleases" or "endonucleases" (such as CRISPR) to introduce gene point mutations to inactivate the peptidase domain of the ClbP protein or to genetically specifically recombinantly replace the peptidase domain of the ClbP protein with an inactive equivalent.

[0024] A specific method for inactivating specific domains of the genes according to the invention is also described in the experimental part (gene mutation by the lambda Red recombinase method (see 37)).

[0025] Thus, typically, the EcN mutants according to the invention (i) have the ability to possess antibacterial activity (through the synthesis of the siderophores - MCC: MccH47 and MccM); ii) they lack the ability to activate the genotoxin colicin (have an inactive ClbP peptidase domain).

[0026] A person skilled in the art can easily determine whether the EcN bacteria according to the present invention have biological activity. For example, the ability to have antibacterial activity can be determined, for example, by any conventional test known to a person skilled in the art: antibiotics, competitive growth test, ...

[0027] The effect of the antimicrobial activity can be measured using an in vitro assay (competitive growth assay) by monitoring the reduction of a target bacterial strain (sensitive to EcN, e.g., E. coli strain LF82) present on a surface before and after application of a mutant EcN composition according to the invention. Both the producer strain and the target strain (i.e., EcN or EcN mutant and LF82, respectively) are inoculated as described above (3) and in the Examples + Figures 1-5.

[0028] The determination of colicin-induced genotoxic effects (for EcN mutant candidate strains) can be measured by monitoring colicin-induced cellular senescence and the associated cell enlargement called megakaryocytosis. HeLa cells were infected with EcN candidate strains for 4 h as previously described (40). The genotoxicity of EcN and EcN mutants (i.e., clbP-S95R) was confirmed by in-cell Western procedures as previously described (36). Briefly, HeLa cells were infected at a given multiplicity of infection (number of bacteria per cell at the beginning of infection) for 4 h. Four hours after the end of infection, cells were fixed, permeabilized, and stained with rabbit monoclonal anti-γ-H2AX and infrared fluorescent secondary antibodies.

[0029] As used herein, a "biologically active" EcN mutant according to the present invention refers to an EcN mutant that exhibits all the biological activities of an EcN mutant, provided that the biologically active mutant retains the wild-type EcN ability to be antibacterial, but lacks the ability to activate the genotoxin colicin. For example, a biologically active EcN mutant according to the present invention may be characterized in that it is capable of having antibacterial activity (by producing the siderophore-MCC); and ii) lacks the ability to activate the genotoxin colicin (having an inactivated ClbP peptidase domain) (see Examples and Figure 5).

[0030] In a specific embodiment of the present invention, the ClbP protein inactivated for the peptidase domain is selected from the following list:

[0031] ClbP protein mutated at position S95,

[0032] ClbP protein mutated at position K98,

[0033] ClbP protein mutated at position Y186,

[0034] ClbP protein without peptidase domain (SEQ ID N° 3).

[0035] In the present invention, the numbering of amino acids with respect to mutations (on-time mutation or reorganization of the peptidase domain) is performed according to the wild-type ClbP protein sequence (SEQ ID NO: 1).

[0036] In a preferred embodiment, the ClpP protein mutated at position S95 is preferably not substituted by an equivalent polar and uncharged amino acid of serine. Therefore, the ClbP protein mutated at position S95 is preferably not substituted by threonine, asparagine or glutamine.

[0037] In a preferred embodiment, the ClbP protein mutated at position K98 is preferably not substituted by an equivalent positively charged amino acid of lysine. Therefore, the ClbP protein mutated at position K98 is preferably not substituted by arginine, asparagine or histidine.

[0038] In a preferred embodiment, the ClbP protein mutated at position Y186 is preferably not substituted by an equivalent amino acid having a hydrophobic side chain of tyrosine. Therefore, the ClbP protein mutated at position Y186 is preferably not substituted by alanine, valine, isoleucine, leucine, methionine, phenylalanine or tryptophan.

[0039] In a preferred embodiment, the ClbP protein does not contain a peptidase domain, and the peptidase domain of ClbP is replaced or substituted by a peptide domain with a molecular weight of 15-150 kDa, preferably 25-100 kDa, more preferably 30-80 kDa, even more preferably 30-50 kDa.

[0040] This replacement with a peptide domain (equal in weight but without any ClbP peptidase activity, which involves the removal of the N-acyl-D-asparagine prodrug scaffold from the colicin precursor to form colicin (24, 25)) allowed the preservation of the three-dimensional structure and organization of the mutant ClbP protein and maintained antibacterial activity.

[0041] Examples of peptide domains that can be used instead of the ClbP peptidase domain are the mature form of the alkaline phosphatase PhoA encoded by the E. coli phoA gene, or the mature form of the enzyme β-lactamase encoded by the bla gene, or the mature form of the enzyme β-galactosidase encoded by the E. coli lacZ gene.

[0042] Examples of these peptide domains (alkaline phosphatase PhoA, enzymes β-lactamase, β-galactosidase) are the most commonly used, especially alkaline phosphatase PhoA (as in this study) as reporter proteins for gene fusion studies in prokaryotes, especially transmembrane fusion proteins with periplasmic domains (see review van Geest M. and. Lolkema JS. Microbiol Mol Biol Rev. 2000 Mar; 64(1): 13–33).

[0043] In a preferred embodiment, the ClbP protein without a peptidase domain inactivated for the peptidase domain is a ClbP protein having a peptidase domain replaced by the alkaline phosphatase enzymatic domain of PhoA (SEQ ID N°11). The ability to produce gene fusions is well known to those skilled in the art. With regard to this ClbP fusion protein, only the mature part of PhoA lacking its signal sequence is fused behind the C-terminal truncated part of the membrane protein (see also review van Geest M. and. Lolkema JS. Microbiol Mol Biol Rev. 2000 Mar; 64 (1): 13–33). More precisely, the PhoA domain is fused to the ClbP N-terminal signal sequence that allows translocation to the periplasm and the ClbP C-terminal sequence from amino acid 390; the residues that form three transmembrane helices are 390-412, 433-455 and 465-485 (see the Examples section).

[0044] As used herein, the term "amino acid" refers to natural or non-natural amino acids in the D and L stereoisomers of chiral amino acids. It should be understood that it refers to amino acids and corresponding amino acid residues, such as residues present in peptidyl structures. Natural and non-natural amino acids are well known in the art. Common natural amino acids include but are not limited to alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).

[0045] Typically, amino acids are divided into one or more types according to their side chains, including polarity, hydrophobicity, acidity, alkalinity and aromaticity. Examples of polar amino acids include those with side chain functional groups (such as hydroxyl, sulfhydryl and amide), as well as acidic and basic amino acids. Polar amino acids include, but are not limited to, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan and tyrosine. Examples of hydrophobic or non-polar amino acids include those with non-polar aliphatic side chain residues, such as, but not limited to, leucine, isoleucine, valine, glycine, alanine, proline, methionine and phenylalanine. Examples of basic amino acid residues include those with basic side chains, such as amino or guanidino. Basic amino acid residues include, but are not limited to, arginine, homolysine and lysine. Examples of acidic amino acid residues include those with acidic side chain functional groups (such as carboxyl). Acidic amino acid residues include, but are not limited to, aspartic acid and glutamic acid. Aromatic amino acids include amino acids with aromatic side chain groups. Examples of aromatic amino acids include, but are not limited to, biphenylalanine, histidine, 2-naphthylalanine, pentafluorophenylalanine, phenylalanine, tryptophan, and tyrosine. It is noteworthy that some amino acids are classified into more than one group, for example, histidine, tryptophan, and tyrosine are classified as polar and aromatic amino acids. Amino acids can be further classified as uncharged or charged (positively or negatively charged) amino acids. Examples of positively charged amino acids include, but are not limited to, lysine, arginine, and histidine. Examples of negatively charged amino acids include, but are not limited to, glutamic acid and aspartic acid. Other amino acids that fall into each of the above groups are known to those of ordinary skill in the art.

[0046] "Equivalent amino acids" refers to amino acids that can be substituted for another amino acid in a peptide compound according to the invention without any significant loss of function. One of ordinary skill in the art will recognize equivalent amino acids. Substitution of similar amino acids is based on the relative similarity of the side chain substituents, e.g., with respect to size, charge, hydrophilicity, and hydrophobicity as described herein. When used in accordance with a list of individual amino acids, the phrase "equivalent amino acids" refers to equivalents of one or more of the individual amino acids included in the list.

[0047] In a more specific embodiment of the present invention, the ClbP protein inactivated for the peptidase domain is selected from the following list:

[0048] ClbP S95A mutant (SEQ ID N° 4),

[0049] ClbP K98T mutant (SEQ ID N° 6),

[0050] ClbP S95R mutant SEQ ID N° 8),

[0051] ClbP Y186G mutant (SEQ ID N° 10),

[0052] ClbP protein with the peptidase domain replaced by the alkaline phosphatase enzymatic domain of PhoA (SEQ ID N° 11).

[0053] The sequences of the mutated ClbP according to the present invention are shown in Table 2 below:

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Mutations (or substitutions) in the ClbP peptidase domain are indicated in bold in the AA sequence. In the present invention, the numbering of amino acids is based on the wild-type ClbP protein sequence ((SEQ ID NO: 1).

[0060] Methods for treating gastrointestinal disorders

[0061] A second object of the present invention relates to the Escherichia coli strain Nissle 1917 (EcN) bacterium as defined above, for use as a medicament.

[0062] As previously noted, EcN was initially studied for its ability to combat bacterial gastrointestinal infections. It has been shown to prevent intestinal colonization of Salmonella enterica serovar Typhimurium (3,4) and exhibit antimicrobial activity against enterohemorrhagic Escherichia coli strains (5). EcN is an excellent colonizer of the human intestine and has shown beneficial effects on a variety of intestinal disorders, such as acute diarrhea in infants and young children (6), chronic constipation (7), and abdominal pain in patients with irritable bowel syndrome (8). It has been widely used to treat inflammatory bowel disease (1) and has been shown to be as effective as the gold standard mesalamine for maintaining remission in children and adults with ulcerative colitis (9). Furthermore, the inventors demonstrated in an in vivo animal model infected with a bacterial enteric pathogen (S. Typhimurium) that administration of an EcN-modified strain having a clbP gene encoding a ClbP protein with an inactivated peptidase domain (i.e., ClbP mutated at position S95) was non-genotoxic (no colicin production) but maintained bacterial antagonist activity and reduced pathogen colonization and virulence by maintaining the production of siderophore-microcins (see FIG8 ).

[0063] Therefore, a third object of the present invention relates to the Escherichia coli strain Nissle 1917 (EcN) bacterium as defined above, for use in the treatment of gastrointestinal diseases.

[0064] Therefore, another object of the present invention relates to a method for treating a gastrointestinal disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the EcN bacteria of the present invention

[0065] Treatment can be for any purpose, including therapeutic treatment for subjects with pain, and preventive treatment for subjects without pain (e.g., subjects identified as having a high risk of gastrointestinal disease). As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, suppressing the progress of a disease or condition as described herein (i.e., gastrointestinal disease), or delaying, eliminating or reducing the incidence or onset of a disease or disease as described herein compared to what would occur if no measures were taken. As used herein, the terms "prevention" or "prophylactic use" and "prophylactic treatment" refer to any medical or public health program whose purpose is to prevent a disease disclosed herein (i.e., gastrointestinal disease). As used herein, the terms "prevention", "prevention" and "preventing" refer to reducing the risk of obtaining or developing a given condition in a subject who is not ill, but has or may be close to suffering from such a disease (i.e., gastrointestinal disease), or reducing or suppressing recurrence or the condition.

[0066] A specific strain of Escherichia coli Nissle 1917 (EcN) bacteria can be used to treat gastrointestinal diseases, such as bacterial gastrointestinal infections, intestinal inflammatory diseases and visceral pain.

[0067] There are a large number of bacteria that can cause gastrointestinal diseases (E. coli, Salmonella, Shigella, Campylobacter, Clostridium). Most often, bacterial infections of the intestines cause diarrhea or dysentery, nausea, vomiting, and abdominal pain or cramping. If the bacterial infection is in the small intestine, symptoms include watery diarrhea and / or vomiting. Bacterial infections in the large intestine usually cause dysentery (small stool volume with mucus and multiple times blood). Certain diseases follow certain predisposing conditions (antibiotic treatment: pseudomembranous colitis). Not all of these diseases occur after infection, but may occur after ingestion of preformed toxins (staphylococcal food poisoning). Symptoms of poisoning (vomiting, diarrhea) usually appear soon after ingestion of the toxin (a few hours). There are several ways to classify this group of diseases. Some classify them by where they are in the intestine (small intestine vs. large intestine), others by how the disease is acquired (food vs. water vs. person-to-person), and still others by the effect of the infectious agent on the host (poisoning vs. gastroenteritis vs. noninflammatory diarrhea vs. inflammatory diarrhea vs. enteric fever). All of these methods of classifying these causes are used to help doctors narrow down the possible causes of symptoms. Gastrointestinal infections are common. Diarrhea is the most common cause of death in developing countries (2.5 million deaths per year). Pathogens that cause diarrhea can be transmitted to humans in three basic ways: food, water, and person-to-person. Many of these infections are self-limited and do not require treatment. Some can spread to other parts of the body and require treatment to prevent further damage. The trick is knowing when to treat and how to treat the patient.

[0068] In specific embodiments, the bacterial gastrointestinal infection is a Salmonella serovar Typhimurium infection or an Enterohemorrhagic Escherichia coli infection.

[0069] In a more specific embodiment, the bacterial gastrointestinal infection is a Salmonella infection.

[0070] In a specific embodiment, Escherichia coli strain Nissle 1917 (EcN) bacteria can be used to treat gastrointestinal diseases, such as inflammatory bowel diseases. Such inflammatory bowel diseases are, for example, inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS).

[0071] As used herein, the term "inflammatory bowel disease (IBD)" is a group of inflammatory diseases of the colon and small intestine.

[0072] In a specific embodiment, the inflammatory bowel disease (IBD) is selected from the following list: Crohn's disease, ulcerative colitis, celiac disease, gluten sensitivity, and pouchitis.

[0073] As used herein, the term "irritable bowel syndrome (IBS)" is a term used for a variety of pathological conditions that cause gastrointestinal discomfort. It is a functional intestinal disorder characterized by chronic abdominal pain, discomfort, bloating, and changes in bowel habits, but without any organic cause. It also includes certain forms of food-related visceral allergies, such as gluten sensitivity (i.e., celiac disease).

[0074] Another gastrointestinal disease is also visceral pain, which includes the pain associated with inflammatory bowel disease (IBD). Visceral pain is the pain associated with viscera, which covers abdominal organs. These organs include the spleen and a part of the digestive system. The pain associated with viscera can be divided into peptic visceral pain and non-digestive visceral pain. Common gastrointestinal (GI) disorders that cause pain include functional bowel disease (FBD) and inflammatory bowel disease (IBD). These gastrointestinal diseases include a variety of disease states that are currently only moderately controlled, including about FBD-, gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS) and functional abdominal pain syndrome (FAPS) and-about IBD-Crohn's disease, ileitis and ulcerative colitis, all of which regularly produce visceral pain.

[0075] In some embodiments, the methods of the present invention are particularly useful for treating visceral pain caused by gastrointestinal diseases, including functional bowel disorder (FBD) and inflammatory bowel disease (IBD), gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS), and functional abdominal pain syndrome (FAPS), as well as for IBD, Crohn's disease, ileitis, ulcerative colitis, dysmenorrhea, cystitis and pancreatitis, and pelvic pain.

[0076] In a specific embodiment, the visceral pain is selected from the group consisting of inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS).

[0077] In some embodiments, the prevention methods of the present invention are particularly suitable for subjects determined to be at high risk for pain. Typically, subjects at risk for pain include patients who are about to undergo surgical procedures.

[0078] The EcN bacteria of the present invention can be used as medicines, in particular as probiotics.

[0079] The term "probiotic" has its ordinary meaning in the art and refers to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (see: Clinical Infectious Diseases, Volume 46, Issue Supplement 2, 1 February 2008, Pages S58–S61, https: / / doi.org / 10.1086 / 523341).

[0080] It should be understood that the daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage for any particular patient will depend on a variety of factors, including the type and severity of the condition to be treated; the activity of the specific compound used; the specific ingredients used, the patient's age, weight, general health, sex and diet; the time and route of administration and the excretion rate of the specific compound used; the duration of treatment; the drugs used in combination or concurrently with the specific bacteria used and other factors well known in the medical field. For example, it is within the skill of the art to recommend starting treatment with a dose of the compound below the level required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved.

[0081] The compounds of the present invention can be administered by any suitable route of administration. For example, the compounds according to the present invention can be administered orally (including buccal and sublingual), rectally, nasally, topically, pulmonary, vaginally or parenterally (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenously).

[0082] In a preferred embodiment of the present invention, the therapeutic composition containing the compounds of the present invention is administered intrarectally, topically or orally. Rectal administration is preferably carried out in the form of suppositories, enemas or foams. Rectal administration is particularly suitable for intestinal diseases affecting the lower intestinal tract (e.g., colon).

[0083] Pharmaceutical composition

[0084] The EcN bacteria of the present invention can be prepared into the form of pharmaceutical compositions and unit dosages together with one or more conventional adjuvants, carriers or diluents.

[0085] "Pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to mammals, especially humans. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid.

[0086] Pharmaceutical compositions and unit dosage forms may contain conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the expected daily dosage range to be used. Pharmaceutical compositions may be used in the form of solids (such as tablets or filled capsules), semisolids, powders, sustained release formulations, or liquids (such as solutions, suspensions, emulsions, elixirs) or filled capsules for oral use; or in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral use. Thus, formulations containing about one (1) milligram of active ingredient per tablet, or more broadly about 0.01 to about one hundred (100) milligrams of active ingredient, are suitable representative unit dosage forms.

[0087] The compounds of the present invention can be formulated into a variety of oral dosage forms. Pharmaceutical compositions and dosage forms can contain a compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that can also serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material. In powders, the carrier is typically a finely divided solid that is a mixture with the finely divided active ingredient. In tablets, the active ingredient is typically mixed with a carrier having the necessary binding capacity in a suitable proportion and compressed into the desired shape and size. Powders and tablets preferably contain about one (1) to about seventy (70) percent of the active compound. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch gelatin, tragacanth, methylcellulose sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like.

[0088] The term "preparation" is intended to include formulating the active compound with encapsulating material as a carrier, providing a capsule in which the active ingredient (with or without carriers) is surrounded by a carrier in association therewith. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be solid forms suitable for oral administration.

[0089] Other forms suitable for oral administration include preparations in liquid form, including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations intended to be converted into liquid form preparations shortly before use. Emulsions can be prepared in solution, for example in propylene glycol aqueous solution, or can include emulsifiers, such as lecithin, sorbitan monooleate, or gum arabic (acacia). The aqueous solution can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavorings, stabilizers, and thickeners. Aqueous suspensions can be prepared by dispersing finely ground active ingredients in water with viscous substances (such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents). Solid form preparations include solutions, suspensions, and emulsions, and in addition to the active ingredient, can also contain colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0090] Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle (eg, sterile, pyrogen-free water) before use.

[0091] The present invention will be further described by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way.

[0092] Attached photos

[0093] Figure 1. Role of the pks / clb island in the antibacterial activity of EcN against LF82. (A) Serial dilutions of a 24-hour co-culture of E. coli LF82 (rifampicin-resistant) with wild-type (WT) E. coli strain Nissle 1917 (EcN) or a mutant of the colicin maturation peptidase ClbP were spotted (10 μL) on LB plates containing rifampicin and incubated overnight at 37°C. (B) Colony forming unit (CFU) counts of Escherichia coli LF82 after 24 h of co-culture with WT EcN, gene-deficient phosphopantetheine transferase ClbA, peptidase ClbP and corresponding complementation mutant (pclbP), polyketide synthases (PKS) ClbC and ClbO, nonribosomal peptide synthases (NRPS) ClbH and ClbN, hybrid PKS-NRPS ClbB, putative amidase ClbL, efflux pump ClbM, and thioesterase ClbQ in M63 medium. LF82 was also cultured alone as a control. Median values ​​and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-test comparison with co-culture with WT; ★★★P<0.001.

[0094] Figure 2 : Role of the microcin gene cluster in the EcN antimicrobial activity against LF82. Colony forming units (CFU) of E. coli LF82 were counted after 24 h of co-culture in M63 medium with the wild-type (WT) E. coli strain Nissle 1917 (EcN), EcN mutants of the microcin M (MccM) precursor gene, EcN mutants of the microcin H47 (MccH47) precursor gene mchB, EcN mutants of the two mcmA mchB genes; EcN mutants and complementation strains of the mchC mchD genes responsible for post-translational modifications, and EcN mutants of the mchE mchF genes encoding the MccM and MccH47 efflux pumps. LF82 was also cultured alone as a control. Median values ​​and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-test comparison with co-culture with WT; ★★★P<0.001.

[0095] Figure 3: Role of siderophores in the antibacterial activity of EcN against LF82. (A) Colony forming unit (CFU) counts of E. coli LF82 after co-culture in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN), EcN mutants of entE encoding enterobactin synthase E, and double mutants of phosphopantetheine transferase ClbA and EntD; EcN mutants of glucosyltransferase IroB, cytoplasmic esterase IroD, periplasmic esterase IroE, and export protein IroC, and complemented strains for 24 h. LF82 was also cultured alone as a control Median values ​​and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-test comparison with co-culture with WT; ★★★P<0.001.

[0096] Figure 4 : Role of the ClbP catalytic domain and transmembrane domain in the EcN antibacterial activity against LF82.

[0097] Colony forming units (CFU) counts of E. coli LF82 after 24 h of co-cultivation in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN); clbP gene deletion and complementation mutants carrying plasmids encoding wild-type ClbP (pclbP), plasmids encoding ClbP with mutations S95A or K98T in the catalytic site, and plasmids encoding a fusion between alkaline phosphatase PhoA and the ClbP C-terminal sequence from amino acid 390 (pclbP-3H). LF82 was also cultured alone as a control Median values ​​and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-test comparison with co-culture with WT; ★★★P<0.001.

[0098] Figure 5: Genomic point mutations that inactivate the ClbP catalytic domain abolish EcN genotoxicity but not antibacterial activity against LF82. (A) HeLa cells were transiently infected with wild-type E. coli Nissle (WT), a genome-edited mutant in which a single chromosomal nucleotide change in the clbP gene inactivates the catalytic site (clbP-S95R), and the genome-edited mutant was complemented with plasmid pclbP. Then, the cells were fixed, permeabilized, and stained with rabbit monoclonal anti-γ-H2AX followed by infrared fluorescent secondary antibody. DNA was counterstained with RedDot2. (B) HeLa cells were transiently infected with wild-type E. coli Nissle (photo B), clbP gene deletion mutant (C), and genome-edited clbP-S95R mutant (D). Then, the cells were washed and incubated with gentamicin for 72 hours and then stained with Giemsa. The control is shown in image A. The bars represent 50 μm. (C) Colony forming unit (CFU) counts of E. coli LF82 after co-culture with wild-type (WT) E. coli strain Nissle 1917 (EcN), a clbP gene deletion mutant (ΔclbP), and a genome-edited mutant with a single nucleotide change in the clbP gene resulting in an S95R mutation in the catalytic site (clbP-S95R) in M63 medium for 24 h. LF82 was also cultured alone as a control Median values ​​and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-test comparison with co-culture with WT; ★★★P<0.001.

[0099] Figure 6 : Gene clusters involved in the production of microcins H47 and M (MccH47 and M) in the E. coli strain Nissle (EcN) represented on the genome map. The loci encoding enterobactin (ent), colicin (pks), yersiniacin (ybt) on EcN genomic island (GEI) IV; salmonellin (iro) and McH47 (mch) and M (mcm) on GEI I are shown. Arrows indicate interactions between different gene clusters involved in the production of MccH47 and M in EcN.

[0100] Figure 7: Model proposed for the biosynthesis of the siderophore-microcins H47 and M in Escherichia coli Nissle. The enterobactin precursor is modified by the enterobactin glucosyltransferase IroB. This siderophore moiety is transferred to the C-terminus of the precursor peptide by the MchCD complex. The active form of the siderophore-microcin is the result of cleavage of the leader peptide by the specific efflux pump MchEF-TolC during its export. The C-terminal domain of ClbP allows the final step in the production of the siderophore-microcins, while its N-terminal enzymatic domain cleaves the enterobactin precursor to produce colicins.

[0101] Figure 8: Deletion of clbP, but not genomic point mutations inactivating the ClbP catalytic domain, impairs the protective effect of EcN against enteric serovar Typhimurium in mice.

[0102] C57BL / 6 female mice were given 20 mg of streptomycin orally, and then 24 hours later, they were treated with 10 9 Oral infection with Salmonella typhimurium (STm) or with 10 9 Salmonella typhimurium and 10 9 EcN wild-type, ΔclbP or clbPS95R strains were co-administered. (A) The clinical symptoms (weight loss, diarrhea, abdominal pain symptoms) of mice were monitored daily for 4 days. Each point corresponds to the average clinical score + / - SEM of 10-15 animals in each group in three independent experiments. Animals were scored blindly (without knowing the infecting bacteria) in the last two of the three experiments. Two-way ANOVA with Bonferonni post-test compared with STm+EcN, a: p<0.05, c: p<0.001. (B) The fecal shedding of STm was examined by calculating the feces collected on the 2nd and 4th days after infection. The median and individual results are shown. One-way ANOVA of log-transformed CFU counts compared with STm+PBS, a: p<0.05 (C) Fecal counts of STm and EcN were used to determine the competition index (CFU STm / CFU EcN). Compared with STm+EcNclbPS95R, one-way ANOVA, a: p<0.05.

[0103] Fig. 9 : Detection of N-acyl-Asn-GABAOH in EcN wild-type, ΔclbN and clbPS95R strains.

[0104] EcN wild-type, ΔclbN or clbPS95R strains were grown in DMEM Hepes for 8 h, and N-acyl-Asn-GABAOH in the bacterial pellets was quantified by HPLC (Agilent 1290 Infinity) coupled to a triple quadrupole mass spectrometer (G6460). Concentrations C are shown as pg per 1E+8 CFU). DETAILED DESCRIPTION

[0105] Materials and methods

[0106] Bacterial strains, mutants, and plasmids

[0107] The bacterial strains and plasmids used in this study are listed in Table 3. Gene mutagenesis was performed by the lambda Red recombinase method (37). Double mutants were constructed sequentially. Mutations and deletions of the FRT cassette were verified by PCR using primers upstream and downstream of the target gene.

[0108] A fusion between the ClbP N-terminal signal sequence, the alkaline phosphatase PhoA, and the three transmembrane helices of ClbP was constructed using the HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA, USA) with primers overlapping between each fragment. The construction was verified by PCR and confirmed by sequencing. Blue-stained colony forming units on LB plates containing 40 mg / L 5-bromo-4-chloro-3-indolyl phosphate revealed that the PhoA alkaline phosphatase domain was present in the periplasm as previously reported (38).

[0109] To construct plasmids pmchEF and pmchCD, the genes were PCR amplified and cloned into pCR-XL-TOPO (Invitrogen, Carlsbad, CA, USA).

[0110] To construct plasmid pIroB, the iroB gene was PCR amplified using EcN genomic DNA as a template and primers IRSDNG7 and IRSDNG8, digested by EcoRI and BamHI and ligated into pBbA5a-RFP (obtained from Addgene) from which the rfp gene had been digested and removed.

[0111] The strain EcN clbP-S95R chromosomal isogenic mutant was constructed using genome editing technology (39). EcN was transformed with pORTMAGE and then grown in LB at 30°C and 300 rpm to reach OD600 = 0.5. The initial mutagenesis cycle was started by inducing the expression of Lambda recombinase and the dominant negative mutLE32K allele at 250 rpm at 42°C for 15 minutes. The culture was then cooled to 0°C, washed with water and electroporated with 50 μM oligonucleotide IRSDNG26, which contains the S95A mutation in the clbP gene sequence. In a control experiment, the lacZ gene was targeted by a specific mutagenic oligonucleotide. After recovery in LB at 30°C and 300 rpm for 1 hour, two additional mutagenesis cycles were performed and the bacteria were finally plated on MacConkey agar without any antibiotics. In the control experiment, approximately 33% of the isolates were LacZ negative. Sixty candidate clbP-S95R mutants were tested for genotoxicity and loss of the macrocytosis phenotype in infected HeLa cells as described previously (40). Non-genotoxic mutants that lost the pORTMAGE plasmid were selected and ultimately verified for removal of the ClaI restriction site by the S95A mutation in the PCR-amplified clbP sequence.

[0112] Determination of colicin-induced genotoxic effects

[0113] Colibactin-induced cellular senescence is accompanied by an associated cell enlargement called macrocytosis, and each EcN mutant constructed in this study in the Mcc gene cluster, iroA locus, and clbP-S95R mutant was assayed. HeLa cells (ATCC, CCL-2) were infected for 4 hours as previously described (40). Then, the cells were washed and incubated with gentamicin for 72 hours and then stained with Giemsa. The genotoxicity of EcN and clbP-S95R chromosomal mutants was confirmed by intracellular Western blotting procedures as previously described (36). Briefly, HeLa cells were infected in 96-well plates at a given multiplicity of infection (the number of bacteria per cell at the beginning of infection) for 4 hours. Four hours after the end of infection, the cells were fixed, permeabilized, and stained with rabbit monoclonal anti-γ-H2AX (Cell Signaling, 20E4, 1:200) and then stained with infrared fluorescent secondary antibodies. DNA was counterstained with RedDot2 (Biotum). Fluorescence was recorded using the Odyssey infrared imaging system (Li-Cor).

[0114] Competitive growth assay

[0115] The strains were grown in lysogeny broth (LB Lennox, Invitrogen) at 37° C. with shaking at 240 rpm overnight. Rifampicin, streptomycin, kanamycin, carbenicillin or chloramphenicol were added to the medium as needed.

[0116] The culture medium used for co-culture experiments was M63 minimal medium with final concentrations of 15 mM ammonium sulfate, 1 mM magnesium sulfate heptahydrate, 100 mM potassium phosphate monobasic, 2.5 g / L glucose, 1 mg / L thiamine, and 1 g / L Bacto tryptone (BD Biosciences, Le Pont de Claix, France) or Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX (Invitrogen) supplemented with 25 mM Hepes, 10% (v / v) fetal calf serum (FCS, Eurobio, Courtaboeuf, France), and 1% (v / v) non-essential amino acids (NEAA, Invitrogen).

[0117] Each 500 μL overnight culture was grown in 9.5 mL of co-culture medium and incubated at 37°C with shaking at 240 rpm for 2 h. As previously described (3), both the production strain and the target strain (EcN and LF82, respectively) were inoculated from these 2-h cultures at 106 CFU / mL in 10 mL of co-culture medium and incubated at 37°C with shaking at 240 rpm for 24 h. For CFU counts, the cultures were serially diluted in PBS and plated on selective LB agar plates containing the desired antibiotic (e.g., rifampicin for LF82 (41)). In the general results section, only the growth of the target strains (mainly LF82) is reported. As controls, the growth of competing strains (mainly EcN and EcN mutants) was systematically examined (data not shown).

[0118] Animal infection

[0119] Animal infections were performed in accordance with the European Directive on the protection of animals used for scientific purposes (2010 / 63 / EU). The protocol was approved by the local ethics committee (protocol number: 2019041710292271). Female C57BL / 6 (Janvier) were housed in ventilated cages with 5 animals per cage and had ad libitum access to food and water.

[0120] Animals were orally administered 20 mg of streptomycin and then 24 hours later, 10 9 Typhimurium strain IR715 (resistant to nalidixic acid) or 10 9 Salmonella typhimurium and 10 9Oral infection was performed by co-administering EcN, EcNΔclbP or EcN clbPS (using the rpsLK42R allele conferring resistance to streptomycin).

[0121] Fecal shedding of S. typhimurium and EcN was determined by homogenization of feces in PBS, serial dilution, and plating on LB agar plates supplemented with nalidixic acid or streptomycin.

[0122] The severity of salmonellosis was assessed by daily scoring of symptoms of weight loss, abdominal pain, fever, and diarrhea.

[0123] The experiment was terminated 4 days after infection to avoid lethality.

[0124] The experiments were repeated three times with five animals per group, and clinical scores were scored blindly (without knowledge of the infecting bacteria) in two of three independent experiments.

[0125] Bioinformatics analysis

[0126] Genes involved in the synthesis of MccH47 and MccM were searched using BLASTn and the CA58 Mcc gene cluster as reference: mchB and mcmA encoding precursor proteins, the immunity genes mchI and mcmI, the genes mchE and mchF encoding specific efflux pumps, and the genes mcmK and mcmL (and their respective homologs in the E. coli H47 Mcc gene cluster, mchS1 and mchA) responsible for post-translational modifications. Query coverage >80%, identity >90% and E-value <1e40 were chosen as critical values ​​for significance. The genes clbB and clbP were identified as markers for the 5' and 3' regions of the pks island, respectively, using the same approach, as were the iroN and iroB genes as markers for the 5' and 3' regions of the saprophytin gene cluster (iroA locus). The phylogroups were determined bioinformatically based on the presence / absence of four genes: arpA, chuA, yjaA and tspE4.C2 (and trpA to distinguish between A and C phylogroups) (42). The phylogenetic tree was constructed using rpoC sequences. Sequences were collected using PATRIC 3.5.8 (43), multiple sequence alignment was performed using the logarithmic expectation (MUSCLE) method using MEGA 7.0.26 software (44), and the phylogenetic tree was constructed using MEGA 7.0.26 according to the maximum likelihood method.

[0127] Statistical analysis

[0128] Statistical analysis was performed using GraphPad Prism 7.0a (GraphPad, San Diego, CA, USA). P values ​​were calculated using one-way ANOVA followed by Bonferroni post-test. CFU / ml was log-transformed for analysis. P values ​​< 0.05 were considered significant and indicated by ★, P < 0.01 by ★★, and P < 0.001 by ★★★.

[0129] result

[0130] EcN antimicrobial activity requires ClbP but not other components of the colicin synthesis pathway

[0131] To specifically separate the genotoxic from the probiotic activity, we tested the antimicrobial activity of a ClbP-deficient EcN mutant that allows proactin maturation in genotoxic colibactins (24,25). We compared it with a pleiotropic ClbA mutant encoding a PPTase (27,35,36). We performed coculture experiments with wild-type EcN, EcNΔclbA and ΔclbP mutants, and the Crohn's disease-associated E. coli strain LF82, which has been previously shown to be sensitive to EcN (45,46). CFU showed that the EcN strains strongly inhibited the growth of LF82. The antimicrobial activity of EcN against LF82 was not altered in the ΔclbA mutant but was completely lost in the ΔclbP mutant (Fig. 1). Kinetic experiments showed that the inhibitory activity of EcN against LF82 began at 6 h post-inoculation at the onset of the stationary phase and reached a maximum at 8 h post-inoculation (data not shown). The growth of LF82 was not altered by the ΔclbP mutant at any time, further demonstrating the clbP-dependence of the antibacterial effect of EcN. This EcN-ClbP-dependent inhibitory activity was also observed in other pathogenic E. coli strains (JJ186 and NRG857c) and closely related bacterial species such as Salmonella enterica subsp. enterica Typhimurium and Enterobacter aerogenes (data not shown).

[0132] To further determine whether other components of the colicin biosynthesis pathway in addition to ClbP are required for the antimicrobial activity of EcN, mutants were evaluated for inhibition of the PKS ClbC and ClbO, the NRPS ClbH and ClbN, the hybrid PKS-NRPS ClbB, the putative amidase ClbL, the efflux pump ClbM, and the thioesterase ClbQ against LF82. The antimicrobial activity of EcN against LF82 was not altered in these mutants (Fig. 1). These results confirm that colicin itself or the cleavage product N-myristoyl-D-asparagine is not required for the antimicrobial activity of EcN against LF82 (and other Gram-negative bacteria, data not shown). Therefore, the probiotic activity of EcN is clearly associated with the presence of the pks / clb island and ClbP, but colicin is not involved in the EcN inhibitory activity.

[0133] EcN-ClbP-dependent antimicrobial activity requires MccH47 and MccM

[0134] Previous studies have linked EcN antimicrobial activity to MccH47 and MccM (4, 11, 14, 15). Therefore, we performed co-culture experiments with LF82, EcN, and mutants in MccH47 and MccM production systems. The antimicrobial activity of EcN against LF82 was not affected by the deletion of the MccM precursor gene mcmA or the MccH47 precursor gene mchB alone ( Figure 2 ). In contrast, deletion of mcmA and mchB almost completely abolished the inhibitory effect of EcN on LF82. Similarly, deletion of the genes encoding mchE and mchF, MccM and the MccH47 efflux pump, resulted in loss of antimicrobial activity ( Figure 2 Compared with the wild-type EcN strain, trans-complementation of mchE and mchF increased the EcN inhibitory activity ( Figure 2 ), probably because Mcc export was increased after overexpression of the MccE-MchF efflux pump. None of these mutations in the Mcc production system affected the ability of EcN to produce active colicin (data not shown).

[0135] To further confirm the role of MccH47 and MccM in the antibacterial activity of EcN, plasmids encoding MccH47 or MccM immunity genes were transformed in LF82, and the resistance of the strains to EcN was evaluated (data not shown). The antibacterial activity of the EcNΔmchB mutant was almost completely abolished on LF82 carrying the MccM immunity gene mcmI (data not shown). Similar results were obtained with the ΔmcmA mutant and LF82 carrying the MccH47 immunity gene mchI (data not shown). In summary, these results confirm that the dependent inhibitory activity of EcN ClbP on LF82 is due to MccH47 and MccM.

[0136] EcN ClbP-dependent antimicrobial activity is due to the production of siderophore-Mcc

[0137] MccH47 and MccM can be post-translationally modified by ligating catechol siderophores to form “siderophore-Mcc” (13). Therefore, we hypothesized that the antimicrobial activity dependent on ClbP might depend on these modified forms of microcins. Indeed, the antimicrobial activity of EcN against LF82 was significantly reduced in a ΔentE mutant that lacks the enzyme 2,3-dihydroxybenzoate-AMP ligase, which is essential for the production of the siderophore enterobactin (47). Similar results were obtained with the EcNΔclbAΔentD double mutant, which is unable to produce enterobactins (36) ( Figure 3 ).

[0138] Two genes (mcmL and mcmK) responsible for the glycosylation and esterification of enterobactin are missing from the EcN Mcc gene cluster (18, 48). Therefore, whether MccH47 and MccM are siderophore-Mcc or unmodified Mcc is still under debate (13). Considering that EcN carries McmL and McmK homologs, glucosyltransferase IroB and esterase IroD, respectively (13), we investigated the interaction between Mcc and the salmochelin production system. The antimicrobial activity of EcN mutants encoding genes for glucosyltransferase IroB, cytoplasmic esterase IroD, periplasmic esterase IroE, and export protein IroC (49, 50) was compared with that of the wild-type EcN strain. Only the loss of iroB resulted in a significant reduction in the antimicrobial activity of EcN ( Figure 3 Complementation of the ΔiroB mutant fully restored antimicrobial activity. None of these mutations in the iroA locus affected the ability of EcN to cause macrocytosis associated with the genotoxic effects of colibactin (data not shown). These results suggest that IroB may be responsible for enterobactin glycosylation, which enables the Mcc precursor protein to be attached to the siderophore-derived moiety in the absence of McmL.

[0139] MchC and MchD are homologous to MceJ and MceI, respectively, of Klebsiella pneumoniae strain E492 (13). These proteins form a complex that is responsible for linking the glycosylated enterobactin derivative to MccE492, the precursor peptide MceA (51). The EcN mutants of mchC and mchD lost their antibacterial activity against LF82, whereas complementation restored the initial phenotype ( Figure 2 ). These results suggest that the antibacterial activity of EcN requires post-translational modification of MccH47 and MccM with enterobactin-derived moieties. In short, the EcNClbP-dependent antibacterial activity is due to the siderophore-Mcc.

[0140] The antimicrobial activity of EcN requires the ClbP transmembrane domain, but not the periplasmic peptidase catalytic site

[0141] To further elucidate the role of ClbP in the production of siderophore-Mcc, we examined whether the antimicrobial activity of EcN requires ClbP catalytic activity. S95 and K98 are key residues for the peptidase activity of ClbP, and mutants of these residues are unable to cleave procolicin to release the mature active genotoxin (24,25). Co-culture experiments were performed with LF82 and the EcNΔclbP mutant complemented with plasmids encoding the wild-type ClbP protein or the ClbP protein containing S95A or K98T substitutions. The EcNΔclbP mutant complemented with ClbP S95A or K98T exhibited antimicrobial activity similar to that of the wild-type ClbP protein ( Figure 4 ), whereas they lost the ability to cause macrocytosis associated with the genotoxic effects of colicin (data not shown).

[0142] To exclude the role of another putative catalytic site of the ClbP enzymatic domain, this enzymatic domain was replaced with the alkaline phosphatase enzymatic domain of PhoA, as previously reported (38). The PhoA domain was fused to the ClbP N-terminal signal sequence that allows translocation to the periplasm and to the ClbP C-terminal sequence from amino acid 390; the residues that form the three transmembrane helices are 390-412, 433-455, and 465-485 (24). The EcNΔclbP mutant transformed with a plasmid carrying this fusion showed LF82 inhibitory activity similar to that of the EcNWT strain ( Figure 4 ), while it did not cause macrocytosis (data not shown). Thus, in contrast to the periplasmic peptidase domain of ClbP, which is essential only for genotoxic activity, the C-terminal domain of ClbP, which contains three transmembrane helices, is essential for the antimicrobial activity of EcN.

[0143] To confirm this observation and prove the concept that a non-genotoxic EcN probiotic strain can be engineered, we used genome editing to construct an EcN mutant strain that exhibits a single nucleotide mutation in the chromosomal clbP gene, resulting in an S95R mutation in the ClbP catalytic site at the amino acid level. This mutant did not produce colicin and was not genotoxic, but still exhibited antimicrobial activity against LF82, which was similar to that of the wild-type genotoxic EcN strain (Figure 5).

[0144] The EcN strain with a point mutation in the clbP gene is non-genotoxic but retains antagonist activity and reduces intestinal colonization and virulence of Salmonella typhimurium

[0145] It is known that EcN probiotics can provide protection against enteric pathogens such as Salmonella by competing for iron and producing siderophores, microcins (3, 4). Therefore, we used an in vivo model to examine whether EcN wild-type, ΔclbP, and clbP-S95R mutants reduce S. Typhimurium intestinal colonization and pathogenesis. We used C57BL / 6 mice treated with streptomycin (to ensure high colonization) and then 24 hours later, infected with S. Typhimurium alone or co-administered with S. Typhimurium and each EcN strain (3, 4, 66). Mice were monitored for clinical signs (signs of weight loss, diarrhea, abdominal pain) and bacterial colonization was examined by fecal counts during 4 days (the time point at which the experiment had to be stopped due to lethality). When administered alone, S. Typhimurium readily colonized the intestine, which was associated with high clinical scores associated with intense enteric salmonellosis (Figure 8). Clinical scores and S. typhimurium fecal colonization were significantly reduced in animals co-administered with wild-type EcN ( Fig. 8A -B). By day 2 post-infection, EcN significantly exceeded that of S. typhimurium ( Figure 8C ). In contrast, animals co-administered with the EcNΔclbP mutant exhibited increased clinical scores and reduced resistance to S. Typhimurium colonization, demonstrating a role for ClbP in the beneficial effects of EcN during acute Salmonella colitis. Similar to wild-type EcN, the EcN clbP-S95R strain significantly reduced fecal shedding and exceeded S. Typhimurium and reduced clinical scores (Figure 8).

[0146] Together, these results suggest that it is possible to separate the genotoxic activity of EcN from its probiotic (antimicrobial) activity, but that the biosynthetic pathways of colicin and the siderophore microprotein are more complex than initially thought.

[0147] ClbP-dependent antibacterial activity was observed in a subset of Escherichia coli strains carrying a truncated Mcc gene cluster and a pks island

[0148] Comparative genomic analysis showed that EcN was closely related to the E. coli pyelonephritis strain CFT073 and the asymptomatic bacteriuria strain ABU83972 (18). These three strains as well as the reference strain 25922 carries the pks island, the iroA locus and a truncated Mcc gene cluster lacking the genes mcmL / mchA and mcmK / mchS1. Therefore, we evaluated whether the siderophore-Mcc antimicrobial effect of these strains is CbP-dependent, as observed in EcN. Two groups of E. coli strains were tested for inhibition in co-culture experiments against LF82, and their respective ΔclbP mutants: i) strains similar to EcN, carrying both a truncated Mcc gene cluster and a pks island: strains CFT073, ABU83972 and 25922; and ii) strains carrying the pks island but lacking Mcc encoding genes: human commensal strain M1 / 5, meningitis-causing strain SP15, mouse commensal strain NC101, and laboratory strain MG1655 carrying a bacterial artificial chromosome (BAC) carrying the pks island. The three wild-type strains carrying a truncated Mcc gene cluster and the pks island showed significant inhibition, as observed in EcN (data not shown). The inhibition of all three corresponding ΔclbP mutants was significantly reduced, while ClbP complementation restored the initial phenotype (data not shown). In contrast, in strains carrying only the pks island, there was no significant difference in the growth of LF82, whether cultured with the wild-type strain or the ΔclbP mutant (data not shown). Cumulatively, these results indicate that the peptidase ClbP is involved in the antibacterial activity of MccH47 and MccM in E. coli strains carrying the pks island and a truncated form of the Mcc gene cluster. Our results also show that this association exists in pathogenic and probiotic strains.

[0149] Distribution of pks, salmochelin, and MccH47 and MccM gene clusters in Escherichia coli

[0150] We demonstrated that Escherichia coli strains carrying truncated Mcc gene clusters exhibit siderophore-Mcc-dependent antibacterial activity (data not shown). This antibacterial activity requires ClbP from the biosynthetic pathway that produces the genotoxin colicin and IroB from the biosynthetic pathway that produces the siderophore sarcosine. Therefore, we examined this association between the pks island, iro locus and Mcc island in E. coli strains using genomes available in GenBank. Interestingly, all strains lacking the mcmL and mcmK genes responsible for post-translational modifications belong to the B2 system group and carry the pks island and iroA (data not shown), except for strain 1105 lacking the pks island. In contrast, strains carrying mcmL / mchA and mcmK / mchS1 belong to the B1, C or D system groups and lack the pks island. These special associations of genetic determinants have led to the hypothesis that truncated islands are almost exclusively present in strains carrying the pks and iroA loci. This suggests that this interaction between colicin, salmochelin, and the siderophore-Mcc biosynthetic pathway is due to co-selection by pathogenic or probiotic strains.

[0151] Production of beneficial compounds potentially related to the probiotic activity of Nissle 1917 by the Nissle clbPS95R strain

[0152] Metabolites other than colibactin synthesized by enzymes encoded on the pks island may contribute to the probiotic properties of Nissle 1917. Recent studies have shown that the metabolite C12AsnGABAOH is produced by the Nissle 1917 wild type (but not by the clbN mutant, suggesting that the pks-encoded machinery plays a role in its production)(34). C12-Asn-GABAOH has been shown to inhibit the activation of nociceptors in neurons. Nociceptor neurons in the intestine play an important role in defense against enteric pathogens and intestinal homeostasis as they regulate M cell density and inflammation. Therefore, it was important to ensure that the non-genotoxic modified strain retained production of C12AsnGABAOH and played a role in the anti-inflammatory properties of Nissle.

[0153] We quantified N-acyl-Asn-GABAOH in bacterial cultures of Nissle 1917 wild-type, Nissle clbN mutant, and Nissle clbPS95R by HPLC-QQQ (34). Nissle clbPS95R still produced the beneficial GABAOH lipopeptide similar to the wild-type strain ( Fig. 9 ).

[0154] Genetic stability of Nissle clbPS95R

[0155] To verify that the Nissle clbPS95R strain is genetically stable, we sequenced its genomic DNA before and after oral gavage in mice (as well as pathogenic Salmonella (67)) and fecal reisolation. The genome was compared with the genome of the wild-type strain, which was also sequenced. The Nissle clbPS95R strain showed only a 2 bp change (5441200 bp) compared to the wild-type. No differences were found in Nissle clbPS95R before and after passage through the mouse intestine (data not shown). Therefore, Nissle clbPS95R appears to be genetically stable.

[0156] discuss

[0157] Since Fleming’s discovery of penicillin in 1928, antibiotics have contributed to an increase in human life expectancy. Many infections that were previously fatal are now curable. Unfortunately, the overuse and misuse of antibiotics, as well as the lack of new antimicrobial agents, have allowed the emergence and spread of multidrug-resistant bacteria (52). According to the World Health Organization (WHO), this phenomenon “poses a major threat to morbidity and mortality worldwide” (53). For Gram-negative bacteria, this trend is particularly worrying. For example, between 2007 and 2015, deaths in Europe due to third-generation cephalosporin-resistant or carbapenem-resistant Escherichia coli increased more than fourfold (54). Of the antibiotics currently under development for intravenous use, only a small fraction (15 of 44) have any activity against Gram-negative bacteria, and all of these molecules are from known antibiotic classes. Therefore, the WHO has identified research and development of new antibiotics against Gram-negative bacteria as a “key priority” (53).

[0158] In the search for new antimicrobials, microcins appear to be a promising alternative to “traditional” antibiotics. Indeed, many microcins exhibit potent narrow-spectrum antimicrobial activity, whereas antibiotics can eliminate beneficial bacteria, alter the microbiota, and promote the selection of resistant strains (55,56). A major challenge in the use of microcins is the delivery of sufficient quantities to the site of infection, especially after oral administration, as they are often degraded in the upper gastrointestinal tract (57,58). Therefore, engineered probiotics have been proposed as in situ producers of microcins to combat enteric pathogens (59) or reduce colonization by multidrug-resistant bacteria (60).

[0159] EcN has been used as a probiotic for over a century, and many therapeutic benefits have been described. However, serious concerns have arisen over the years regarding the safety of EcN administration. EcN has been reported to be the cause of severe sepsis in infants (61), and its genome has been shown to possess the pathogenicity island pks (21, 35), which encodes colicin, the true virulence factor of E. coli strains responsible for extraintestinal infections (26, 27). In addition, E. coli harboring colicin-producing strains may also be detrimental to intestinal homeostasis. In adult rats, it increases intestinal epithelial permeability, causes signs of genotoxic damage to enterocytes, such as crypt fission, and increases cell proliferation (28). In mice susceptible to colorectal cancer, pks-positive E. coli increased tumor size and number (31, 62). In humans, several studies have reported that pks-positive E. coli are overrepresented in colorectal cancer biopsies compared with controls (31, 32, 63). Collectively, these studies suggest that colicin-producing bacteria can promote tumorigenesis. Therefore, our goal was to understand the interplay between the production of the genotoxin colicin and the beneficial effects associated with the pks island in the probiotic activity of EcN. We therefore sought to disarm EcN while maintaining its probiotic properties.

[0160] In a previous attempt, our group constructed a non-genotoxic mutant of the EcN PPTase ClbA that also lost its probiotic activity (35). Subsequently, the PPTase ClbA was found to contribute to the synthesis of enterobactins (and therefore also salmonellins) and yersinib (36). In this study, we demonstrated the existence of a collaboration between the Salmochelin (iroB) and Mcc gene clusters, both of which are located in the EcN genomic island I and pks island (clbP) ( Figure 6 ). The intertwining of these determinants is so strong that a single protein, ClbP, is involved in the production of both colicin and Mcc. So far, ClbP has only been described as a peptidase that removes the N-acyl-D-asparagine prodrug scaffold from the colicin precursor (24,25). Although an intact C-terminal domain with three transmembrane helices is required for the biological activity of ClbP, catalytic activity is carried out by the N-terminal periplasmic domain (25,38). In this study, we demonstrate that the C-terminal domain of ClbP, which lacks known enzymatic functions, is required for the antimicrobial activity of EcN due to MccH47 and MccM. This suggests that the ClbP C-terminal transmembrane domain can facilitate the export of MccH47 and MccM from EcN through the MchE-MchF efflux pump ( Figure 7 ).

[0161] Through functional and bioinformatic analyses, we demonstrated the interplay between the siderophore Mcc, salmochelin, and the colicin assembly line. Strikingly, two groups of E. coli strains emerged. On the one hand, all strains carrying a “truncated” MccH47 and MccM gene cluster (i.e., strains such as EcN lacking mcmL / mchA and mcmK / mchS1) were B2 strains that also carried the pks island and iroA locus. Remarkably, in this group of strains, isolates from urine were overrepresented (CFT073, clones D i14 and D i2, UPEC 26-1, and ABU 83972). On the other hand, non-B2 strains carrying a “complete” MccH47 and MccM gene cluster lacked the pks island and iroA locus. All of these strains were isolated from feces (except ACN002, whose origin is unknown). Therefore, we can assume that these strains with a “complete” Mcc gene cluster are specialized to produce Mcc in order to survive in the competitive intestinal environment, which is their only ecological niche. In contrast, extraintestinal pathogenic E. coli (ExPEC) must be efficient intestinal colonizers in order to emerge from the intestinal niche and infect other body sites (such as the urinary tract) to which they must subsequently adapt. This is why ExPEC are considered “generalists” rather than specialists (64). The strains examined in our study fit this model. They can express a variety of virulence factors depending on the environment: for example, MccH47 and M, siderophores, and analgesic lipopeptides derived from the colicin pathway. In order to be able to produce so many virulence or fitness factors in a genome of limited size (65), the elements of the assembly line that produces these determinants must be multifunctional and intervene in several apparently independent metabolic pathways.

[0162] In conclusion, we found that the pks island is more closely linked to the probiotic activity of EcN than expected. This entanglement reflects the co-evolution of probiotics and pathogenicity determinants to adapt to various environments. The separation of probiotics from genotoxic activity by specifically targeting the enzymatic domain of ClbP opens the way for the safe use of EcN.

[0163] Table 3. Strains and plasmids used in this study.

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] refer to:

[0170] Throughout this application, various references describe prior art related to the present invention. The disclosures of these references are hereby incorporated into the present disclosure by reference.

[0171] 1. Wassenaar TM. Insights from 100 years of research with probiotic E. coli. Eur J Microbiol Immunol. 2016 Sep 29; 6(3): 147–61.

[0172] 2.Sonnenborn U.Escherichia colistrain Nissle 1917—from bench tobedside and back:history of a special Escherichia coli strain with probioticproperties.FEMS Microbiol Lett[Internet].2016 Oct 1[cited 2017 Dec 15]; 363(19).Available from:https: / / academic.oup.com / femsle / article / 363 / 19 / fnw212 / 2236266

[0173] 3. Deriu E, Liu JZ, Pezeshki M, Edwards RA, Ochoa RJ, Contreras H, et al. Probiotic bacteria reduce Salmonella Typhimurium intestinal colonization by competing for iron. Cell Host Microbe. 2013 Jul 17;14(1):26.

[0174] 4. Sassone-Corsi M, Nuccio SP, Liu H, Hernandez D, Vu CT, Takahashi AA, et al. Microcins mediate competition among Enterobacteriaceae in the inflamedgut. Nature. 2016 Dec 8;540(7632):280–3.

[0175] 5.Rund SA,Rohde H,Sonnenborn U,Oelschlaeger TA.Antagonistic effectsof probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4and O157:H7.Int J Med Microbiol IJMM.2013 Jan;303(1):1–8.

[0176] 6.Henker J,Laass M,Blokhin BM,Bolbot YK,Maydannik VG,Elze M,et al.Theprobiotic Escherichia coli strain Nissle 1917(EcN)stops acute diarrhoea ininfants and toddlers.Eur J Pediatr.2007 Apr;166(4):311–8.

[0177] 7. M,Bruckschen E.[Treatment of chronic constipationwithphysiologic Escherichia coli bacteria.Results of a clinical study of theeffectiveness and tolerance of microbiological therapy with the Escherichiacoli Nissle 1917 strain (Mutaflor)].Med Klin Munich Ger 1983.1994 Nov 15;89(11):587–93.

[0178] 8.Kruis W,Chrubasik S,Boehm S,Stange C,Schulze J.A double-blindplacebo-controlled trial to study therapeutic effects of probioticEscherichia coli Nissle 1917 in subgroups of patients with irritable bowelsyndrome.Int J Colorectal Dis.2012 Apr;27(4):467–74.

[0179] 9.Losurdo G,Iannone A,Contaldo A,Ierardi E,Di Leo A,PrincipiM.Escherichia coli Nissle 1917 in ulcerative colitis treatment:systematicreview and meta-analysis.J Gastrointest Liver DisJGLD.2015 Dec;24(4):499–505.

[0180] 10.Sonnenborn U,Schulze J.The non-pathogenic Escherichia coli strainNissle 1917–features of a versatile probiotic.Microb Ecol Health Dis.2009Sep;21(3 / 4):122–58.

[0181] 11.Papavassiliou J.Biological characteristics of colicineX.Nature.1961 Apr 1;190:110.

[0182] 12.Patzer SI.The colicin G,H and X determinants encode microcins Mand H47,which might utilize the catecholate siderophore receptors FepA,Cir,Fiu and IroN.Microbiology.2003 Sep 1;149(9):2557–70.

[0183] 13.Vassiliadis G,Destoumieux-Garzón D,Lombard C,Rebuffat S,PeduzziJ.Isolation and characterization of two members of the siderophore-microcinfamily,microcins M and H47.Antimicrob Agents Chemother.2010 Jan;54(1):288–97.

[0184] 14.Asensio C,Pérez-Díaz JC,Martínez MC,Baquero F.A new family of lowmolecular weight antibiotics from enterobacteria.Biochem Biophys ResCommun.1976 Mar 8;69(1):7–14.

[0185] 15.Duquesne S,Destoumieux-Garzón D,Peduzzi J,Rebuffat S.Microcins,gene-encoded antibacterial peptides fromenterobacteria.NatProd Rep.2007Jul25;24(4):708–34.

[0186] 16.Thomas X,Destoumieux-Garzón D,PeduzziJ,Afonso C,Blond A,BirlirakisN,et al.Siderophore peptide,a new type ofpost-translationally modifiedantibacterial peptide with potent activity.J Biol Chem.2004 Jul2;279(27):28233–42.

[0187] 17.Nolan EM,FischbachMA,Koglin A,Walsh CT.BiosynthetictailoringofmicrocinE492m:post-translational modification affords anantibacterial siderophore-peptide conjugate.J Am Chem Soc.2007 Nov 21;129(46):14336–47.

[0188] 18.Grozdanov L,Raasch C,Schulze J,Sonnenborn U,Gottschalk G,Hacker J,et al.Analysis of the genome structure of the 40arbour40g40enic probioticEscherichia coli strain Nissle 1917.J Bacteriol.2004 Aug;186(16):5432–41.

[0189] 19.Vejborg RM,Friis C,Hancock V,Schembri MA,Klemm P.A virulent parentwith probiotic progeny:comparative genomics ofEscherichia coli strainsCFT073,Nissle 1917 and ABU 83972.Mol Genet Genomics.2010 Mar 31;283(5):469–84.

[0190] 20.Reister M,Hoffmeier K,Krezdorn N,Rotter B,LiangC,Rund S,et al.Complete genome sequence of the gram-negative probiotic EscherichiacolistrainNissle 1917.J Biotechnol.2014Oct 10:187:106–7.

[0191] 21.Nougayrède JP,HomburgS,Taieb F,Boury M,BrzuszkiewiczE,GottschalkG,et al.Escherichia coli induces DNA double-strand breaks in eukaryotic cells.Science.2006 Aug 11,313(5788):848–51.

[0192] 22.Cuevas-Ramos G,Petit CR,Marcq I,Boury M,Oswald E,Nougayrède JP.Escherichia coli induces DNA damage in vivo and triggers genomic stability in mammals.Proc Natl Acad Sci US A.2010 Jun 22:107(25):11537–42.

[0193] [ PMC free article ] [ PubMed ] 23. Mousa JJ, Yang Y, Tomkovich S, Shima A, Newsome RC, Tripathi P, et al.

[0194] 24.Dubois D,Baron O,Cougnoux A,Delmas J,Pradel N,Boury M,et al.ClbPis a prototype of a peptidase subgroup involved in biosynthesis ofnonribosomal peptides.J Biol Chem.2011 Oct 14;286(41):35562–70.

[0195] 25.Brotherton CA,Balskus EP.A prodrug resistance mechanism isinvolved in colibactin biosynthesis and cytotoxicity.J Am Chem Soc.2013 Mar6;135(9):3359–62.

[0196] 26.Marcq I,Martin P,Payros D,Cuevas-Ramos G,Boury M,Watrin C,etal.The genotoxin colibactin exacerbates lymphopenia and decreases survivalrate in mice infected with septicemic Escherichia coli.J Infect Dis.2014 Jul15;210(2):285–94.

[0197] 27.McCarthy AJ,Martin P,Cloup E,Stabler RA,Oswald E,Taylor PW.Thegenotoxin colibactin is a determinant of virulence in Escherichia coli K1experimental neonatal systemic infection.Infect Immun.2015 Sep 1;83(9):3704–11.

[0198] 28.Payros D,Secher T,Boury M,Brehin C,Ménard S,Salvador-Cartier C,etal.Maternally acquired genotoxic Escherichia coli alters offspring’sintestinal homeostasis.Gut Microbes.2014 May 1;5(3):313–25.

[0199] 29.Bossuet-Greif N,Vignard J,Taieb F,Mirey G,Dubois D,Petit C,etal.The colibactin genotoxin generates DNA interstrand cross-links in infectedcells.mBio[Internet].2018 Mar 20[cited 2018 Nov 21];9(2).Available from:https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5874909 /

[0200] 30.Xue M,Shine E,Wang W,Crawford JM,Herzon SB.Characterization ofnatural colibactin–nucleobase adducts by tandem mass spectrometry andisotopic 41 arbour41 g.Support for DNA alkylation by cyclopropane ringopening.Biochemistry.2018 Nov 13;57(45):6391–4.

[0201] 31.Arthur JC,Perez-Chanona E,Mühlbauer M,TomkovichS,Uronis JM,Fan T-J,et al.Intestinal inflammation targets cancer-inducing activity ofthemicrobiota.Science.2012 Oct 5;338(6103):120–3.

[0202] 32.Buc E,Dubois D,Sauvanet P,Raisch J,Delmas J,Darfeuille-Michaud A,et al.High prevalence of mucosa-associated E.coli producing cyclomodulin andgenotoxin in colon cancer.PloS ONE [Internet].2013 Feb 14[cited 2018 Oct 24];8(2).Available from:https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3572998 /

[0203] 33.Cougnoux A,Dalmasso G,Martinez R,Buc E,Delmas J,Gibold L,etal.Bacterial genotoxin colibactin promotes colon tumour growth by inducing asenescence-associated secretory phenotype.Gut.2014 Dec;63(12):1932–42.

[0204] 34.Pérez-Berezo T,Pujo J,Martin P,Faouder P,Galano J-M,Guy A,etal.Identification of an analgesic lipopeptide produced by the probioticEscherichia colistrainNissle 1917.Nat Commun.2017 Nov 3;8(1):1314.

[0205] 35.Olier M,Marcq I,Salvador-Cartier C,Secher T,DobrindtU,Boury M,etal.Genotoxicity of Escherichia coli Nissle 1917 straincannotbe dissociatedfromits probiotic activity.GutMicrobes.2012 Nov 1;3(6):501–9.

[0206] 36.Martin P,Marcq I,Magistro G,Penary M,Garcie C,Payros D,etal.Interplay between siderophores and colibactingenotoxin biosyntheticpathways in Escherichia coli.PloS Pathog [Internet].2013 Jul[cited 2015 Dec8]9(7).Available from:http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3708854 /

[0207] 37.Datsenko KA,Wanner BL.One-step inactivation of chromosomal genesin Escherichia coli K-12 using PCR products.Proc Natl Acad Sci US A.2000Jun697(12):6640–5.

[0208] 38.Cougnoux A,Gibold L,Robin F,Dubois D,Pradel N,Darfeuille-MichaudA,et al.Analysis of structure–function relationships in the colibactin-maturating enzyme ClbP.J Mol Biol.2012Dec;424(3–4):203–14.

[0209] 39.Gallagher RR,Li Z,Lewis AO,Isaacs FJ.Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA.Nat Protoc.2014 Oct:9(10):2301–1

[0210] 40.Bossuet-GreifN,Belloy M,Boury M,Oswald E,Nougayrede J-P.Protocolfor HeLa cells infection with Escherichia coli strains producing colibactinand quantification of the induced DNA-damage.BIO-Protoc[Internet].2017[cited2019 Jan 21];7(16).Available from:https: / / bio-protocol.org / e2520

[0211] 41.Boudeau J,Glasser A-L,Masseret E,Joly B,Darfeuille-MichaudA.Invasive ability of an Escherichia coli strain isolated from the ilealmucosa ofa patient with Crohn’s disease.Infect Immun.1999 Sep;67(9):4499–509.

[0212] 42.Clermont O,Christenson JK,Denamur E,Gordon DM.The ClermontEscherichia coli phylo-typing method revisited:improvement of specificity anddetection of new phylo-groups.Environ Microbiol Rep.2013 Feb 1;5(1):58–65.

[0213] 43.Wattam AR,Davis JJ,Assaf R,Boisvert S,Brettin T,Bun C,etal.Improvements to PATRIC,the all-bacterial bioinformatics database andanalysis resource center.Nucleic Acids Res.2017Jan 4;45(D1):D535–42.

[0214] 44.Kumar S,Stecher G,Tamura K.MEGA7:Molecular Evolutionary GeneticsAnalysis Version 7.0for Bigger Datasets.Mol Biol Evol.2016 Jul 1;33(7):1870–4.

[0215] 45.Boudeau J,Glasser A-L,Julien S,Colombel J-F,Darfeuille-MichaudA.Inhibitory effect of probiotic Escherichia coli strain Nissle 1917onadhesion to and invasion of intestinal epithelial cells by adherent–invasive Escherichia coli strains isolated from patients with Crohn’sdisease.Aliment Pharmacol Ther.2003 juillet;18(1):45–56.

[0216] 46.Huebner C,Ding Y,Petermann I,Knapp C,Ferguson LR.The probioticEscherichia coli Nissle 1917 reduces pathogen invasion and modulates cytokineexpression in Caco-2 cells infected with Crohn’s disease-associatedE.coliLF82.Appl Environ Microbiol.2011 Apr 1;77(7):2541–4.

[0217] 47.Rusnak F,Faraci WS,Walsh CT.Subcloning,expression,and purificationof the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase:demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylateproduct.Biochemistry.1989 Aug 22;28(17):6827–35.

[0218] 48.Poey ME,Azpiroz MF, M.Comparative analysis of chromosome-encoded microcins.Antimicrob Agents Chemother.2006 Apr;50(4):1411–8.

[0219] 49.Lin H,Fischbach MA,Liu DR,Walsh CT.In vitro characterization ofsalmochelin and enterobactin trilactone hydrolases IroD,IroE,and Fes.J AmChem Soc.2005 Aug 10;127(31):11075.

[0220] 50.Zhu M,Valdebenito M,Winkelmann G,Hantke K.Functions of thesiderophore esterases IroDand IroE in iron-salmochelinutilization.Microbiology.2005;151(7):2363–72.

[0221] 51.Nolan EM,Walsh CT.Investigations of the MceIJ-catalyzedposttranslational modification of the microcin E492 C-terminus:linkage ofribosomal and nonribosomal peptides to form“Trojan Horse”antibiotics.Biochemistry.2008 Sep 2;47(35):9289–99.

[0222] 52.Ventola CL.The Antibiotic Resistance Crisis.Pharm Ther.2015 Apr;40(4):277–83.

[0223] 53.Tacconelli E,Carrara E,Savoldi A,Harbarth S,Mendelson M,Monnet DL,et al.Discovery,research,and development of new antibiotics:the WHO prioritylist of antibiotic-resistan tbacteriaand tuberculosis.LancetInfectDis.2018Mar 1;18(3):318–27.

[0224] 54.Cassini A, LD,Plachouras D,Quattrocchi A,Hoxha A,SimonsenGS,et al.Attributable deaths and disability-adjusted life-years caused byinfections with antibiotic-resistant bacteria in the EU and the EuropeanEconomic Area in2015:a population-level modelling analysis.Lancet Infect Dis[Internet].2018 Nov 5[cited 2018 Nov 26];0(0).Available from:https: / / www.thelancet.com / journals / laninf / article / PIIS1473-3099(18)30605-4 / abstract

[0225] 55.Cotter PD,Ross RP,Hill C.Bacteriocins—a viable alternative toantibiotics?Nat Rev Microbiol.2013 Feb;11(2):95–105.

[0226] 56.Raffatellu M.Learning from bacterialcompetition inthe host todevelop antimicrobials.Nat Med.2018 Aug;24(8):1097–103.

[0227] 57.Gardiner GE,Rea MC,O’Riordan B,O’Connor P,Morgan SM,Lawlor PG,etal.Fate of the two-component lantibiotic lacticin 3147 in thegastrointestinal tract.Appl Environ Microbiol.2007 Nov;73(21):7103–9.

[0228] 58.Naimi S,Zirah S,Hammami R,Fernandez B,Rebuffat S,Fliss I.Fate andbiological activity of the antimicrobial lasso peptide microcin J25 undergastrointestinal tract conditions.Front Microbiol[Internet].2018 Aug 3[cited2018 Nov 27];9.Available from:https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6085462 /

[0229] 59.Forkus B,Ritter S,Vlysidis M,Geldart K,Kaznessis YN.AntimicrobialProbiotics Reduce Salmonella enterica in Turkey Gastrointestinal Tracts.SciRep.2017 Jan 17;7:40695.

[0230] 60.Geldart KG,Kommineni S,Forbes M,Hayward M,Dunny GM,Salzman NH,etal.Engineered E.coli Nissle 1917 for the reduction ofvancomycin-resistantEnterococcus in the intestinaltract.Bioeng Transl Med.2018 Sep 8;3(3):197–208.

[0231] 61.Guenther K,Straube E,Pfister W,Guenther A,Huebler A.Sever sepsisafter probiotic treatment with Escherichia coliNissle 1917.Pediatr InfectDisJ.2010 Feb 1;29(2):188–9.

[0232] 62.Bonnet M,Buc E,Sauvanet P,Darcha C,Dubois D,Pereira B,etal.Colonization of the human gut by E.coliand colorectal cancer risk.ClinCancer Res OffJ AmAssoc Cancer Res.2014 Feb 15;20(4):859–67.

[0233] 63.Dejea CM,Fathi P,Craig JM,Boleij A,Taddese R,Geis AL,etal.Patients with familial adenomatous polyposis 43arbour colonic biofilmscontaining tumorigenic bacteria.Science.2018Feb 2;359(6375):592–7.

[0234] 64.Johnson JR,Russo TA.Extraintestinal pathogenic Escherichia coli:“The other bad E.coli”.J Lab Clin Med.2002 Mar 1;139(3):155–62.

[0235] 65.Hendrickson H.Order and disorder during Escherichia colidivergence.PloS Genet.2009 Jan;5(1):e1000335.

[0236] 66.Barthel M,Hapfelmeier S,Quintanilla-Martínez L,Kremer M,Rohde M,Hogardt M,et al.Pretreatment of mice with streptomycin provides a Salmonellaenterica serovar Typhimurium colitis model that allows analysis of bothpathogen and host.Infect Immun.2003 May;71(5):2839–58.

[0237] 67.Massip C.et al.Deciphering the interplay between the genotoxic andprobiotic activities of Escherichia coli Nissle 1917.PLoS Pathog.2019 Sep 23;15(9):e1008029.

Claims

1. E. coli EcN bacteria carrying a gene encoding a ClbP protein that inactivates the peptidase domain, The amino acid sequence of the ClbP protein is shown in SEQ ID NO:

1. The peptidase domain of the ClbP protein whose amino acid sequence is shown in SEQ ID NO: 3 participates in the activation of the genotoxin colicin, and wherein the EcN bacteria (i) have the ability to possess antibacterial activity; and (ii) lack the ability to activate the genomic toxin colicin, and The ClbP protein that inactivates the peptidase domain is selected from any one of the following lists: The amino acid sequence of ClbP S95A mutant is shown in SEQ ID NO: 4, The ClbP K98T mutant with an amino acid sequence as shown in SEQ ID NO: 6, The ClbP S95R mutant with an amino acid sequence as shown in SEQ ID NO: 8, The ClbP Y186G mutant with an amino acid sequence as shown in SEQ ID NO: 10, or ClbP proteins without peptidase domains.

2. Use of a therapeutically effective amount of the Escherichia coli EcN bacteria according to claim 1 in the preparation of a medicament for treating a gastrointestinal disease in a subject, The gastrointestinal disease is selected from Crohn's disease or Salmonella typhimurium infection.

3. Use of the Escherichia coli EcN bacteria according to claim 1 in the preparation of probiotics.

Images