WO2015001053A1 - Methods for the screening of substances that may be useful for the prevention and treatment of infections by enterobacteriaceae family - Google Patents

Abstract

The present invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and/or clbA of the Enterobacteriaceae or to inhibit the activity of the protein encoded by the gene entD and/or the gene clbA.

Description

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METHODS FOR THE SCREENING OF SUBSTANCES THAT MAY BE USEFUL FOR THE PREVENTION AND TREATMENT OF INFECTIONS BY ENTEROBACTERIACEAE FAMILY FIELD OF THE INVENTION:

The invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and/or clbA of the Enterobacteriaceae or to inhibit the activity of the protein encoded by the gene entD and/or the gene clbA.

BACKGROUND OF THE INVENTION: Escherichia coli is a normal resident of the lower-gut of humans and animals.

Although usually a commensal, E. coli can be also a pathogen, associated with diarrheal disease and extra-intestinal infections [Kaper JB et al., 2004]. The majority of E. coli strains can be assigned to one of five main phylogenetic groups: A, Bl, B2, D and E. Strains of the distinct phylogenetic groups differ in their phenotypic and genotypic characteristics. Extra- intestinal pathogenic E. coli (ExPEC), which display enhanced ability to cause infection outside the intestinal tract, carry specific genetic determinants or virulence factors that are clustered on different pathogenicity islands. These virulence factors associated with extraintestinal infections are nonrandomly distributed, and strains of the E. coli phylogenetic group B2 harbor the greatest frequency and diversity of virulence traits.

As iron bioavailability is limited in the host, ExPEC are known to synthesize up to four types of siderophores involved in iron uptake: enterobactin, salmochelins, yersiniabactin and aerobactin [Gao Q et al. 2012]. The biosynthesis of the first three requires a 4'- phosphopantetheinyl transferase (PPTase). These enzymes activate polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) by catalyzing the transfer of a phosphopantetheinyl (P-pant) moiety from coenzyme A to conserved serine residues on PKSs and NRPSs. In organisms containing multiple P-pant-requiring pathways, each pathway generally involves a dedicated cognate PPTase. In E. coli, the EntD PPTase is involved in the synthesis of enterobactin and salmochelins, which are glycosylated forms of enterobactin. The IroA locus responsible for salmochelins production is located either on a chromosomal pathogenicity island or on a transmissible plasmid. Contrary to enterobactin, salmochelins are able to evade the mammalian innate immune response protein lipocalin 2 (siderocalin) and are therefore more potent virulence factors. The other siderophore necessitating a PPTase is yersiniabactin. This siderophore is encoded by the high-pathogenicity island (HPI) that was acquired through horizontal transfer. The HPI core region was detected in more than 70% of ExPEC isolated from blood cultures, urine samples and cerebrospinal fluid. While yersiniabactin production in Yersinia requires the YbtD PPTase encoded outside the HPI, no gene homologous to ybtD has been identified in the genome of E. coli strains producing yersiniabactin. The PPTase committed to the synthesis of yersiniabactin in E. coli remains unknown.

The inventors have shown that a majority of E. coli strains from phylogenetic group B2 display also the pks island, which codes for the production of colibactin, a polyketide-non ribosomal peptide genotoxin [Nougayrede JP et al, 2006]. Colibactin is known to induce DNA double-strand breaks, cell cycle arrest in G2-phase and megalocytosis in infected eukaryotic cells [Nougayrede JP et al, 2006]. E. coli strains harboring the pks island can induce DNA damage in enterocytes in vivo and trigger genomic instability in mammalian cells. In a rodent model of colon inflammation, colibactin was demonstrated to potentiate the development of colon cancer. Surprisingly, colibactin is also required for the colonic antiinflammatory properties of the probiotic E. coli strain Nissle 1917. The synthesis of colibactin requires a PPTase encoded by the clbA gene located on the pks island [Nougayrede JP et al, 2006]. Epidemiological studies revealed that the majority (73.1%) of the colibactin-positive E. coli strains was clinical ExPEC and that the pks island was significantly associated with a highly virulent subset of ExPEC isolates. Strikingly, an analysis of the prevalence of the colibactin island among Enterobacteriaceae revealed that the pks island was constantly associated with the yersiniabactin gene cluster.

SUMMARY OF THE INVENTION:

In this work, the inventors investigated a potential interplay between the biosynthetic pathways leading to the production of siderophores and of the colibactin genotoxin, through a possible functional interchangeability between PPTases in E. coli. They demonstrated that ClbA can contribute to the synthesis of siderophores both in vitro and in vivo. They proved in a mouse model of sepsis that the presence of either functional EntD or ClbA is required to maintain full virulence of ExPEC. This evidenced the interconnection between pathways leading to the synthesis of distinct secondary metabolites, via the PPTase ClbA. Therefore, the strict association of the pks island with HPI could have been selected in highly virulent E. coli isolates because ClbA can contribute to the synthesis of both the genotoxin and yersiniabactin.

Thus, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and/or clbA of the Enterobacteriaceae or to inhibit the activity of the protein encoded by the gene entD and/or the gene clbA.

DETAILED DESCRIPTION OF THE INVENTION:

Screening method A first object of the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and/or the gene clbA of Enterobacteriaceae or the activity of the protein encoded by said genes.

Substances that are inhibitors of EntD and/or ClbA may be indeed very useful for inhibiting the virulence of Enterobacteriaceae family in the whole organism during extraintestinal infections and moreover for limiting or inhibiting the colonisation and virulence of the Enterobacteriaceae family in the gut and notably in the lower-gut. Said substances may represent very efficient tools for prevention of Enterobacteriaceae family, and more particularly of the prevention of diarrheal diseases and extra-intestinal infections. Finally said substances may be also useful in therapeutic treatment preferably in combination with antibiotics. As used herein the term "gene entD" denotes a gene which encodes a for the EntD phosphopantetheinyl transferase (PPTase) required for synthesis of the iron-chelating and transport molecule enterobactin (Ent), which is express by Enterobacteriaceae family. „

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As used herein the term "gene clbA" denotes a gene which encodes the ClbA phosphopantetheinyl transferase (PPTase) required for colibactin biosynthesis, a genotoxin. This gene is localized on the pks island. The locus of the pks island is accessible under accession number GENBANK: AM229678.

As used herein, the term "protein encoded by the gene entD" denotes the EntD PPTase involved in the synthesis of siderophores that is to say the enterobactin, the salmochelins, and the yersiniabactin [Gehring AM et al, 1997]. For example, the 4'-phosphopantetheinyl transferase EntD from E. coli IHE3034 is accessible under accession number GENBANK: ADE91656.1.

As used herein, the term "protein encoded by the gene clbA" denotes the ClbA PPTase involved in the synthesis of colibactin [Nougayrede JP et al, 2006]. For example, the putative 4'- phosphopantetheinyl transferase ClbA from E. coli is accessible under accession number GENBANK: CAJ76300.1.

As used herein, the term "Enterobacteriaceae family" denotes a large family of Gram- negative bacteria that includes, along with many harmless symbionts, many of the more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella. This family is the only representative in the order Enter obacteriales of the class Gammaproteobacteria in the phylum Proteobacteria. Bacteria classified as members of Enterobacteriaceae may be Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cronobacter, Dickeya ,Edwardsiella, Enterobacter, Enterobacter aerogenes, Erwinia, e.g. Erwinia amylovora, Erwinia tracheiphila, Erwinia carotovora, etc., Escherichia, e.g. Escherichia coli, Ewingella, Grimontella, Hafnia, Klebsiella, e.g. Klebsiella pneumonia, Kluyvera, Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium see Erwinia, Candidatus Phlomobacter, Photorhabdus, e.g. Photorhabdus luminescens, Plesiomonas, e.g. Plesiomonas shigelloides, Pragia, Proteus, e.g. Proteus vulgaris, Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia, e.g. Serratia marcescens, Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia, e.g. Yersinia pestis and Yokenella.

In one embodiment, the Enterobacteriaceae is an Escherichia coli or an extra-intestinal pathogenic E. coli (ExPEC). In one embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD of Enterobacteriaceae.

In another embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene clbA of Enterobacteriaceae.

In another embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and the gene clbA of Enterobacteriaceae.

In another embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the activity of the protein encoded by the gene entD of Enterobacteriaceae.

In another embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the activity of the protein encoded by the gene clbA of Enterobacteriaceae.

In another embodiment, the invention relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the activity of the protein encoded by the genes entD and clbA of Enterobacteriaceae.

In one embodiment, the method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family which comprises the steps consisting of:

a) providing a composition comprising a Enterobacteriaceae e.g. Escherichia coli; _r

- 6 - b) adding the candidate substance to be tested to the composition provided at step a), whereby providing a test composition;

c) comparing the activity of entD and/or elbA genes or the activity of proteins encoded by said genes in said test composition with the activity of the same entD and/or clbA genes or proteins in the absence of said candidate substance; and

d) selecting positively the candidate substance that inhibits the expression of entD and/or clbA genes or the activity of the proteins encoded by said genes.

The method according to the invention wherein the inhibitor substance is selected for its in vivo activity.

In one embodiment, the inhibition of the gene expression according to the invention may be monitored by the production of the protein encoded by this gene. The invention also encompasses methods for the screening of candidate substances that are based on the ability of said candidate substances to inhibit the activity of the proteins encoded by the genes entD and/or clbA.

In other word, the invention consists to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family which comprises the steps consisting of:

a) providing a composition comprising the proteins encoded by the genes entD and/or clbA;

b) adding the candidate substance to be tested to the composition provided at step a), whereby providing a test composition;

c) comparing the activity of said proteins in said test composition with the activity of the proteins in the absence of said candidate substance; and

d) selecting positively the candidate substance that inhibits the activity of said proteins.

Candidate substances that have been positively selected with the method below at the end of any one of the in vitro screening methods of the invention may then tested in various in vitro assays. Said in vitro assays may consist in testing the ability of the positively selected candidate substance to impact the uptake of iron by the Enterobacteriaceae in an in vitro test. In another embodiment, the candidate substances may be tested in a test of production of siderophores or colibactin in a CAS plates or sideroTec kit (for example see Schwyn B., 1987). In another embodiment, a candidate substance to be tested inhibits the catalytic activity of the protein encoded by the gene entD and/or the gene clbA, when the candidate substance is present, is lower than when said enzyme is used without the candidate substance under testing. This invention also encompasses methods for the screening of candidate substances, that are based on the ability of said candidate substances to bind to a protein encoded by the gene entD and/or the gene clbA as defined herein, thus methods for the screening of potentially substances that may be useful for the prevention and treatment of infections by

Enterobacteriaceae family.

The binding assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

All binding assays for the screening of candidate substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family are common in that they comprise a step of contacting the candidate substance with a protein as defined herein, under conditions and for a time sufficient to allow these two components to interact.

These screening methods also comprise a step of detecting the formation of complexes between said protein encoded by the gene entD and/or the gene clbA and said candidate substances.

Thus, screening for substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family includes the use of two partners, through measuring the binding between two partners, respectively a protein as defined herein and the candidate substance.

In binding assays, the interaction is binding and the complex formed between a protein encoded by the gene entD and/or the gene clbA as defined above and the candidate substance that is tested can be isolated or detected in the reaction mixture. In a particular embodiment, the protein as defined above or alternatively the anti- Enterobacteriaceae family candidate substance is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non- covalent attachments. Non-covalent attachment generally is accomplished by coating the solid - o - surface with a solution of the protein of the invention and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the protein of the invention to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non- immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

The binding of the anti- Enterobacteriaceae family candidate substance to a protein of the invention may be performed through various assays, including traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co -purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, 1989; Chien et al, 1991) as disclosed by Chevray and Nathans, 1991. Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for .beta.- galactosidase. A complete kit (MATCHMAKER.TM.) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. Thus, another object of the invention consists of a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family, wherein said method comprises the steps of:

(i) providing a candidate substance;

(ii) assaying said candidate substance for its ability to bind to a protein of the invention.

The same method may also be defined as a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family, wherein said method comprises the steps of:

(i) contacting a candidate substance with a protein of the invention;

(ii) detecting the complexes eventually formed between said protein and said candidate substance. Thus, any substance that has been shown to behave like an inhibitor of the genes or proteins according to the invention, after positive selection at the end of any one of the screening methods that are disclosed previously in the present specification, may be further assayed for his in vivo activity.

Consequently, any one of the screening methods that are described above may comprise a further step of assaying the positively selected inhibitor substance for its in vivo activity. The assay may be done in a mouse model of sepsis for example. However, other non human mammals may be used.

Non human mammals encompass rodents like mice, rats, rabbits, hamsters, guinea pigs. Non human mammals and also cats, dogs, pigs, veals, cows, sheep, goats. Non human mammals also encompass primates like macaques and baboons.

Thus, another object of the present invention consists of a method for the in vivo screening of a candidate substance that may be useful for the prevention and treatment of infections by Enterobacteriaceae family which comprises the steps of:

a) performing a method for the in vitro screening of a substance as disclosed in the present specification, with a candidate substance; and

b) assaying a candidate substance that has been positively selected at the end of step a) for its in vivo activity. In other words, the invention consists to a method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family which comprises the steps consisting of:

a) using an animal model with a disease induced by Enterobacteriaceae for example a mouse model of sepsis as explained in the examples;

b) adding the candidate substance to be tested to the animal model;

c) comparing the effect of the candidate substance on the animal model with another animal model which has not received any candidate substance or which has received a placebo; and

d) selecting positively the candidate substance that improve the survival of the animal model.

Candidate substances that have been positively selected at the end of any one of the in vitro screening methods of the invention may then tested in various in vivo assays. Said in vivo assays may consist in testing the ability of the positively selected candidate substance to impact the virulence of Enterobacteriaceae in a mouse model of sepsis as explained in examples. Thus, any substance that has been shown to behave like an inhibitor of a protein, after positive selection at the end of any one of the in vitro screening methods that are disclosed previously in the present specification, may be further in vivo assayed.

With a method as explained below, the survival of the animal may be monitored as explained in the examples thanks to a animal model of sepsis ( see for example Martin et al, Plos Pathogens in press) or thank to a model of vertical colonization (see for example Payros et al, 2012).

Before in vivo administration to a mammal, the inhibitor substances selected through any one of the in vitro screening methods above may be formulated under the form of pre- pharmaceutical compositions. The pre-pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically acceptable, usually sterile, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the test composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. ^ ^

Compositions comprising such carriers can be formulated by well known conventional methods. These test compositions can be administered to the mammal at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by taking into account, notably, clinical factors. As is well known in the medical arts, dosages for any one mammal depends upon many factors, including the mammal's size, body surface area, age, the particular substance to be administered, sex, time and route of administration and general health. Administration of the suitable pre-pharmaceutical compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. If the regimen is a continuous infusion, it should also be in the range of 1 ng to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The pre-pharmaceutical compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-oxidants, chelating agents, and inert gases and the like.

The inhibitor substances may be employed in powder or crystalline form, in liquid solution, or in suspension.

The injectable pre-pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder (lyophilized or non-lyophilized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In injectable compositions, the carrier is typically comprised of sterile water, saline, or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included. Topical applications may be formulated in carriers such as hydrophobic or hydrophilic base formulations to provide ointments, creams, lotions, in aqueous, oleaginous, or alcoholic liquids to form paints or in dry diluents to form powders.

Oral pre-pharmaceutical compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize carriers such as conventional formulating agents and may include sustained release properties as well as rapid delivery forms.

Generally, all animals are sacrificed at the end of the in vivo assay. For determining the in vivo activity of the inhibitor substance that is tested, blood or tissue samples of the tested animals such as brain samples are collected at determined time periods and bacteria counts are performed, using standard techniques, such as staining fixed slices of the collected tissue samples or plating the collected blood samples and counting the bacterial colonies formed. Then, the values of the bacteria counts found for animals having been administered with increasing amounts of the inhibitor substance tested are compared with the value(s) of bacteria count(s) obtained from animals that have been injected with the same number of bacteria cells but which have not been administered with said inhibitor substance.

Inhibitors and use thereof A second object of the invention relates to i) an inhibitor of entD gene expression or of the activity of the protein encoded by the entD gene and ii) an inhibitor of clbA gene expression or of the activity of the protein encoded by the clbA gene according to the invention as a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of infections by Enterobacteriaceae family in a patient.

In one embodiment, the combined preparation according to the invention is useful against infections by Enterobacteriaceae family, for example diarrheal disease or an extraintestinal infection provoked by Enterobacteriaceae family, typically Escherichia coli or extraintestinal pathogenic E. coli.

In another embodiment, the combined preparation according to the invention is administered concomitantly with antibiotics.

In one embodiment, the invention relates to i) an inhibitor of entD gene expression and ii) an inhibitor of clbA gene expression according to the invention as a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of infections by Enterobacteriaceae family in a patient.

In another embodiment, the invention relates to i) an inhibitor of the activity of the protein encoded by the entD gene and ii) an inhibitor of the activity of the protein encoded by the clbA gene according to the invention as a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of infections by Enterobacteriaceae family in a patient.

In one embodiment of the invention, the inhibitors according to the invention may be selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies, aptamers or nucleic acids. For example the inhibitors according to the invention may be selected from a library of substances previously synthesised, or a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesised de novo.

In a particular embodiment, the inhibitors according to the invention may be selected form small organic molecules.

As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.

In one embodiment, the inhibitors according to the invention are described in Beld J. et al, 2014.

In one embodiment, the inhibitors according to the invention may be an inhibitor of Surfactin phosphopantetheinyl transferase (Sfp) PPTases.

In one embodiment, the inhibitors according to the inventions may be the Calmidazolium chloride, the 6-Nitroso-l,2-benzopyrone, PD404,182, sanguinarine chloride, SCH-202676 HBr or guanadinyl-naltrindole ditrifluoroacetate (see Beld J. et al, 2014). In another embodiment, the inhibitors according to the invention are an inhibitor of the entD gene and/or clbA gene expression.

Small inhibitory R As (siR As) can also function as inhibitors of entD and/or clbA gene expression for use in the present invention. entD and/or clbA gene expression can be reduced by contacting a subject or cell with a small double stranded R A (dsR A), or a _{Λ Λ}

- 14 - vector or construct causing the production of a small double stranded RNA, such that entD and/or clbA gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of entD and/or clbA gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of EntD and/or ClbA mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of entD and/or clbA gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone. Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably bacteria expressing entD and/or clbA genes. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing _{Λ r}

- 16 - multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In another embodiment, the inhibitors may be an antibody directed against the protein encoded by the gene entD and/or the gene clbA that is disclosed in the present specification, or to a biologically active peptide fragment thereof. Any one of these antibodies may be useful for purifying or detecting the corresponding protein.

There is no particular limitation on the antibodies encompassed by the present invention, as long as they can bind specifically to the desired protein or the desired biologically active fragment thereof. It is possible to use mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, chimeric antibodies, humanized antibodies, human antibodies and the like, as appropriate. Such antibodies may be polyclonal or monoclonal, but are preferably monoclonal because uniform antibody molecules can be produced stably. Polyclonal and monoclonal antibodies can be prepared in a manner well known to those skilled in the art.

In principle, monoclonal antibody-producing hybridomas can be prepared using known techniques, as follows. Namely, the desired antigen or the desired antigen-expressing cell is used as a sensitizing antigen and immunized in accordance with conventional procedures for immunization. The resulting immunocytes are then fused with known parent cells using conventional procedures for cell fusion, followed by selection of monoclonal antibody-producing cells (hybridomas) through conventional screening procedures. Preparation of hybridomas may be accomplished according to, for example, the method of Milstein et al. (Kohler, G. and Milstein, C, Methods Enzymol. (1981) 73 :3-46). If an antigen used is less immunogenic, such an antigen may be conjugated with an immunogenic macromolecule (e.g., albumin) before use in immunization.

In addition, antibody genes are cloned from hybridomas, integrated into appropriate vectors, and then transformed into hosts to produce antibody molecules using gene recombination technology. The genetically recombinant antibodies thus produced may also be used in the present invention (see, e.g., Carl, A. K. Borrebaeck, James, W. Larrick, «Therapeutic monoclonal antibodies», Published in the United Kingdom by MacMillan Publishers Ltd, 1990). More specifically, cDNA of antibody variable domains (V domains) is synthesized from hybridoma mRNA using reverse transcriptase. Upon obtaining DNA encoding the target antibody V domains, the DNA is ligated to DNA encoding desired antibody constant domains (C domains) and integrated into an expression vector. Alternatively, the DNA encoding the antibody V domains may be integrated into an expression vector carrying the DNA of the antibody C domains. The DNA construct is integrated into an expression vector such that it is expressed under control of an expression regulatory region, e.g., an enhancer or a promoter. Host cells are then transformed with this expression vector for antibody expression. - lo in a case where antibody genes are isolated and then transformed into appropriate hosts to produce antibodies, any suitable combination of host and expression vector can be used for this purpose. When eukaryotic cells are used as hosts, animal cells, plant cells and fungal cells may be used. Animal cells known for this purpose include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa and Vera, (2) amphibian cells such as Xenopus oocytes, and (3) insect cells such as sf9, sf21 and Tn5. Plant cells include those derived from Nicotiana plants (e.g., Nicotiana tabacum), which may be subjected to callus culture. Fungal cells include yeasts such as Saccharomyces (e.g., Saccharomyces serevisiae) and filamentous fungi such as Aspergillus (e.g., Aspergillus niger). When prokaryotic cells are used, there are production systems employing bacterial cells. Bacterial cells known for this purpose are E. coli and Bacillus subtilis. Antibodies can be obtained by introducing target antibody genes into these cells via transformation and then culturing the transformed cells in vitro.

A further aspect of the invention relates to a method for treating or preventing infections by Enterobacteriaceae family a subject in need thereof with a therapeutically effective amount of an inhibitors or a combined preparation according to the invention.

By a "therapeutically effective amount" is meant a sufficient amount of the inhibitors or the combined preparation to treat or prevent infections by Enterobacteriaceae family at a reasonable benefit/risk ratio applicable to any medical treatment.

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the agent for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the agent, preferably from 1 mg to about 100 mg of the agent. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

A further aspect of the invention relates to method for treating diarrheal disease provoked by Enterobacteriaceae family and especially E. Coli comprising administering in a subject in need thereof a therapeutically effective amount of inhibitors or a combined preparation according to the invention.

The inhibitors or the combined preparation according to the invention may be incorporated into a pharmaceutical composition.

Pharmaceutical composition used in the present methods comprise a therapeutically effective amount of an inhibitor of entD gene expression or an inhibitor of the activity of the protein encoded by the gene entD and an inhibitor of clbA or an inhibitor of the activity of the protein encoded by the gene clbA according to the invention and a pharmaceutically acceptable carrier.

In one embodiment, the composition comprises a relatively inert carrier. Many such carriers are routinely used and can be identified by reference to pharmaceutical texts. Examples include polyethylene glycols, polypropylene copolymers, and some water soluble gels. Such a composition may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other pharmaceutically acceptable materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the anti-microbial activity of the inhibitors.

In practicing the present method, a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor according to the invention is applied to a potential or actual site of infection in the host subject before or after the host subject is exposed to the bacterium. Such composition may be used prophylactically to prevent or reduce the severity of diseases. In the case of oral administration, dentrifices, mouthwashes, tooth paste or gels, or mouth sprays are used. Vaginal or rectal administration may be by the usual carriers such as douches, foams, creams, ointments, jellies, and suppositories, the longer _2Q lasting forms being preferred. Ocular administration is preferably by ophthalmic ointments or solutions.

The pharmaceutical composition may further contain other agents which either enhance the activity of the inhibitors or complement its activity or use in inhibiting growth of the Enterobacteriaceae family. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with the inhibitors of the invention, or to minimize side effects.

Preferably the pharmaceutical composition comprises a solvent for the inhibitors such as, for example, an alcohol. A liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, corn oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain a physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. The preparation of such pharmaceutical composition having suitable pH, isotonicity, and stability, is within the skill in the art.

Administration of the pharmaceutical composition to an uninfected subject is via local administration to a site which has been or may be contacted with the pathogenic organism. It is preferred that the pharmaceutical composition be applied prior to exposure to the targeted pathogen or preferably within 1-24 hours, more preferably within 1-12 hours after exposure of the uninfected subject to the pathogenic organism.

The present invention also relates to compositions or kits for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family.

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

FIGURES: Figure 1. Both EntD and ClbA can support the yersiniabactin siderophore synthesis in vitro. Siderophore production by the enterobactin and yersiniabactin siderophores producer Escherichia coli strain SE15 and derivatives. A. Quantification of total siderophore production in supernatants of E. coli strain SE15 and derivatives determined by the CAS assay. The data are the means and standard deviations of 5 independent experiments. B. Quantification of the yersiniabactin siderophore production in E. coli strain SE15 and derivatives. E. coli strains HB101, MG1655 and DH5a were used as negative controls (K12). RLU: relative light units. ***: PO.001, **: P<0.01, *: P<0.05.

Figure 2. Colibactin synthesis cannot be sustained by EntD in vitro. Colibactin production by Escherichia coli strain Ml/5 and derivatives determined by megalocytosis (A) and by quantification of DNA double strand breaks (B) in infected HeLa cells. A. Live E. coli wild type strain Ml/5, mutants and complemented derivatives were added directly onto HeLa cells [multiplicity of infection (MOI) = 100], cocultivated for 4 h, then washed as described in Nougayrede et al. [21]. The cells were incubated for 72 h with gentamicin before protein staining with methylene blue. The quantification of staining was measured at OD 660 nm. **: P<0.01, *: P<0.05, ns: not significant. B. Quantification of DNA double strand breaks through the quantification of phosphorylated H2AX (γ-Η2ΑΧ) using In Cell Western method [27]. HeLa cells were infected 4 h with strain Ml/5 and derivatives [MOI = 50 to 6] fixed, and examined 8 h post infection for quantification of γ-Η2ΑΧ.

Figure 3. Colibactin synthesis can be sustained by exogenous PPTases in vitro.

Colibactin production by Escherichia coli strain Ml/5 and derivatives determined by megalocytosis (A) and by quantification of DNA double strand breaks (B), as in Fig. 3. P<0.001, **: P<0.01, ns: not significant. E. coli strain SE15, which is devoid of colibactin locus, was used as a negative control. The ybtD gene encodes the YbtD PPTase in Yersinia pestis, the pptT gene the PptT PPTase in Mycobacterium tuberculosis, and the sfp gene the Sfp PPTase in Bacillus subtilis. Figure 4. ClbA is more promiscuous in its substrate specificity than EntD. The synthesis of the single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae resulting in the production of indigoidine was qualitatively and quantitatively (A) assessed as previously described. E. coli strain MG1655 AentD + p-BpsA was transformed with the plasmids p-ybtD, p-pptT, p-sfp, p-clbA (1), p-entD and pUC19 (left). E. coli strain ^

MG1655 BAC pks+ and MG1655 BAC pksAclbA were transformed with p-BpsA (right). A. Quantification of indigoidine in the different strains. The data are the means and standard deviations of 3 independent experiments. P<0.001, **: P<0.01, ns: not significant. Figure 5. Both EntD and ClbA must be inactivated to abolish virulence of

ExPEC. Mice underwent footpad injection with 108 CFU of E. coli SP15 wild type strain or derivatives. A. The percentage of mice survival was monitored. 10 to 25 mice were used per group. B. 18 h post infection 4 to 10 mice per group were sacrificed. Bacteria were quantified in spleen and blood collected from each animal. For statistical analysis, two-factor ANOVA and Bonferroni's multiple comparison test was performed. P<0.001, **: P<0.01.

Figure 6. The presence of either EntD or ClbA is required to maintain full virulence of ExPEC. Mice underwent footpad injection with 108 CFU of E. coli SP15 entD clbA strain and complemented derivatives. A. The percentage of mice survival was monitored. 10 to 25 mice were used per group. B. 18 h post infection 3 to 5 mice per group were sacrificed. Bacteria were quantified in spleen and blood collected from each animal. For statistical analysis, two-factor ANOVA and Bonferroni's multiple comparison test was performed. P<0.001, ns: not significant.

Table 1: Strains and plasmids used in the study

Strain or plasmid Genotype or Source or reference phenotype

E. coli strains

DH10B Enterobactin

siderophore producer

DH5a Enterobactin

siderophore producer

HB101 Enterobactin

siderophore producer

MG1655 Enterobactin

siderophore producer

WR1542 + Tc^r, Ap^r, Kan^r, Cm¹ ; Gift from W. Rabsch pACYC5.3L fepAv.TnlOdlc,

iroN: :pGP704, czV: :MudJ

carrying pACYC5.3L

plasmid

MG1655 entE entE mutant of strain This study

MG1655; Kan^r

MG1655 entE + BAC entE mutant of strain This study

pks+ MG1655 carrying BAC

EXAMPLE: Material & Methods

Bacterial Strains, Mutagenesis Procedures and Growth Conditions

Bacterial strains used in this study are listed in Table 1. E. coli SE15 (O150:H5) is a human commensal bacterium isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2. Strain SE15 is devoid of the pks island. E. coli Ml/5 is a human commensal bacterium isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2. Strain Ml/5 harbors of the pks island. Strain SP15 is an extraintestinal pathogenic E. coli strain (ExPEC) of serotype 018:K1 :H7 isolated from neonatal ^ _r

- 26 - meningitis. Strain SP15 harbors the pks island. The repertoire of siderophores the E. coli strains possess is indicated in Table 1. Gene inactivations were engineered by using the lambda Red recombinase method [Datsenko KA, et al, 2000] using primers. For complementation, the clbA gene was cloned into plasmid pASK75, a cloning vector that harbors a pBR322 origin of replication and therefore is low copy number plasmid (p-clbA (1), table 1) or PCR-Script, a cloning vector that harbors a ColEl origin of replication and therefore is high copy number plasmid (p-clbA (2), table 1). For complementation, the entD gene was cloned into PCR-Script (p-entD, table 1).

Before injection to mice, all E. coli strains were grown overnight in LB broth supplemented with antibiotics if required, at 37°C with shaking. These cultures were diluted 1 : 100 in LB broth with antibiotics when necessary and grown for 3 h at 37°C with shaking. Bacterial cells were resuspended in sterile PBS to the appropriate concentration (2 x 109 CFU/mL). All the strains were shown to display similar growth kinetics in vitro in LB broth (data not shown).

Detection and quantification of total siderophores

Chrome azurol S (CAS) assay was used to detect siderophores produced by E. coli. The CAS solution was prepared according to Schwyn and Neilands. E. coli strains were grown on CAS agar plates and incubated at 37°C overnight in the dark. The colonies with orange zones were siderophore-producing strains.

To quantify siderophore synthesis, 500 of CAS indicator solution containing 4 mM sulfosalicylic acid was mixed with the same volume of supernatant. The reaction mixtures were incubated for 60 min at room temperature to allow complex formation, and the siderophore-dependent color change was determined at OD630nm. For quantification, the iron chelating agent 8 hydroxy quino line (8HQ, sigma-aldrich) was used as the standard.

Quantification of yersiniabactin

The expression of the fyuA gene encoding the yersiniabactin receptor (FyuA) is known to be up-regulated in the presence of extracellular yersiniabactin. Thus, yersiniabactin- dependent up-regulation of fyuA expression can be monitored by means of a fyuA-reporter fusion in the indicator strain.

Bacterial strains were cultivated in NBD medium, i.e. Nutrient Broth (NB) medium supplemented with 200 μΜ α,α'-dipyridyl (Sigma), for 24 h at 37°C. Bacteria were pelleted by centrifugation and the supernatant was added to the indicator strain WR1542 carrying plasmid pACYC5.3L (kind gift of W. Rabsch, Wernigerode). The plasmid encodes all genes necessary for yersiniabactin uptake; i.e. irp6, irp7, irp8, fyuA and ybtA. Additionally, the fyuA promoter region fused to the luciferase reporter gene is included on pACYC5.3L. After further 24 h of incubation at 37°C the indicator strain was pelleted and resuspended in bacterial lysis buffer (100 mM potassium phosphate buffer [pH 7.8], 2 mM EDTA, 1% [wt/vol] Triton X-100, 5 mg/ml bovine serum albumin, 1 mM dithiothreitol, 5 mg/ml lysozyme). Complete lysis was performed by incubation at room temperature for 20 min and repeated mixing. The samples were centrifuged and supernatants of lysates were analyzed by addition of luciferase reagent (20 mM Tricine-HCl (pH 7.8), 1.07 mM (MgC03)4 Mg(OH)2, 100 μΜ EDTA, 470 μΜ D(-) luciferin, 33.3 mM dithiothreitol, 270 μΜ Li3 coenzyme A, 530 μΜ Mg-ATP). Luciferase activities were determined in triplicates using the multimode reader Berthold Tristar LB 941. Values were corrected by relating luciferase activity to the OD600 of bacterial cultures grown 24 h in NBD medium. K12 E. coli strain DH5a served as negative control. The experiments were repeated at least three times.

Detection and quantification of indigoidine

After overnight cultures in LB broth supplemented with the appropriate antibiotics, bacteria were diluted 1 : 10 in M9 minimal medium supplemented with 100 mM L-glutamine and 1 mM IPTG, and cultivated 16 h at 18-20°C under shaking. Bacteria were then collected by centrifugation at 900 x g for 5 min. At this speed, bacterial cells were pelleted while indigoidine still remained in the supernatant. Indigoidine production was quantified by measuring the absorbance of blue-colored supernatant (OD612nm). The bacterial pellet was resuspended in PBS, and biomass was quantified by measuring the absorbance (OD450nm). Finally, the indigoidine production was normalized with the ratio Indigoidine/Biomass (e.g. ratio OD612nm/OD45 Onm) .

Determination of the megalocytosis induced by colibactin

HeLa cells were maintained by serial passage in DMEM supplemented with 10% FCS, non-essential amino acids and 50 μg/mL gentamicin. HeLa cells were dispensed in 96-well cell culture plate (5x 103 cells/well). For bacterial infections, overnight LB broth cultures of E. coli were diluted in interaction medium (DMEM, 5% FCS, 25 mM HEPES) and cell cultures (-70% confluent) were infected with a multiplicity of infection (number of bacteria per HeLa cell at the onset of infection) of 3 to 400. Four hours post-inoculation, cells were washed 3 times with HBSS and incubated in cell culture medium 72 h with 200 μg/mL gentamicin before protein staining with methylene blue (1% w/v in Tris-HCl 0.01M). The methylene blue was extracted with HCl 0.1N. The quantification of staining was measured at OD660nm. Determination of the genotoxic effect induced by colibactin

The In Cell Western procedure was performed as described previously . Briefly, HeLa cells were dispensed in 96-well cell culture plate (1.5 ^x 105 cells/200 μΙ,ΛνεΙΙβ). Twenty four hours later, cells were infected with E. coli strains for 4 h. Eight hours post-infection the cells were directly fixed in the plate with 4% paraformaldehyde. Paraformaldehyde was neutralized, and cells were permeabilized as previously described. Cells were blocked with MAXblock Blocking medium (Active Motif, Belgium) supplemented with phosphatase inhibitor PHOSTOP (Roche), followed by overnight incubation with rabbit monoclonal anti γ-Η2ΑΧ (Cell Signaling) (1 :200). An infrared fluorescent secondary antibody absorbing at 800 nm (IRDyeTM 800CW, Rockland) was then applied (dilution 1 :500). For DNA labeling, RedDot2 (Biotium) was used (dilution 1 :500) together with the secondary antibody. The DNA and the γ-Η2ΑΧ were simultaneously visualized using an Odyssey Infrared Imaging Scanner (Li-Cor ScienceTec, Les Ulis, France) with the 680 nm fluorophore (red color) and the 800 nm fluorophore (green dye). Relative fluorescence units from the scanning allowed a quantitative analysis. Relative fluorescent units for γ-Η2ΑΧ per cell (as determined by γ- H2AX divided by DNA content) were divided by vehicle controls to determine percent change in phosphorylation of H2AX levels relative to control. All experiments were carried out in triplicate.

Mouse sepsis model

Animal experimentations were carried out in accordance with the European directive for the protection of animals used for scientific purposes. The protocols were validated by the local ethics committee on animal experiment "Comite d'ethique Midi Pyrenees pour l'experimentation animale" which is affiliated to "Comite National de Reflexion Ethique sur l'Experimentation Animale" and linked to the french ministry of research (Referenced protocols: PX-ANI-A2-94, 95, 96, 99, 100, and 101). Nine week old female C57BL/6J mice (JANVIER) were injected into the footpad with 108 ExPEC WT, clbA mutants, entD mutant, entD clbA mutant and entD clbA mutant complemented with clbA (entD clbA + p-clbA(2)) and entD (entD clbA + p-entD), together with intraperitoneal injection of 100 of carbenicillin (1.6 mg/mL) or PBS. When required, mice were sacrificed by lethal anaesthesia (rompun / ketamine in 0.9% NaCl) 18 h post injection. The abdominal cavity of anesthetized mouse was opened. The widest part of the posterior vena cava was localized and sectioned. Blood was collected by aspiration from the abdominal cavity. Spleens were surgically removed. Bacteria located in spleen cells were isolated from the mechanical dissociation of the splenic tissue using Precellys tissue homogenizer. Bacteria were quantified by plating of serial dilutions of blood and dissociated spleen on appropriate selective MacConkey agar. The antibiotics used to supplement the medium correspond to the resistance displayed by the different strains and are indicated in Table 1. Statistical analysis

Statistical analyses were conducted using GraphPad Prism 5.0d. The mean ± standard deviation (SD) is shown in figures, and P values were calculated using a one-way or two-way ANOVA followed by a Bonferroni post-test unless otherwise stated. For bacterial quantification, CFU by mg of spleen or mL of blood were log transformed for the analysis. A P value of less than 0.05 was considered statistically significant and is denoted by *. P < 0.01 is denoted by ** and P < 0.001 by

Results The pks island does not code for the biosynthesis of a siderophore in vitro

Because colibactin and siderophores belong to the same family of chemical compounds, we investigated first whether the pks island could not only allow the production of a genotoxin, but also of a siderophore. The entE gene, that encodes the ligase component of synthase multienzyme complex necessary for the enterobactin biosynthesis, was inactivated in the enterobactin producer E. coli strain MG1655. The resulting MG1655 entE mutant strain was shown not to produce any siderophore, as detected on CAS plate (data not shown). The wild type (WT) and entE derivative of strain MG1655 were transformed with the bacterial artificial chromosome (BAC) harboring the entire pks island (BAC pks+). Both strains MG1655 + BAC pks+ and MG1655 entE + BAC pks+ were shown to produce the genotoxin, as evidenced by the induction of double-strand breaks in eukaryotic cells (data not shown). The production of siderophore was qualitatively investigated in the resulting strains by plating on CAS plates (data not shown). A yellow halo was not observed surrounding the bacterial colonies of strain MG1655 entE + BAC pks+. This showed that the pks island did not code for the biosynthesis of a siderophore. _3Q

ClbA, the PPTase encoded on the pks island, can support the enterobactin siderophore synthesis in vitro

In order to test whether the ClbA PPTase was functionally capable of participating to the biosynthesis of enterobactin, the entD gene was disrupted in E. coli strain MG1655. The resulting MG1655 entD mutant strain was subsequently transformed with BAC pks+ and with the BAC harboring the entire pks island where the clbA gene was deleted (BAC pksAclbA). The production of siderophore was investigated by plating the resulting strains on CAS medium (data not shown). This revealed that disruption of the entD gene in strain MG1655 resulted in the abrogation of the production of enterobactin (data not shown). The introduction of the intact pks island in strain MG1655 entD restored the production of yellow pigmentation surrounding the colonies. This was not observed upon the introduction of the pks island disrupted for the clbA gene (data not shown). Introduction of a functional plasmidic clbA gene in strain MG1655 entD + BAC pksAclbA and in strain MG1655 entD restored the production of enterobactin (data not shown).

These data evidenced that the ClbA PPTase can contribute to the enterobactin siderophore synthesis in vitro.

Both EntD and ClbA can support the yersiniabactin siderophore synthesis in vitro

Yersiniabactin is a siderophore the biosynthesis of which requires the PPTase YbtD in Yersinia pestis. Although numerous E. coli strains were shown to produce yersiniabactin, an in silico analysis of the genome of all the E. coli strains available to date did not reveal any gene homologous to the ybtD gene.

In order to test whether the ClbA PPTase was functionally proficient to participate to the biosynthesis of yersiniabactin, we analyzed the enterobactin and yersiniabactin producer E. coli strain SE15. The entD gene was disrupted in E. coli strain SE15. The resulting SE15 entD mutant was subsequently transformed with plasmids carrying wild type entD gene or clbA gene. The production of total siderophores was qualitatively (data not shown) and quantitatively (Fig. 2A) investigated using the CAS assay. This revealed that disruption of the entD gene resulted in the abrogation of the production of siderophores in strain SE15 (Fig. 2A). As expected, complementation with entD gene restored the production of siderophores. Remarkably, complementation with clbA gene also resulted in the synthesis of siderophores (Fig. 2A). The synthesis of yersiniabactin was specifically quantified in the different SE15 derivatives (Fig. 2B). This revealed that in the entD mutant, the yersiniabactin biosynthesis ^ was abolished. The introduction of entD or clbA genes in SE15 entD mutant strain resulted in the restoration of yersiniabactin production.

These data showed that in E. coli strain SE15, EntD is the PPTase dedicated to the synthesis of yersiniabactin. Moreover, the EntD function can be substituted by ClbA. This suggests that both EntD and ClbA are involved in the synthesis of yersiniabactin in E. coli strains producing endogenously EntD and ClbA.

Colibactin synthesis cannot be sustained by EntD in vitro

As our data demonstrated that ClbA could complement EntD for the synthesis of enterobactin and yersiniabactin, we investigated whether EntD could rescue a clbA mutant for the production of colibactin. The entD gene was disrupted alone or in combination with the clbA gene in the colibactin producing E. coli strain Ml/5. The Ml/5 entD clbA double mutant was transformed with multicopy plasmids harboring wild type entD or clbA genes. The production of colibactin was quantified in the resulting strains through the quantification of megalocytic cells (Fig. 2 A) and phosphorylation of H2AX histone (Fig. 2B) which correlate with DNA double strand breaks resulting from the genotoxic effect of colibactin.

HeLa cells were infected with the different strains for 4 hours, fixed and stained with methylene blue in order to quantify the megalocytosis effect, as previously described. This revealed that the megalocytosis effect observed with the Ml/5 entD mutant strain was similar to the effect measured with the wild type Ml/5 strain (Fig. 2A). Inactivation of the clbA gene in the Ml/5 entD mutant abrogated the colibactin effect (Fig. 2A). Transformation of the Ml/5 entD clbA mutant strain with plasmids carrying the functional wild type clbA gene resulted in the restoration of the megalocytosis. A partial complementation of the double mutation was observed with plasmid p-clbA (1) whereas the double mutant was fully complemented with p-clbA (2). The different copy number of the plasmids can account for the quantitative differences observed below. A complementation was not observed when the wild type entD gene was expressed from a multicopy plasmid in the double mutant (Fig. 2A).

Genotoxicity of colibactin was also examined in HeLa cells using H2AX assay based on indirect DNA double strand break detection using In Cell Western (ICW) with infrared fluorescence for H2AX phosphorylation (γ-Η2ΑΧ) quantification. HeLa cells were infected with strains Ml/5, Ml/5 entD, Ml/5 entD clbA or M 1/5 entD clbA complemented with entD. Following the quantification of the γ-Η2ΑΧ (green) and the DNA (red) signals (Fig. 2B), respectively, the fold induction of γ-Η2ΑΧ per cell was calculated. This revealed a genotoxic dose-response depending on the multiplicity of infection (MOI, Fig. 2B). No difference of γ- ^

H2AX per cell was observed between WT and entD mutant strains. Infection of HeLa cells with mutant Ml/5 entD clbA did not induce phosphorylation of H2AX. Moreover, the introduction of the functional entD gene did not result in the generation of DNA double strand breaks in strain Ml/5 entD clbA (Fig. 2B).

Altogether, these data evidenced that EntD does not contribute to the colibactin synthesis, even when highly expressed on a multicopy plasmid.

Colibactin synthesis can be sustained by exogenous PPTases in vitro

We then investigated whether other PPTases, originated from other bacterial species, could rescue a clbA mutant for the production of colibactin. The clbA gene was disrupted in E. coli strain Ml/5. The Ml/5 clbA mutant was transformed with plasmids harboring wild type ybtD gene that encodes the YbtD PPTase in Yersinia pestis, pptT gene the PptT PPTase in Mycobacterium tuberculosis, sfp gene the Sfp PPTase in Bacillus subtilis, and clbA gene. PptT is involved in biosynthesis of the mycobactin siderophore and is essential for mycobacterial viability. Sfp is required for production of the peptide antibiotic surfactin. The production of colibactin was quantified in the resulting strains through the quantification of megalocytic cells (Fig. 3 A) and phosphorylation of H2AX histone (Fig. 3B). This revealed that both the megalocytosis and the H2AX phosphorylation were restored in the clbA mutant upon introduction of ybtD, pptT and sfp genes.

These data evidenced that ClbA can be xeno-complemented for the colibactin synthesis.

ClbA is more promiscuous in its substrate specificity than EntD

In order to confirm that EntD and ClbA have narrow and broad substrate-specificity, respectively, we investigated whether EntD and ClbA had the capacity to activate the carrier protein involved in a reporter biosynthetic pathway. When activated by a PPTase, the single- module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae synthesizes a colored product (indigo idine), from a single substrate (L-glutamine). Plasmid p-BpsA that encodes BspA was transformed into strain MG1655 entD. The resulting MG1655 AentD + p- BspA strain was subsequently transformed with plasmids carrying ybtD, pptT, sfp, clbA, or entD genes. In addition, E. coli strain MG1655 BAC pks+ and MG1655 BAC pksAclbA were transformed with p-BpsA. The resulting strains that carry both the NRPS and a functional PPTase were grown in auto -induction medium, as previously described. A blue coloration was detectable in cultures after overnight incubation for all strains but strain MG1655 AentD + p-BspA + p-entD (data not shown). A quantification of the indigoidine production was determined for all the strains (Fig. 4). This confirmed that contrary to EntD, the PPTases Y ), PptT, Sfp and ClbA were able to participate to the synthesis of the blue pigment .

This strengthens the fact that ClbA is more promiscuous in its substrate specificity than EntD in E. coli.

Both EntD and ClbA must be inactivated to abolish virulence of ExPEC in a mouse model of sepsis

In order to address the consequences, on the virulence of E. coli, of the cross talk between the synthesis pathways of colibactin and siderophores demonstrated in vitro, we investigated E. coli strain SP 15, an extra-intestinal pathogenic E. coli strain (ExPEC) of serotype 018:K1 :H7 isolated from neonatal meningitis, in a mouse model of sepsis. E. coli strain SP 15 produces colibactin and four different siderophores (aerobactin, yersiniabactin, enterobactin and salmochelin). The entD or clbA genes were disrupted individually and in combination. The strains were injected individually into the mice footpad; and the mice survival was monitored (Fig. 5 A). This revealed that all the strains but SP I 5 entD clbA induced 70% mortality within 40 hours after injection. In contrast, virulence of strain SP 15 entD clbA was completely attenuated in this mouse model of sepsis (Fig. 5A).

The bacterial dissemination in the mice was analyzed (Fig. 5B). Mice were sacrificed 18 hours post injection with PBS, WT strain, single or double mutants. Spleens and blood samples were collected, and bacteria were quantified by plating on selective medium (Fig. 5B). We observed that in both spleen and blood of infected animals the bacterial loads were similar with all the strains, but strain SP15 entD clbA. No bacteria were recovered from spleen or blood of mice injected with the double mutant SP15 entD clbA (Fig. 5B).

This demonstrated that both EntD and ClbA must be inactivated to abolish virulence of ExPEC in a mouse model of sepsis.

The presence of either functional EntD or ClbA is required to maintain full virulence of ExPEC in a mouse model of sepsis

In order to investigate the relative importance of EntD and ClbA in the virulence of £. coli, the SP15 entD clbA mutant strain was transformed with plasmids harboring clbA or entD functional genes. The resulting complemented strains were injected in mice (Fig. 6). This showed that complementation of strain SP15 entD clbA with either clbA or entD totally restored the virulence of the strain (Fig. 6A). A slight but statistically significant delay in ^ survival kinetics was observed when strain SP15 entD clbA complemented with the clbA gene was used for the injections (Fig. 6A). The quantification of bacteria in spleen and blood of the infected animals was determined (Fig. 6B). This revealed that complementation with clbA or entD allowed the survival of strain SP15 entD clbA in vivo, in a statistically significant manner at least in blood (Fig. 6B).

This evidenced that the presence of either functional EntD or ClbA is required to maintain full virulence of ExPEC in a mouse model of sepsis.

Validation of the screening method using commercially available PPTases inhibitors

Among the few molecules proposed to act as PPTases inhibitors (Beld et al, 2014), the inventors select Calmidazolium chloride and 6-Nitroso-l,2-benzopyrone, which are not antibiotics and which do not induce DNA damage in eukaryotic cells. Appropriate concentrations of inhibitors not altering _E. coli_ growth kinetics and HeLa cells survival is determined. The production of colibactin and siderophores (that rely on PPTases) is quantified subsequently to growth of E. coli in the presence of appropriate concentrations of inhibitory molecules. Both inhibitors are tested as previously described (Martin et al., 2013). Production of both colibactin and siderophores is altered showing the capability of the substances (Calmidazolium chloride and 6-Nitroso-l,2-benzopyrone) to prevent and treat infections by Enterobacteriaceae family.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Beld Joris, Eva C. Sonnenschein, Christopher R. Vickery, Joseph P. Noelb, nd Michael D. Burkart. The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. Nat. Prod. Rep., 2014.

Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640-6645. ^

Gao Q, Wang X, Xu H, Xu Y, Ling J, et al. (2012) Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model. BMC Microbiol 12: 14.

Gehring AM, Bradley KA, Walsh CT (1997) Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate. Biochemistry 36: 8495-8503.

Kaper JB, Nataro JP, Mobley HL (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 2: 123-140.

Martin Patricia, Ingrid Marcq, Giuseppe Magistro, Marie Penary, Christophe Garcie,

Delphine Payros, Michele Boury, Ma^'iwenn Olier, Jean-Philippe Nougayrede, Marc Audebert, Christian Chalut, Soren Schubert, Eric Oswald. Interplay between siderophores and colibactin genotoxin biosynthetic pathways in escherichia coli. PLoS Pathog. 2013;9(7):el003437.

Nougayrede JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, et al. (2006) Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science 313: 848- 851.

Payros Delphine, Maiwenn Olier, Pascale Plaisancie, Thomas Secher, Gabriel Cuevas- Ramos, Sandrine Menard, Michele Boury, Christel Cartier, Adelie Balguerie, Ingrid Marcq, Jean-Philippe Nougayrede, Jean Fioramonti, Eric Oswald. The Genotoxicity of Commensal Escherichia coli Colonizing Newborns Influences the Intestinal Barrier at Adulthood. Gastroenterology. Volume 142, Issue 5, Supplement 1 , Pages S-45-S-46, May 2012.

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Claims

CLAIMS:

1. A method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family comprising a step of determining the ability of a candidate substance to inhibit the expression of the gene entD and/or clbA of the Enterobacteriaceae or to inhibit the activity of the protein encoded by the gene entD and/or the gene clbA.

2. The method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family according to claim 1 which comprises the steps consisting of: a) providing a composition comprising a Enterobacteriaceae e.g. Escherichia coli; b) adding the candidate substance to be tested to the composition provided at step a), whereby providing a test composition;

c) comparing the activity of entD and/or clbA genes or the activity of proteins encoded by said genes in said test composition with the activity of the same entD and/or clbA genes or said proteins in the absence of said candidate substance; and d) selecting positively the candidate substance that inhibits the expression of entD and/or clbA genes or the activity of the proteins encoded by said genes.

3. The method for the screening of substances that may be useful for the prevention and treatment of infections by Enterobacteriaceae family according to claims 1 to 2 which comprises a step of assaying the positively selected inhibitor substance for its in vivo activity.

4. A i) inhibitor of entD gene expression or of the activity of the protein encoded by the entD gene and ii) an inhibitor of clbA gene expression or of the activity of the protein encoded by the clbA gene according to any of claims 1 to 3 as a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of infections by Enterobacteriaceae family in a patient.

5. The combined preparation for use according to claim 4 wherein the infection by Enterobacteriaceae family is a diarrheal disease or an extra-intestinal infection provoked by Enterobacteriaceae family, typically Escherichia coli or extra-intestinal pathogenic E. coli.

6. The combined preparation for use according to any of claim 4 to 5 wherein antibiotics may be administered concomitantly with the combined preparation.

7. A pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of entD gene expression or an inhibitor of the activity of the protein encoded by the gene entD and an inhibitor of clbA or an inhibitor of the activity of the protein encoded by the gene clbA according to any of claim 4 to 6 and a pharmaceutically acceptable carrier.

8. A method for treating or preventing infections by Enterobacteriaceae family a subject in need thereof with a therapeutically effective amount of inhibitor of entD gene expression or of the activity of the protein encoded by the entD gene and/or an inhibitor of clbA gene expression or of the activity of the protein encoded by the clbA gene.