TITLE

IMMUNOGENIC PROTEIN AND METHOD OF USE TECHNICAL FIELD

THIS INVENTION relates to Escherichia coli. More particularly this invention relates to an immunogenic protein that is capable of eliciting an immune response to Escherichia coli.

BACKGROUND

Escherichia coli is a bacterium that exists in many different guises. On one hand it is a common commensal organism of the human small intestine. On the other, it is a rapidly evolving pathogen that is able to acquire and combine different genetic elements into novel and complex gene repertoires. The latter has led to the evolution of E. coli as a multi-faceted pathogen, highlighted by aggressive disease outbreaks (1) and the emergence of multidrug resistant (MDR) lineages (2, 3).

E. coli can be classified into different pathotypes according to a common set of virulence factors and specific clinical manifestations (4). Despite these phenotypic associations, strains from a single pathotype are not restricted to one phylo group; such strains can share the same genomic profile with other pathotypes (5) and be distributed over the entire span of the E. coli phylogenetic diversity (6, 7). These observations indicate a common evolutionary origin and divergence into different pathotypes as a result of the independent acquisition of specific virulent genes via multiple events of horizontal gene transfer (8).

The 2011 E. coli O104:H4 German outbreak provided a new perspective to our understanding of evolution and genome plasticity in the species. The outbreak strain had acquired key virulence genes from two different E. coli pathotypes (enteroaggregative E. coli [EAEC] and enterohaemorrhagic E. coli [EHEC]) and, combined with genes encoding resistance to antibiotics, emerged as a highly virulent lineage that infected nearly 4000 people and caused 54 deaths (9). Since this outbreak, it has been proposed that targeting accessory components encoded by the E. coli genome may be insufficient to prevent the emergence of new pathogenic lineages, and that broader strategies directed against conserved features of all strains may be more effective (10).

SUMMARY

The invention relates to an E. coli YncE protein suitable for eliciting an immune response to E. coli in an animal. Preferably, the immune response is capable of being directed to a plurality of different E. coli strains, serotypes and pathotypes. The immune response may be a protective immune response.

Accordingly, one form of the invention is broadly directed to an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative, preferably for use in eliciting an immune response in an animal.

Suitably, the immune response may be a protective immune response and/or prevent or treat an E. co/z-associated disease, disorder or condition in the animal.

In a first aspect, the invention provides a method of eliciting an immune response in an animal, said method including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby elicit an immune response to E. coli in the animal.

Preferably, said immune response is a protective immune response and/or confers passive immunity.

In a second aspect, the invention provides a method of immunizing an animal including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby induce immunity to E. coli in the animal.

In a third aspect, the invention provides a method of treating an E. coli- associated disease, disorder or condition in an animal, said method including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO:l; one or more immunogenic fragments variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby treat the disease, disorder or condition in the animal.

In some embodiments of the aforementioned aspects, the isolated protein of SEQ ID NO:l, or one or more immunogenic fragments variants and/or derivatives thereof, or the isolated nucleic acid may be administered in combination with one or more additional immunogens that may be specific to certain strains/pathotypes of E coli, or are conserved across a number of different E coli strains/pathotypes. Non-limiting examples of other such immunogens include iron-receptor proteins, fimbrial proteins, flagella, capsular antigens and O antigens.

In a fourth aspect, the invention provides a method of detecting an E. coli bacterium, or one or more molecular components thereof, in a sample, said method including the step of detecting: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; in said biological sample, which indicates the presence of E. coli, or one or more molecular components thereof, in the sample.

The animal according to the aforementioned aspects is preferably a mammal, or more preferably a human.

In a fifth aspect, the invention provides an immunogenic fragment of an isolated protein comprising the amino acid sequence of SEQ ID NO: l. Suitably, the immunogenic fragment comprises one or more T or B epitopes.

In a sixth aspect, the present invention provides an isolated nucleic acid that comprises a nucleotide sequence that encodes the immunogenic fragment of the fifth aspect, or a nucleotide sequence complementary thereto.

In a seventh aspect, the invention provides a genetic construct comprising the isolated nucleic acid of the sixth aspect; operably linked or connected to one or more regulatory sequences in an expression vector.

In an eighth aspect, the invention provides a host cell transformed or transfected with the genetic construct of the seventh aspect.

In a ninth aspect, the invention provides a composition comprising: (a) an isolated protein comprising the amino acid sequence of SEQ ID NO: l; (b) the immunogenic fragment of the fifth aspect; (c) a variant or derivative of (a) or (b); (d) an antibody or antibody fragment that binds (a), (b) or (c); (e) an isolated nucleic acid encoding (a), (b) or (c); (f) the genetic construct of the seventh aspect; or (g) the host cell of the eighth aspect; and a carrier, diluent or excipient.

Suitably, the composition is for use according to the method of any one of the first to fourth aspects.

In one embodiment, the composition for use according to the first to third aspects further comprises a carrier, diluent or excipient.

In another embodiment, the composition is suitable for detection, such as according to the fourth aspect.

Throughout this specification, unless otherwise indicated, "comprise", "comprises ^' " and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

By "consist essentially of is meant in this context that the isolated protein or immunogenic fragment has one, two or no more than three amino acid residues in addition to the recited amino acid sequence. The additional amino acid residues may occur at the N- and/or C-termini of the recited amino acid sequence, although without limitation thereto.

It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" protein includes one protein, one or more proteins or a plurality of proteins.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Immunoreactivity of plasma from urosepsis patients to E. coli vaccine antigens, and of mice to YncE. (A) Blood plasma was collected from 47 urosepsis patients (U) at least 4 days post admittance to hospital. IgG-specific antibodies levels were compared to 47 healthy volunteers with no recent history of UTI (C). (B) Level of infection in immunized (I) and control (C) groups of mice at 24 h following intravenous challenge with UPEC strain CFT073. Symbols represent individual mice, and bars show the medians. The limit of detection was 200 CFU/g or CFU/ml. Statistically significant P values (***<0.001, *<0.05) are indicated.

Figure 2. Western blot analysis of total cell lysate (TL) and supernatant (SN) fractions prepared from E. coli strains representing different phylogroups. (A) The YncE protein was detected in all TL and SN samples. The specificity of the antiserum was confirmed by the absence of a cross-reacting band in the CVT0T3yncE mutant. (B) Overexpression of YncE in fur knockout. OmpA was used as an expression control. Figure 3. Classification of the database according to the region and year of isolation, source and disease.

Figure 4. A. Expression of selected targets. TL, total cell lysates; SF, soluble fraction. B. Murine YncE-specific IgG response following subcutaneous immunization. Median titres with range plotted in logarithmic scale.

Figure 5. Primers used herein. The nucleotide sequences of these primers are designated herein as SEQ ID NOS:3-12, respectively.

BRIEF DESCRIPTION OF THE AMINO ACID AND NUCLEOTIDE

SEQUENCES

SEQ ID NO: l = amino acid sequence of E. coli protein YncE

SEQ ID NO:2 = nucleotide sequence encoding SEQ ID NO: l

SEQ ID NO:3 = nucleotide sequence of primer 4636.

SEQ ID NO:4 = nucleotide sequence of primer 4637.

SEQ ID NO:5 = nucleotide sequence of primer 4640.

SEQ ID NO:6 = nucleotide sequence of primer 4641.

SEQ ID NO:7 = nucleotide sequence of primer 4642.

SEQ ID NO:8 = nucleotide sequence of primer 4643.

SEQ ID NO:9 = nucleotide sequence of primer 4650.

SEQ ID NO: 10 = nucleotide sequence of primer 4651.

SEQ ID NO: l l : = nucleotide sequence of primer 5600.

SEQ ID NO: 12 : = nucleotide sequence of primer 5601.

DETAILED DESCRIPTION

The present invention is at least partly predicated on work defining core and accessory E. coli genes that identified YncE as a highly conserved vaccine antigen that is protective against acute E. coli bacteremia. The invention therefore provides methods of immunizing against, preventing and/or treating E. coli infections in animals by administering YncE protein to the animal. This also extends to administering immunogenic YncE fragments, variants, encoding nucleic acids and/or antibodies that bind YncE protein. A particular feature of the invention is that because YncE is "universally" expressed by a plurality of different E. coli strains, serotypes and pathotypes, immunity to YncE may provide immunity to a plurality of different E. coli strains, serotypes and pathotypes.

Accordingly, a broad form of the invention is broadly directed to an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative, for use in eliciting an immune response in an animal.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be enriched, partially purified or in a substantially pure form. Isolated material may be in native, chemical synthetic or recombinant form.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.

The term "protein" includes and encompasses "peptide", which is typically used to describe a protein having no more than fifty (50) amino acids and "polypeptide", which is typically used to describe a protein having more than fifty (50) amino acids.

A non-limiting example of an E. coli YncE protein comprises the amino acid sequence of SEQ ID NO: 1 :

mhlrhlfssr lrgslllgsl l vssfstqa aeemlrkavg kgayemaysq qenalwlats qsrkldkggv vyrldpvtle vtqaihndlk pfgatinntt qtlwfgntvn savtaidakt gevkgrlvld drkrteevrp lqprelvadd atntvyisgi gkesviw vd ggniklktai qntgkmstgl aldsegkrly ttnadgelit idtadnkils rkkllddgke hffinisldt arqrafitds kaaevl vdt rngnilakva apeslavlfn parneayvth rqagkvsvid aksyk vktf dtpthpnsla lsadgktlyv svkqkstkqq eatqpddvir ial

(SEQ ID NO: 1)

A "fragment" is a segment, domain, portion or region of a protein, which constitutes less than 100% of the amino acid sequence of the protein.

In general, fragments may comprise, consist essentially of, or consist of, up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 280, 300 or 320 amino acids of an amino acid sequence. Suitably, the fragment is an immunogenic fragment. In the context of the present invention, the term "immunogenic " as used herein indicates the ability or potential of a protein, fragment or variant to elicit or generate an immune response to one or a plurality of strains of E. coli, or molecular components thereof, upon administration of the protein to an animal. It is envisaged that the immune response may be either B -lymphocyte or T-lymphocyte mediated, or a combination thereof. Advantageously, by "immunogenic " is meant capable of eliciting a B- lymphocyte response, although is not limited thereto, preferably including an antibody response, such as an IgG response.

Accordingly, in a preferred form a fragment may comprise at least one T cell epitope and/or at least one B cell epitope.

As used herein, a protein "variant" shares a definable amino acid sequence relationship with an isolated protein or immunogenic fragment disclosed herein. Preferably, protein variants share at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:l, or with a fragment of SEQ ID NO: l as hereinbefore described. Suitably, the variant is immunogenic, as hereinbefore defined.

The "variant" proteins or fragments disclosed herein have one or more amino acids deleted, added or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the immunogenic fragment and/or protein (conservative substitutions).

The term "variant" also includes isolated proteins or fragments thereof disclosed herein, produced from, or comprising amino acid sequences of, naturally occurring allelic variants and orthologs {e.g. from various strains, pathotypes or serotypes of E. coli) and synthetic variants, such as produced in vitro using mutagenesis techniques.

Suitably, protein variants are immunogenic, as hereinbefore described.

Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window ^' " refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions {i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment {i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1991, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

Derivatives of SEQ ID NO:l and fragments and variants are also provided. Suitably, said derivatives are immunogenic, as hereinbefore described.

As used herein, "derivative" proteins have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification {e.g. phosphorylation, acetylation and the like), modification of glycosylation {e.g. adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as would be understood in the art.

Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags.

Other derivatives contemplated by the invention include, but are not limited to, modification to amino acid side chains, incorporation of non-natural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention.

In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

The isolated immunogenic proteins, fragments and/or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.

Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444.

Recombinant proteins and immunogenic fragments may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995- 2008), in particular Chapters 1, 5 and 6.

Alternatively, fragments can be produced by digestion of a polypeptide with proteinases such as endoLys-C, endoArg-C, endoGlu-C and V8-protease. The digested fragments can be purified by chromatographic techniques as are well known in the art.

As used herein, an "antibody" may be, or comprise, an antigen-binding protein encoded by the immunoglobulin gene complex inclusive of IgA, IgD, IgE and IgG antibodies. Antibodies may be polyclonal or monoclonal, native or recombinant. Well- known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of the invention. For example, the antibodies may be polyclonal antibodies. Such antibodies may be prepared for example by administering an E. coli YncE protein, fragment, variant or derivative to a production species, such as mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et ah, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibody fragments, such as Fc, Fab or F(ab)2 fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293. Antibodies may also include multivalent recombinant antibody fragments, such as diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g., WO/2007/062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90:6444-6448; or in Kipriyanov, 2009 Methods Mol Biol 562: 177-93 and herein incorporated by reference in their entirety.

For aspects of the invention relating to detection of E. coli and/or YncE proteins such as in biological samples, the antibody or antibody fragment may be labelled. By way of example only, the antibody or antibody fragment may be labeled with biotin, an enzyme, fluorophore, radionuclide or other label. Alternatively, the antibody or antibody fragment is unlabeled and a secondary antibody comprises a label. By way of example, the enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or glucose oxidase, although without limitation thereto. In embodiments where the antibody or antibody fragment or the secondary antibody is labeled with an enzyme, an appropriate substrate substrate may include diaminobanzidine (DAB), permanent red, 3-ethylbenzthiazoline sulfonic acid (ABTS), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 3,3 ',5,5'- tetramethyl benzidine (TNB) and 4-chloro-l-naphthol (4-CN), although without limitation thereto. A non-limiting example of a chemiluminescent substrate is Luminol™, which is oxidized in the presence of HRP and hydrogen peroxide to form an excited state product (3-aminophthalate). By way of example, the fluorophore may be, for example, fluorescein isothiocyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or R- Phycoerythrin (RPE) as are well known in the art.

An aspect of the invention provides a method of eliciting an immune response in an animal, said method including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby elicit an immune response to E. coli in the animal.

Another aspect of the invention provides a method of immunizing an animal including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby induce immunity to E. coli in the animal.

As used herein, "elicits an immune response" indicates the ability or potential of a protein, fragment or variant, antibody or encoding nucleic to elicit or generate an immune response to one or a plurality of strains of E. coli, or molecular components thereof, upon administration of to an animal. Suitably, the immune response may be either B -lymphocyte or T-lymphocyte mediated, or a combination thereof. Advantageously, the immune response includes a B-lymphocyte response, although is not limited thereto, preferably including an antibody response, such as an IgG response.

As used herein, "immunize" and "immunization" refer to administering a protein, immunogenic fragment, variant, derivative, antibody or encoding nucleic acid to elicit or potentiate a protective immune response to E. coli. By "protective immune response " is meant an immune response that is sufficient to prevent or at least reduce the severity or symptoms of an E. coli infection in an animal. It will be appreciated that this definition includes "passive immunization" typically elicited or conferred by the administration of an antibody or antibody fragment to an animal. As will be described hereinafter in the Examples, the presence of an IgG response by the protein of SEQ ID NO: l was an indicator that a protective immune response had been elicited by the protein. It is therefore proposed that administration of antibodies {e.g. IgG) that bind the protein of SEQ ID NO: l may confer passive immunity.

A third aspect of the invention provides a method of treating an E. coli- associated disease, disorder or condition in an animal, said method including the step of administering to the animal: an isolated protein comprising the amino acid sequence of SEQ ID NO:l; one or more immunogenic fragments variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; to thereby treat the disease, disorder or condition in the animal.

As used herein, "treating" (or "treat" or "treatment") refers to a therapeutic intervention that ameliorates a sign or symptom of an E. co/z-associated disease, disorder or condition after it has begun to develop. The term "ameliorating " with reference to an E. co/z-associated disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, "preventing" (or "prevent" or "prevention") refers to a course of action (such as administering a composition comprising a therapeutically effective amount of one or more immunogenic proteins and/or a fragment, variant or derivative thereof of the present invention) initiated prior to the onset of a symptom, aspect, or characteristic of an E. co/z-associated disease, disorder or condition, so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of an E.co/z-associated disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or clinical characteristic or outcome of the E. co/z-associated disease, disorder or condition.

In the context of the present invention, by "E.coli-associated disease, disorder or condition " is meant any clinical pathology, illness, trauma or adverse health consequence resulting from exposure to, or infection by, any strain, pathotype or serotype of E. coli. Non-limiting examples of E co/z-associated diseases, disorders or conditions include extra-intestinal infections such as urinary tract infection, sepsis and meningitis; and intestinal infections that lead to watery/bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome.

Pathogenic E. coli can be classified into two (2) major subgroups: extraintestinal pathogenic E. coli (ExPEC) and intestinal pathogenic E. coli (InPEC). ExPEC strains are responsible for diseases, disorders and conditions such as urinary tract infections (UTIs), sepsis and meningitis and are classified as uropathogenic E. coli (UPEC) or neonatal meningitis-associated E. coli (NMEC). There are many pathotypes within the InPEC group (e.g. entertoxigenic E. coli (ETEC) and entero aggregative E. coli (EAEC)) which cause diarrheagenic infections and infections of the intestinal tract.

It will therefore be appreciated that in some embodiments the immunization and/or treatment methods disclosed herein may be efficacious against a plurality of different E. coli strains, pathotypes or serotypes such as described above. In this context, the plurality of different E. coli strains, pathotypes or serotypes may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200, 300, 400, 450, 500, 1000, 5,000, 10,000 or more different E. coli strains, pathotypes or serotypes. A full listing of E. coli strains that express YncE may be determined with reference to Tables 2-5.

In another aspect, the invention provides a composition comprising: (a) an isolated protein comprising the amino acid sequence of SEQ ID NO: l; (b) the immunogenic fragment of the fifth aspect; (c) a variant or derivative of (a) or (b); (d) an antibody or antibody fragment that binds (a), (b) or (c); (e) an isolated nucleic acid encoding (a), (b) or (c); (f) the genetic construct of the seventh aspect; or (g) the host cell of the eighth aspect..

In some embodiments, the composition may be a "vaccine" for eliciting a protective immune response to E. coli.

Typically, the composition comprises a carrier, diluent or excipient.

By "carrier, diluent or excipient" is generally meant a solid or liquid filler, diluent, solvent, vehicle or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful general reference describing carriers, diluents and excipients is

Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.

In particular embodiments, the carrier diluent or excipient may include carriers, diluents and/or excipients that have immunological activity, or facilitate immunological activity. For example, these may include: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VPl and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a T cell epitope of a bacterial toxin, toxoid or CRM may be used. In this regard, reference may be made to U.S. Patent No 5,785,973 which is incorporated herein by reference.

The composition may further comprise an adjuvant as is well known in the art. As will be understood in the art, an "adjuvant" is, or comprises, one or more substances that enhance the immunogenicity and efficacy of a composition, such as a vaccine. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-denwed adjuvants such as Corynebacterium parvum; Propionibacterium-deriwed adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N', N'bis(2-hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil- A aluminium hydroxide; liposomes; ISCOM ^® and ISCOMATRIX ^® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); water in oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof and immuno stimulatory DNA such as CpG oligonucleotides and Toll receptor agonists.

Any suitable procedure is contemplated for producing ompositions, such as vaccine compositions. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

Any safe route of administration may be employed for providing an animal with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intranasal, intra- articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular and transdermal administration may be employed. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Compositions of suitable for administration may be presented as discrete units such as capsules, caplets, sachets, functional foods/feeds or tablets, or as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.

The composition may be administered in a manner compatible with the dosage formulation, and in such amount as is immunologically effective. The dose administered to an animal should be sufficient to effect a beneficial response in an animal over an appropriate period of time. The quantity of agent(s) to be administered may depend on the animal to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

It will also be appreciated that the methods of immunization, prevention and/or treatment of an E. co/z-associated disease, disorder or condition, and compositions therefor, may include administration of an isolated nucleic acid encoding an isolated protein having the amino acid sequence of SEQ ID NO: l, or a fragment, variant or derivative thereof.

The term "nucleic acid" as used herein designates single- or double- stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

An embodiment of an isolated nucleic acid encoding an isolated protein having the amino acid sequence of SEQ ID NO: l comprises the nucleotide sequence of SEQ ID NO:2.

atgcatttac gtcatctgtt ttcatcgcgc ctgcgtggtt cattactgtt aggttcattg

cttgttgctt catcattcag tacgcaggcc gcagaagaaa tgctgcgtaa agcggtaggt

aaaggtgcct acgaaatggc ttatagccag caagaaaacg cgctgtggct tgccacttcg

caaagccgca aactggataa aggcggcgtg gtttatcgtc ttgatccggt tactctggaa

gtgacgcagg cgatccataa cgatctcaag ccgtttggtg ccaccatcaa taacacgact

cagacgttgt ggtttggtaa caccgtaaat agtgcggtca cggcgataga tgccaaaacg

ggcgaagtga aaggtcgtct ggtgctggat gatcgtaagc gcacggaaga ggtacgcccg

ttgcaaccac gtgagctggt agctgatgat gccacgaaca ccgtttacat cagtggtatt

ggtaaagaga gcgtgatttg ggtcgttgat ggcgagaata tcaaactgaa aaccgccatc

cagaacaccg gtaaaatgag taccggtctg gcgctggata gcaaaggcaa acgtctttac

accactaacg ctgacggcga attgattacc atcgacaccg ccgacaataa aatcctcagc

cgtaaaaagc tgctggatga cggcaaagag cacttcttta tcaacatcag ccttgatacc

accaggcagc gtgcatttat caccgattct aaagcggcag aagtgttagt ggtcgatacc

cgtaatggca atattctggc gaaggttgcg gcaccggaat cactggctgt gctgtttaac

ccagcgcgta atgaagccta cgtgacgcat cgtcaggcag gtaaagtcag tgtgattgac

gcgaaaagct ataaagtggt gaaaacgttc gatacgccga ctcatccgaa cagcctggcg

ctgtctgccg atggcaaaac gctgtatgtc agtgtgaaac aaaaatccac taaacagcag

gaagctaccc agccggacga tgtgattcgt attgcgctgtaa

(SEQ ID N0:2)

It will also be understood that nucleic acids may be in the form of nucleic acid variants or fragments of SEQ ID NO:2. In some embodiments, nucleic acid variants and fragments of SEQ ID NO:2 respectively encode protein variants and fragments of SEQ ID NO: l. In other embodiments, nucleic acid variants have at least 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to SEQ ID NO:2. In yet other embodiments, nucleic acid variants hybridize under high stringency conditions with the nucleotide sequence of SEQ ID NO:2. Non-limiting examples of high stringency hybridization and wash conditions may be found in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), supra and Chapter 2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., supra.

In further embodiments, nucleic acid variants are allelic variants, orthologues or other polymorphic variants of the E. coli YncE nucleotide sequence of SEQ ID NO:2.

In some embodiments, nucleic acid fragments may comprise at least 20, 30, 40, 50, 100, 150, 100, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and up to 1000 contiguous nucleotides of SEQ ID NO:2.

For the purposes of delivery of a nucleic acid, it is preferable that the nucleic acid is in a genetic construct that comprises the isolated nucleic acid operably linked or connected to one or more other genetic components.

Broadly, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and/or expression of the nucleic acid or an encoded protein of the invention.

For the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the invention operably linked to one or more additional sequences in an expression vector. An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

By "operably linked" is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention.

The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein is expressed as a fusion protein, as hereinbefore described.

The expression construct may also include an additional nucleotide sequence encoding a selection marker such as amp R , neo R or kan R , although without limitation thereto. In particular embodiments relating to delivery of isolated nucleic acids (e.g. "DNA vaccination"), the expression construct may be in the form of plasmid DNA, suitably comprising a promoter operable in an animal cell (e.g. a CMV promoter). A review that discusses DNA plasmid vaccination is Williams, 2013, Vaccine 1 225. Reference is also made to DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

In other embodiments, the nucleic acid may be in the form of a viral construct such as an adenoviral, vaccinia, lentiviral or adeno-associated viral vector. Non-limiting examples of viral constructs for immunization are provided in Hu et al., 2011, Immunol. Rev. 239 45 and Nieto & Salvetti, 2014, Front. Immunol. 5 5.

In a further aspect, the invention provides a host cell transformed with a nucleic acid molecule or a genetic construct described herein.

Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells (e.g. HeLa, HEK293T, Jurkat cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g. Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.

It will also be understood that the isolated protein of SEQ ID NO: l, or one or more immunogenic fragments, variants and/or derivatives thereof, or the isolated nucleic acid may be administered in combination with one or more additional immunogens that may be specific to certain strains/pathotypes of E coli, or are conserved across a number of E coli strains/pathotypes. Non-limiting examples of other such immunogens include iron-receptor proteins, fimbrial proteins, flagella, capsular antigens and O antigens.

A further aspect of the invention provides a method of detecting an E. coli bacterium, or one or more molecular components thereof, in a sample, said method including the step of detecting: an isolated protein comprising the amino acid sequence of SEQ ID NO: l; one or more immunogenic fragments, variants and/or derivatives thereof; an isolated nucleic acid encoding said isolated protein, immunogenic fragment, variant and/or derivative; and/or an antibody or antibody fragment that binds said isolated protein, immunogenic fragment, variant and/or derivative; in said biological sample, which indicates the presence of E. coli, or one or more molecular components thereof, in the sample.

The sample may be obtained from an animal, such as a fluid or cellular sample inclusive of blood, serum, plasma, urine, feces, biopsy, swab or smear, although without limitation thereto. The presence of E. coli, or one or more molecular components thereof, in the sample may indicate that the animal is, or has been, infected with E. coli.

In other embodiments, the sample may be an environmental sample such as water or effluent or a food or beverage sample. The presence of E. coli, or one or more molecular components thereof, in the sample may indicate that the environmental, food or beverage sample, or has been, contaminated with E. coli.

The method may use protein- and/or nucleic acid-based detection methods as are well known in the art. Protein-based detection preferably includes using antibodies or antibody fragments that bind SEQ ID NO: l, one or more immunogenic fragments, variants and/or derivatives thereof. Alternatively, protein-based detection may identify antibodies (e.g serum IgG) to SEQ ID NO: l, one or more immunogenic fragments, variants and/or derivatives thereof in a sample obtained from an animal, which indicates that the animal is, or has been, infected with E. coli. Non-limiting examples of antibody-based detection includes ELISA, radioimmunoassay, immunoblotting, and immunoprecipitation, although without limitation thereto.

By way of example, nucleic acid detection may be performed using one or more of nucleic acid sequence amplification (e.g polymerase chain reaction, rolling circle amplification, strand displacement amplification, helicase-dependent amplification), nucleic acid hybridization and/or nucleotide sequencing as are well known in the art. Suitable primers and probes may readily be designed according to the nucleotide sequence set forth in SEQ ID NO:2.

It will also be appreciated that detection may be performed using a composition comprising the isolated protein or fragment thereof, nucleic acids (e.g. primers and/or probes), antibodies or antibody fragments that bind the isolated protein in combination with one or more molecules or reagents that facilitate detection. These one or more molecules or reagents may include enzymes, enzyme substrates, DNA polymerases (e.g for PCR detection), secondary antibodies (which may be labeled such as hereinbefore described), buffers and blocking agents, although without limitation thereto. In some embodiments, the invention may provide a detection kit comprising, the isolated protein, nucleic acids (e.g primers and/or probes), antibodies, antibody fragments, secondary antibodies., the one or more molecules or reagents that facilitate detection and/or compositions comprising same, although without limitation thereto.

As generally used herein, animals may include any species of the animal kingdom susceptible to E. coli infections inclusive of humans and non-human mammals and avians. Non-human mammals may include domestic pets (e.g. cats, dogs), performance animals (e.g racehorses, camels) and livestock (e.g cattle, pigs, sheep), although without limitation thereto. Avians may include commercially important poultry such as chickens, turkeys and ducks.

So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting examples.

EXAMPLE 1

MATERIALS AND METHODS

The E. coli dataset and MLST analysis. The E. coli database was represented by 62 complete and 1638 draft genomes available on the NCBI public database as of the 1 ^st January, 2014 (Table 3 provides a listing of 62 complete genome strains). Bioinformatic analysis. Sequence comparisons were performed using FASTA36 package (38). Subcellular localization was predicted by PSORTb 3.0 (39) and signal sequence was predicted by LipoP 1.0 (40). Structural analysis was performed by PHYRE2 (41). Prevalence of genes was determined by amino acid sequence identity, using a cut-off of >75% over a 75% alignment. Phylogenetic trees were drawn using MEGA6 (42). MLST analysis was conducted following the scheme previously described (8). Circular representations were drawn using Circos (43).

Bacteria and growth conditions. E. coli strains were routinely grown at 37 C on solid or in liquid Luria-Bertani (LB) medium supplemented with appropriate antibiotics; chloramphenicol (30 μg/ml), kanamycin (50 μg/ml) and ampicillin (100 μg/ml). For the generation of total cell lysates and supernatant fractions, bacteria were inoculated in liquid LB to a starting optical density at 600 nm (OD ₆ oo) equal to 0.050 and cultures were grown overnight at 37 C with shaking (180 rpm). Cells were harvested at 10,000 g for 10 minutes at 4 C to generate the whole cell lysate sample. Supernatant fractions were generated as previously described (44). To obtain EDTA-heat induced OMVs, E. coli EC958 was grown in minimal M9 medium supplemented with casamino acids at 37°C under shaking conditions (180 rpm) and harvested at a final OD ₆ oo=0.5. Cells were centrifuged at 10,000 g for 10 minutes at 4°C and the pellet was used for OMV heat- induction.

Molecular methods, proteomic analyses and immunoblotting. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). PCR was performed using Phusion High-Fidelity DNA Polymerase (New England Labs) and primers described in FIG. 5. PCR products were purified from agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and cloned into pMCSG7 using the ligation- independent cloning (LIC) method (45). DNA was transformed into chemically competent E. coli TOP 10 (Invitrogen) for plasmid propagation. Primers used for cloning are described in FIG. 5. Isolation of plasmid DNA was carried out using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced using Big Dye Terminator v3.1 Cycle Sequencing Kit (Invitrogen). Correct constructs were used to transform E. coli BL21 (DE3) pLysS (Invitrogen) for protein expression. A CFT073 yncE mutant was constructed by λ-Red mediated homologous recombination using primers 5600 and 5601 (Figure 5) as previously described (46). The formation of OMVs was induced by incubation in 0.01M EDTA at 56°C and samples were analyzed by HPLC-MS/MS as previously described (37, 44). The peptide fingerprint was evaluated using ProteinPilot software 4.0 in combination with the EC958 protein database. SDS-PAGE and immunoblotting were performed as previously described (47) using a YncE-specific antibody. Rabbit YncE antibodies were raised against purified YncE by the Walter and Eliza Hall Institute Antibody Facility as previously described (48).

Expression and purification of vaccine targets. Polyhistidine-tagged recombinant proteins were obtained by auto -induction (49) and purified by affinity and gel filtration chromatography. Briefly, BL21 (DE3) pLysS harboring the LIC vectors were grown in ZYP-5052 (1 L) overnight at 28°C, 250 rpm. Cells were harvested by centrifugation at 18,600 x g, resuspended in 25 mM Tris-HCl pH 7, 150 mM NaCl, 0.5% Triton X-100, Protease Inhibitor Cocktail (Sigma) and DNasel (Roche) and lysed by sonication (Misonix XL- 2000, QSonica). Cell debris was removed by centrifugation and histidine- tagged proteins were purified using Talon Metal Affinity resin (Clontech) pre- equilibrated with buffer A (25 mM HEPES pH 7, 150 mM NaCl). Proteins bound to the resin were washed extensively with buffer B (25 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole) and eluted with buffer C (25 mM Tris-HCl pH 7, 150 mM NaCl, 250 mM imidazole). Fractions containing the eluted protein were pooled and dialyzed overnight in buffer A and further purified by gel filtration chromatography (AKTA, GE Healthcare) using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) pre- equilibrated in buffer A.

Plasma collections and immunoassays. Blood plasma was collected from 47 urosepsis patients admitted to the Princess Alexandra Hospital (Brisbane, Australia) and 42 healthy volunteers with no recent history of UTI. Recombinant proteins (10 μg/ml) were coated onto Nunc MaxiSorp flat-bottom 96 well plates (Thermo Scientific) in carbonate coating buffer (18 mM Na ₂ C0 ₃ , 450 mM NaHC0 ₃ , pH 9.3) at 4°C overnight. Plates were then washed twice with 0.05% Tween20 PBS (PBST) and blocked on 5% skim milk in PBST (150 μΐ) for 90 min at 37°C. Plates were washed four times with PBST and then plasma samples were added to the wells at 1: 10 dilution. Plates were incubated for 90 min at 37°C and washed four times with PBST. Peroxidase-conjugated anti- human IgG (1:30,000 dilution in 0.5% skim milk) was applied as secondary antibody and incubated for 90 min at 37°C. Plates were washed four times with PBST before development with 3,3',5,5'-tetramethylbenzidine. Reactions were stopped with 1 M HC1. Intensity was determined using SpectraMax 190 Absorbance Microplate Reader at 450 nm. Statistical analysis between patient and healthy plasma was performed using an unpaired two-sample i-test. A statistical significance threshold was set at P<0.05. A rabbit polyclonal antiserum was raised against purified YncE using four immunizations (400 μg recombinant protein/dose) at the Walter and Eliza Hall Institute Antibody Facility. For immunoblotting, samples were subjected to SDS-PAGE using 12% Bis-Tris gels and subsequently transferred to polyvinylidene difluoride (PVDF) microporous membrane. YncE antiserum was used as primary serum and the secondary antibody was alkaline phosphatase-conjugated anti-rabbit IgG. Sigma Fast ^® BCIP/NBT was used as the substrate in the detection process.

Bacteremia model of infection. A murine model of E. coli bacteremia, as previously described (26), was used to assess the protective efficacy of YncE as a vaccine immunogen against systemic infection. Groups of twelve C57BL/6 mice (8-12 weeks old) were immunized subcutaneously (s.c.) with 100 μg of YncE in 100 μΐ of an emulsification of PBS and Complete Freund's Adjuvant (Sigma) (2: 1) on Day 0. Booster doses of 25 μg of antigen in 100 μΐ of an emulsification of PBS and Incomplete Freund's Adjuvant (Sigma) (1 : 1) were administered s.c. on Days 7 and 14, essentially as previously described (50). Mice were challenged intravenously (i.v.) with approximately 6.4 x 10 ⁶ CFU of E. coli CFT073 in 200 μΐ of PBS via the lateral tail vein on Day 21. The burden of disease was assessed at 24 h post-challenge by quantitating the bacterial loads in liver, blood, kidney, and spleen. The experiment included mock-immunized controls (received PBS and adjuvant only) and was repeated independently.

Blood samples were also collected on Days 0, 7, 14, 21, and 22 to measure YncE- specific IgG antibody titers by enzyme-linked immunosorbent assay (ELISA). Sera from immunized mice were separated from blood at 1,500 g for 10 min. Plates were coated, blocked and washed as detailed above. Mice serum samples were applied at a 1:2 serial dilution, starting from a 1:10 dilution. Subsequent wash, secondary antibody and developing steps were performed as previously described (47), with the exception that peroxidase-conjugated anti-mouse antibodies were applied as the secondary antibody. YncE IgG titres were defined as the logarithmic dilution that produced a significant absorbance (450 nm) in comparison to a blank sample.

RESULTS EcoDS: a comprehensive dataset of E. coli genome sequenced strains. An E. coli dataset (EcoDS) was generated from 1700 genome sequences available on the NCBI public database (Strains listed in Table 2). EcoDS contained 62 complete (Table 3) and 1638 draft genome sequences, and represents the most diverse composition of E. coli strains examined to-date (Tables 1, 2 and Fig. 3). Information regarding the source, disease, origin or year of isolation was available for approximately 69% (n=1174) of strains. Analysis of this strain subset revealed the most common sources as human (n=747) or cattle (n=76) and the most common disease associations as bacteremia (n=222) or diarrhea (n=114). The majority of strains originated from North America (n=327) or Asia (n=265) (Table 1). Overall, EcoDS is represented by commensal E. coli strains as well as strains from all known E. coli pathotypes. This includes the intestinal pathotypes EAEC, EHEC, enteroinvasive E. coli (EIEC), adherent-invasive E. coli (AIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and the recently emerged enteroaggregative and hemorrhagic E. coli (EAHEC) pathotype, as well as extraintestinal pathotypes comprising avian -pathogenic E. coli (APEC), neonatal meningitis E. coli (NMEC) and uropathogenic E. coli (UPEC).

Phylogenetic and pathotype relationship of E. coli strains in EcoDS. In order to determine the relationship of the E. coli strains in EcoDS, a phylogenetic tree was constructed based on 435 unique multi-locus sequence types (MLST) (8) identified in EcoDS. To determine the phylogenetic group of the E. coli strains in EcoDS, we used an in silico triplex analysis of the chuA, yjaA and TSPE4.C2 loci, which allowed the classification of strains into the major phylogroups A, B l, B2 and D (11, 12). This strategy revealed a strong correlation between E. coli ST's and the established multi- locus enzyme electrophoresis (MLEE)-based E. coli phylogeny, and described a comprehensive distribution of ST's within the four major phylogroups in EcoDS.

To establish a correlation between the E. coli phylogeny and pathotypes, the major phylogroups were analyzed in greater detail by a comparison of multiple factors, including year of isolation, origin, source and disease association (Table 1). No clear geographic, host, or disease- specific component correlated with this clonal phylogenetic framework, highlighting a limitation for analysis of large collections with incomplete clinical sampling information (13). EcoDS contained a highly diverse combination of ST's and pathotypes. The composition of phylogroup A includes K-12 (MG1655, W3110, MC4100, MDS42, c321.deltaA, BW2952 and DH10B) and B (BL21 [DE3] and BL21-Gold[DE3]pLysS AG) strains as well as most of the complete ETEC genome sequences and the commensal strain HS. The K-12 and UMNF18 strains belong to ST10, which is also represented by strains from UPEC (ATCC 23506), EAEC (C43/90) and APEC (S 17) pathotypes. Phylogroup AxB l is a hybrid group that comprehends strains from phylogroups A and B l. It contains the ST678 EAHEC strains involved in the 2011 German outbreak (1, 14-16) and strains from progenitor EHEC and EAEC ST's that contributed to the emergence of this highly virulent lineage (e.g. the EAEC strain 55989 and the EHEC strains 2009EL-2050 and 2009EL-2071). Interestingly, phylogroup AxB l is also represented by ST675, recently described as EHEC strains involved in UTI cases (17). Moreover, this group also contains non-pathogenic strains and strains from APEC and ETEC pathotypes. Phylogroup B2 contains a large number of strains from ST73 (n=116) and the recently emerged and globally disseminated MDR ST 131 clonal lineage (n=38) (18, 19). This phylogroup predominantly contains strains associated with extraintestinal human infections, although strains from AIEC and EPEC pathotypes are also present. Phylogroup D is represented by the clonal lineages including subgroup E, the previously described subgroup F (6), and the recently identified cryptic lineages referred to as C-I to C-V (20, 21). Phylogroup E is the least diverse and is a clonal lineage predominantly represented by EHEC and EPEC strains belonging to ST11 (235/272 strains) and from 0157 and 055 serogroups. Phylogroup D also comprehends the subgroup F, which is represented by the environmental isolate SMS-3-5, the NMEC strain CEIO and the UPEC strain IAI39. The cryptic lineages C-I to C-V include EHEC, ETEC, commensal and the environmental isolates previously described (20, 21). Moreover, phylogroup D is also represented by strains from ST69 (n=80), which includes the UPEC reference strain UMN026, and the EAEC strain 042. Together, our analysis shows that strains from the same phylogroup, and even from the same ST, can be involved in different clinical manifestations and belong to different pathotypes.

Determination of the E. coli core and accessory genome. The majority of genomes within EcoDS comprised draft genome sequences, and we predicted that they would vary significantly in quality and coverage. Thus, we devised a strategy based on the prevalence of E. coli essential genes to remove genome sequences with poor or low coverage. First, we used two recent studies (22, 23) to define a set of 362 E. coli essential genes (). We then screened for the prevalence of these essential genes in the 62 complete genomes (Table 3), which should represent the best quality genome sequences in EcoDS. In total, 318 genes were present in all 62 completely sequenced strains. These 318 genes were therefore used to filter the 1700 genomes in EcoDS, and our analysis revealed a prevalence of 99.64+3.0% essential genes per strain (which allows a mean tolerance of one missing essential gene per strain). Therefore, strains missing more than one essential gene were discarded (n=144, Table 4), leaving a total of 1556 strains in EcoDS (i.e. Table 2 strains not in Table 4). The least prevalent essential gene in EcoDS was b3119 (tdcR), which was present in 99% (n=1541) of strains ().

We used the 1556 genome sequences in EcoDS to define the conserved set of E. coli genes. The genome of the well-characterised K-12 strain MG1655 was used as a reference (24) and, based on the essential gene data, a cut-off value for gene prevalence was set at 99%. Pairwise comparison of the 4319 ORFs defined in MG1655 with the genome sequence of the 1556 strains in EcoDS led to the identification of 3042 genes present in more than 99% of strains (protein identity >75% over a 75% sequence overlap), of which 1037 genes were present in 100% of the strains. These 3042 genes define a conserved subset of E. coli genes in the 1556 strains that make up EcoDS .

In order to define the E. coli accessory genome, 298,563 annotated ORFs (which comprised 290,776 chromosomal ORFs and 7787 plasmid ORFs) from the 62 completely sequenced strains were compared to each other. This pairwise comparison identified 12,722 unique ORFs based on >75% protein identity that displayed a prevalence of <99% in EcoDS (10,513 chromosomal, 2209 plasmid), and reflects the enormous genetic diversity that exists in the pan E. coli genome. We refer to this gene set as the accessory E. coli genome. Identification of novel vaccine targets. The definition of a conserved set of 3042 E. coli genes provides a framework for the development of new tools in epidemiology, diagnostics and vaccine antigen discovery. In order to evaluate the expression of core components as potential antigens that could be included in a broadly protective vaccine against E. coli, we examined the surface-associated outer membrane proteome of the MDR ST131 strain EC958. Proteomic analysis of outer membrane vesicles (OMVs) induced from EC958 led to the identification of 115 proteins. Among them, 23 were predicted to have an extracellular (n=l), outer membrane (n=15) or unknown (n=7) subcellular localization. To refine this list, we focused our attention on proteins encoded by genes that were prevalent in more than 99% of EcoDS. This led to a panel of 17 potential surface-exposed antigens, which was even further reduced by removing outer membrane proteins predicted to have a transmembrane β-barrel structure based on analysis by PHYRE2 (i.e. OmpA, OmpC, OmpF, OmpX, MipA, Fiu, FepA, Tsx and CirA) and proteins with an unknown subcellular localization and no predicted signal sequence based on LipoP (WrbA, SodA, TrmL and LysC). This left a final list of four potential surface-associated proteins, namely BamC, OsmE, SlyB and YncE.

YncE is a highly immunogenic and protective antigen. To examine these four proteins further, each respective gene was PCR amplified and cloned in frame with an N-terminal 6xHis-tag sequence. Expression studies using these constructs revealed only the BamC and YncE proteins were produced as soluble recombinant proteins (Fig. 4A). Therefore, these two proteins were purified and tested for immunogenicity using plasma obtained from convalescent urosepsis patients and plasma from an age- and sex- matched healthy control group. As a positive control and correlate of immunogenicity, we also included SslE (ecokl_3385) and EsiB (c5321) in our analysis, both of which have previously been shown to be protective against extraintestinal pathogenic E. coli (ExPEC) infection in a mouse sepsis model (25). Both SslE (P<0.001) and EsiB (P=0.022) showed higher reactivity to the plasma from urosepsis patients compared to healthy individuals (Fig. 1A). Among the targets identified in this study, BamC showed no significant reactivity with plasma from urosepsis patients. However, in contrast, YncE was strongly reactive with plasma from urosepsis patients compared to healthy individuals (P<0.001), suggesting it is expressed during human infection.

We investigated the ability of YncE to elicit a protective immune response against acute systemic E. coli infection using an established murine model of bacteremia (26). YncE was highly immunogenic inducing a strong IgG response (Fig. 4B) and mice immunized with YncE were significantly protected against infection as evidenced by lower blood and liver E. coli loads following systemic challenge (Fig. IB). Taken together, these data identify YncE as a novel, highly conserved and strongly immunogenic E. coli antigen that is able to provide protection against acute systemic infection when administered by vaccination.

YncE is broadly expressed and secreted by different E. coli pathotypes. The yncE gene was highly conserved among strains in EcoDS (n=1550/1556, 99.6%). Strains lacking yncE gene were not related by ST or phylogroup: DEC13A, DEC13B, DEC13D, IAI39, Nl, UMEA 3687-1. To verify the expression of YncE by strains belonging to different pathotypes and phylogroups, western blot analysis was performed using rabbit polyclonal antibodies generated against recombinant YncE (Fig. 2A). It was observed that YncE is broadly expressed by E. coli strains from different pathotypes. Moreover, western blot analysis of strains representing the E. coli phylogroups determined in this study revealed the presence of YncE in the supernatant after overnight growth (Fig. 2A). YncE was also overexpressed in an E. coli fur mutant during in vitro growth, demonstrating a role for iron in its regulatory control (Fig. 2B).

DISCUSSION

Current vaccination strategies against E. coli have focused on individual pathotypes and targeted major virulence determinants. Examples include colonization factor antigens and heat-labile toxin from ETEC (27), components and effectors of the type III secretion system from EPEC (28), Shiga toxin from EHEC (29) and fimbrial adhesins and siderophore receptors from UPEC (30). In this study we used an essential gene strategy to generate a curated dataset of E. coli genomes (EcoDS), and used this comprehensive and diverse collection (representing 435 STs) to define core and accessory elements of the pan E. coli genome. This information, together with proteomic data, led to the identification and validation of YncE as a highly conserved and protective E. coli vaccine antigen.

We observed that the clustering of different strains according to the seven housekeeping MLST genes was consistent with a modern framework composed of phylogroups A, AxB l, B2, and D. Moreover, we observed that single subpopulations comprehended strains involved in multiple diseases, supporting previous studies that demonstrated strains from the same pathotype can be distributed over the entire span of phylogenetic diversity and are not restricted to one specific phylogroup (5-7). Therefore, despite the high diversity, complexity and low prevalence of the accessory elements in EcoDS, E. coli strains possess a defined set of highly prevalent genes irrespective of pathotype, phylogroup and associated clinical disease.

Comparative analysis of the genomes in EcoDS showed that genes comprising the E. coli accessory genome were highly variable in prevalence. The accessory genome is considered a flexible gene pool shaped by mobile genetic elements and represents a major driver of E. coli evolution (31). In this study, we observed that the E. coli accessory genome comprises a broad set of 12,722 ORFs (prevalence <99% in EcoDS) and that approximately 90% of the entire accessory genome is present in less than 90% of strains in EcoDS, which poses an enormous challenge for broad therapeutic interventions. Potential E. coli universal vaccine targets such as FimH and SslE, for example, were present in approximately 89% and 70% of the strains in EcoDS, respectively.

A smaller subset of 3042 genes present in more than 99% of strains in EcoDS was also defined. These genes were used to trace novel vaccinology strategies. In order to identify potential surface-exposed and secreted proteins from the E. coli core genome that could represent new vaccine targets, we investigated the OMV-associated proteome of the MDR ST131 strain EC958. Among the 115 proteins identified, we selected four proteins for cloning and expression based on literature analysis, predicted subcellular localization and structural conformation. Among them, YncE showed increased reactivity to a plasma collection of urosepsis convalescent patients, higher than the titers obtained for SslE, a type II secreted-mucinase originally identified from ExPEC (25) that provides broad protection against E. coli infection in different animal models (32). BamC, a surface-associated lipoprotein (33), showed no significant reactivity with the urosepsis plasma collection. Moreover, YncE was also shown to be highly immunogenic and decreased the level of infection in a murine model of bacteremia, confirming the immunogenicity of this antigen and its potential as a broad vaccine candidate against E. coli.

YncE is a seven-bladed beta-propeller (34) transported by the Sec machinery (35) and associated with binding to single stranded DNA (36). We have previously shown that YncE is present in the OMV proteome of a large collection of urosepsis strains (37) and, in this study, we demonstrated that YncE is present in the OMV proteome of EC958 (phylogenetic group B2) and in the secretome of strains representing all other phylogenetic groups. Moreover, we confirmed the regulation of YncE by Fur, indicating its potential role during infection in iron-limiting environments such as blood and the urinary tract. Taken together, our results indicate that YncE fulfills many prerequisites required for a vaccine candidate; YncE is (i) immunogenic, (ii) highly prevalent, (iii) highly conserved, (iv) soluble, (v) stable, and (vi) expressed during infection.

In conclusion, we have demonstrated the genome complexity and plasticity of E. coli and dissected the difficulty associated with targeting the accessory genome for broadly therapeutic interventions. Moreover, we confirmed the close association between different pathogenic and non-pathogenic E. coli lineages. We also designed a strategy based on the E. coli core genome for the identification of novel potential vaccine targets, which led to the discovery of YncE as an immunogenic and protective antigen. Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Table 1, Distribution of strains in EcoDS according to source, disease, year and place of isolation

Source % Disease % Year % Place

Bat 0.1 Asymptomatic 0.8 < 1940s 0.1 Africa (

Buffalo 0.1 Bacteremia 13.1 1950s 0.5 Asia 1

Cow 4.5 Bacteriuria 0.1 1960s 0.6 Europe -

Dog 0.2 Crohn's disease 0.2 1970s 0.7 North America 1

Environment 0.9 Diarrhea 6.7 1980s 2.2 South America

Fish 0.1 HUS 1.1 1990s 5.5 Oceania (

Food 1.0 Mastitis 0.4 2000s 12.2 Unknown 5

Goat 0.1 Meningitis 0.2 2010s 9.6

Horse 0.1 Omphalitis 0.1 Unknown 68.6

Human 43.9 Peritonitis 0.1

Marsupial 0.9 RTI 0.1

Mouse 0.1 Septicemia 0.2

Pig 1.1 UTI 2.1

Poultry 0.8 Unknown 74.8

Rabbit 0.2

Reptile 0.1

Sheep 0.1

Wild bird 0.6

Unknown 45.1

RTI, respiratory tract infections; UTI, urinary tract infections; HUS, hemolytic -uremic syndrome Table 2. 1700 E. coli strains used in this study

BL21-Gold; 042; 536; 55989; ABU 83972; APEC Ol; APEC 078; ATCC 8739; REL606; BL21; BW2952; c321.deltaA; CFT073; DH1; DH1 (ME8569); E24377A; ED la; H10407; HS; IAI1; IAI39; IHE3034; JJ1886; KOllFL; LF82; LY180; NA114; 12009; 2009EL-2050; 2009EL-2071; 2011C-3493; 11128; E2348/69; EC4115; EDL933; Sakai; TW14359; EC958; 11368; CB9615; RM12579; CE10; NRG857c; P12b; PMV-1; S88; SE11; SE15; SMS-3-5; clone D il4; clone D i2; DH10B; MC4100; MDS42; MG1655; W3110; UM146; UMN026; UMNF18; UMNK88; UTI89; Xuzhou21; 0.1288; 0.1304; 3.4870; 3.4880; 5.2239; 6.0172; 7.1982; 8.0416; 8.0566; 8.0569; 8.0586; 8.2524; 10.0821; 10.0833; 10.0869; 38.16; 38.27; 38.34; 38.52; 75; 88.0221; 88.1042; 88.1467; 89.0511; 90.0039; 90.0091; 90.2281; 93.0055; 93.0056; 94.0618; 95.0083; 95.0183; 95.0943; 95.1288; 96.0107; 96.0109; 96.0427; 96.0428; 96.0932; 96.0939; 97.0003; 97.0007; 97.0010; 97.1742; 99.067; 99.0672; 99.0678; 99.0713; 99.0814; 99.0815; 99.0816; 99.0839; 99.0848; 99.1753; 99.1762; 99.1775; 99.1781; 99.1793; 99.1805; 148; 320; 328; 435; 597; 606; 668; 681; 897; 1044; 1047; 1125; 1180; 1240; 1350; 1357; 1365; 3003; 3006; 3431; 5412; 5905; 07798; 8624; 1.2264; 1.2741; 2.3916; 2.4168; 3.2303; 3.2608; 3.3884; 4.0522; 4.0967; 5.0588; 5.0959; 53638; 83972; 9.0111; 9.1649; 96.154; 110957; 113290; 113302; 113303; 174750; 174900; 178200; 178850; 178900; 179100; 179550; 180050; 180200; 180600; 199900.1; 201600.1; 11-3677; 900105; 907357; 907391; 907446; 907672; 907700; 907701; 907710; 907713; 907715; 907779; 907889; 907892; 908519; 908521; 908522; 908524; 908525; 908541; 908555; 908573; 908585; 908616; 908624; 908632; 908658; 908675; 908691; 909957; 11-4404; 93.0624; 95.0941; 11- 4522; 96.0497; 97.0246; 97.0259; 97.0264; 99.0741; 11-4632; 09-7901; 04-8351; 2719100; 2720900; 2722950; 2726800; 2726950; 2729250; 2730350; 2730450; 2731150; 2733950; 2735000; 2741950; 2747800; 2749250; 2756500; 2762100; 2770900; 2780750; 2785200; 2788150; 2845350; 2845650; 2846750; 2848050; 2850400; 2850750; 2851500; 2853500; 2854350; 2860050; 2860650; 2861200; 2862600; 2864350; 2865200; 2866350; 2866450; 2866550; 2866750; 2867750; 2871950; 2872000; 2872800; 2875000; 2875150; 112469215; 112469218; 01-09591; 03-EN-705; 08BKT055439; 08BKT77219; 09BKT024447; 09BKT076207; 09BKT078844; 101-1; 11-02030; 11-02033-1; 11-02092; 11-02093; 11-02281; 11-02318; 11-02913; 11-03439; 11-03943; 11-04080; 11-4632 CI; 11-4632 C2; 11- 4632 C3; 11-4632 C4; 11-4632 C5; 14A; 1827-70; 1A; 2362-75; 2534-86; 27A; 2886-75; 3030-1; 3256- 97; 4_1_47FAA; 4865/96; 493/89; 53A; 53C; 541-1; 541-15; 576-1; 85B; 87A; 909945-2; 910096-2; 93- 001; 95JB1; 95NR1; 9B; A25922R; A35218R; AA86; AB42410445; AB42554418; AB42602061; AB43739056; AD30; AI27; ARS4.2123; ATCC 23502; ATCC 23506; ATCC 25922; ATCC 35150; ATCC 700728; B088; B093; B102; B103; B104; B105; B106; B107; B108; B109; B112; B113; B114; B15; B17; B171; B175; B185; B26-1; B26-2; B28-1; B28-2; B29-1; B29-2; B2F1; B354; B36-1; B36-2; B367; B40; B40-1; B40-2; B41; B49-2; B5-2; B574; B671; B7-1; B7-2; B706; B799; B7A; B83; B84; B85; B86; B89; B90; B921; B93; B94; B95; BAA-2192; BAA-2193; BAA-2196; BAA-2209; BAA- 2215; BAA-2219; BCE001_MS16; BCE002_MS12; BCE006_MS-23; BCE007_MS-11; BCE008_MS- 01; BCE008_MS-13; BCE011_MS-01; BCE019_MS-13; BCE030_MS-09; BCE032_MS-12; BCE034_MS-14; Bd5610_99; BIDMC 19C; BIDMC 37; BIDMC 38; BIDMC 39; BWH 24; BWH 32; C-34666; C12_92; C1214_90; CI 244 _. 91; C154_ll; C155_ll; C157_ll; C161_ll; C166_ll; C170_ll; C213_10; C2139_99; C227-11; C236-11; C238_91; C260_92; C262_10; C283_09; C295_10; C341_10; C343_08; C347_93; C353_09; C354_03B; C40_ll; C418_89; C43/90; C458_10; C48/93; C488_07; C496_10; C497_10; C527_94; C58_ll; C581_05; C586_05; C639_08; C652_10; C654_09; C717_10; C725_88; C732_98; C743_03; C751_03; C78_09C; C79_08; C792_92; C796_10; C799_92; C80_08; C807_09; C82_ll; C824_10; C842_97; C844-97; C87_ll; C887_10; C9_92; C900_01; C93_ll; CB7326; CFSAN001629; CFSAN001630; CFSAN001632; CFSAN002236; CFSAN002237; chi7122; CL-3; clone A il; CUMT8; CVDNalr; CVM10021; CVM10026; CVM10030; CVM10224; CVM9340; CVM9450; CVM9455; CVM9534; CVM9545; CVM9553; CVM9570; CVM9574; CVM9602; CVM9634; CVM9942; CVM9952; DECIOA; DECIOB; DEC10C; DEC10D; DECIOE; DEC10F; DEC11A; DEC11B; DEC11C; DEC11D; DEC11E; DEC12A; DEC12B; DEC12C; DEC 12D; DEC12E; DEC13A; DEC13B; DEC13C; DEC13D; DEC13E; DEC 14 A; DEC14B; DEC14C; DEC14D; DEC 15 A; DEC15B; DEC15C; DEC15D; DEC15E; DEC1A; DEC1B; DEC1C; DEC1D; DEC IE; DEC2A; DEC2B; DEC2C; DEC2D; DEC2E; DEC3A; DEC3B; DEC3C; DEC3D; DEC3E; DEC3F; DEC4A; DEC4B; DEC4C; DEC4D; DEC4E; DEC4F; DEC5A; DEC5B; DEC5C; DEC5D; DEC5E; DEC6A; DEC6B; DEC6C; DEC6D; DEC6E; DEC7A; DEC7B; DEC7C; DEC7D; DEC7E; DEC8A; DEC8B; DEC8C; DEC8D; DEC8E; DEC9A; DEC9B; DEC9C; DEC9D; DEC9E; DORA_A_5_14_21; DORA_B_14; E1002; E101; E110019; E1114; E1118; E112/10; E1167; E128010; E1492; E1520; E1777; E22; E2265; E267; E482; E560; E704; E92/11; Ecll-4984; Ecll-4986; Ecll-4987; Ecll-4988; Ecll-5536; Ecll- 5537; Eel 1-5538; Eel 1-5603; Eel 1-5604; Eel 1-6006; Eel 1-9450; Eel 1-9941; Eel 1-9990; Ecl2-0465; Ecl2-0466; EC1212; EC1734; EC1735; EC1736; EC1737; EC1738; EC1845; EC1846; EC1847; EC1848; EC1849; EC1850; EC1856; EC1862; EC1863; EC1864; EC1865; EC1866; EC1868; EC1869; EC1870; EC302/04; EC4009; EC4013; EC4024; EC4042; EC4045; EC4076; EC4084; EC4100B; EC4113; EC4127; EC4191 ; EC4192; EC4196; EC4203; EC4205; EC4206; EC4401 ; EC4402; EC4421 ; EC4422; EC4436; EC4437; EC4439; EC4448; EC4486; EC4501 ; EC508; EC536; EC869; EC96038; ECA-0157; ECA-727; ECC-1470; ECC-Z; Envira 10/1 ; Envira 8/11 ; EPEC C342-62; EPECal2; EPECal4; Fl l ; FAH1 ; FAH2; FAP1 ; FAP2; FBH1 ; FBP1 ; FCH1 ; FCP1 ; FDA504; FDA505; FDA506; FDA507; FDA517; FRIK1985; FRIK1990; FRIK1996; FRIK1997; FRIK1999; FRIK2000; FRIK2001 ; FRIK523; FRIK920; FRIK966; FVEC 1465; FVEC1302; FVEC1412; G5101 ; G58-1; GOS 1 ; GOS2; GSK2022; GSK2023; GSK2024; GSK202B; GSK202U; H001; H093800014; HI 12180280; HI 12180282; HI 12180283; HI 12180540; HI 12180541 ; H120; H185; H218; H220; H223; H252; H263; H2687; H288; H296; H299; H30; H305; H378; H383; H386; H397; H413; H420; H442; H454; H461 ; H489; H494; H504; H588; H591 ; H593; H605; H617; H660; H730; H736; HM26; HM27; HM46; HM605; HM65; HM69; HVH 1 ; HVH 10; HVH 100; HVH 102; HVH 103; HVH 104; HVH 106; HVH 107; HVH 108; HVH 109; HVH 110; HVH 111 ; HVH 112; HVH 113; HVH 114; HVH 115 (4- 4465989); HVH 115 (4-4465997); HVH 116; HVH 117; HVH 118; HVH 119; HVH 12; HVH 120; HVH 121 ; HVH 122; HVH 125; HVH 126; HVH 127; HVH 128; HVH 13; HVH 130; HVH 132; HVH 133; HVH 134; HVH 135; HVH 136; HVH 137; HVH 138; HVH 139; HVH 140; HVH 141 ; HVH 142; HVH 143; HVH 144; HVH 145; HVH 146; HVH 147; HVH 148; HVH 149; HVH 150; HVH 151; HVH 152; HVH 153; HVH 154; HVH 155; HVH 156; HVH 157; HVH 158; HVH 159; HVH 16; HVH 160; HVH 161 ; HVH 162; HVH 163; HVH 164; HVH 167; HVH 169; HVH 17; HVH 170; HVH 171 ; HVH 172; HVH 173; HVH 175; HVH 176; HVH 177; HVH 178; HVH 18; HVH 180; HVH 182; HVH 183; HVH 184; HVH 185; HVH 186; HVH 187; HVH 188; HVH 189; HVH 19; HVH 190; HVH 191 ; HVH 192; HVH 193; HVH 194; HVH 195; HVH 196; HVH 197; HVH 198; HVH 199; HVH 2; HVH 20; HVH 200; HVH 201 ; HVH 202; HVH 203; HVH 204; HVH 205; HVH 206; HVH 207; HVH 208; HVH 209; HVH 21 ; HVH 210; HVH 211 ; HVH 212; HVH 213; HVH 214; HVH 215; HVH 216; HVH 217; HVH 218; HVH 22; HVH 220; HVH 221 ; HVH 222; HVH 223; HVH 225; HVH 227; HVH 228; HVH 23; HVH 24; HVH 25; HVH 26; HVH 27; HVH 28; HVH 29; HVH 3; HVH 30; HVH 31 ; HVH 32; HVH 33; HVH 35; HVH 36; HVH 37; HVH 38; HVH 39; HVH 4; HVH 40; HVH 41 ; HVH 42; HVH 43; HVH 44; HVH 45; HVH 46; HVH 48; HVH 5; HVH 50; HVH 51 ; HVH 53; HVH 55; HVH 56; HVH 58; HVH 59; HVH 6; HVH 61 ; HVH 63; HVH 65; HVH 68; HVH 69; HVH 7; HVH 70; HVH 73; HVH 74; HVH 76; HVH 77; HVH 78; HVH 79; HVH 80; HVH 82; HVH 83; HVH 84; HVH 85; HVH 86; HVH 87; HVH 88; HVH 89; HVH 9; HVH 90; HVH 91 ; HVH 92; HVH 95; HVH 96; HVH 98; IMT8073; J53; J96; JB 1-95; Jurua 18/11 ; Jurua 20/10; Kl; K2; KD1 ; KD2; KOEGE 10; KOEGE 118; KOEGE 131 ; KOEGE 3; KOEGE 30; KOEGE 32; KOEGE 33; KOEGE 40; KOEGE 43; KOEGE 44; KOEGE 56; KOEGE 58; KOEGE 61 ; KOEGE 62; KOEGE 68; KOEGE 7; KOEGE 70; KOEGE 71; KOEGE 73; KOEGE 77; KTE1 ; KTE10; KTE100; KTE101 ; KTE102; KTE103; KTE104; KTE105;

KTE106 KTE107 KTE108 KTE109 KTE11 ; KTE111 ; KTE112; KTE113 KTE114; KTE115;

KTE116 KTE117 KTE118 KTE119 KTE12; KTE120; KTE121 ; KTE122 KTE123; KTE124;

KTE125 KTE126 KTE127 KTE128 KTE129 KTE13; KTE130; KTE131 KTE132; KTE133;

KTE134 KTE135 KTE136 KTE137 KTE138 KTE139; KTE14; KTE140 KTE141 ; KTE142;

KTE143 KTE144 KTE145 KTE146 KTE147 KTE148; KTE15; KTE150 KTE153; KTE154;

KTE155 KTE156 KTE157 KTE158 KTE159 KTE16; KTE160; KTE161 KTE162; KTE163;

KTE165 KTE166 KTE167 KTE168 KTE169 KTE17; KTE170; KTE171 KTE172; KTE173;

KTE174 KTE175 KTE176 KTE177 KTE178 KTE179; KTE18; KTE180 KTE181 ; KTE182;

KTE183 KTE184 KTE185 KTE186 KTE187 KTE188; KTE189; KTE19 KTE190; KTE191 ;

KTE192; KTE193; KTE194; KTE195; KTE196; KTE197; KTE198; KTE199; KTE2; KTE20; KTE200; KTE201 ; KTE202; KTE203; KTE204; KTE205; KTE206; KTE207; KTE208; KTE209; KTE21 ; KTE210; KTE211 ; KTE212; KTE213; KTE214; KTE215; KTE216; KTE217; KTE218; KTE219; KTE22; KTE220; KTE221; KTE222; KTE223; KTE224; KTE225; KTE226; KTE227; KTE228; KTE229; KTE23; KTE230; KTE231 ; KTE232; KTE233; KTE234; KTE235; KTE236; KTE237; KTE24; KTE240; KTE25; KTE26; KTE27; KTE28; KTE29; KTE3; KTE31 ; KTE33; KTE34; KTE35; KTE36; KTE37; KTE38; KTE39; KTE4; KTE40; KTE41 ; KTE42; KTE43; KTE44; KTE45; KTE46; KTE47; KTE48; KTE49; KTE5; KTE50; KTE51 ; KTE52; KTE53; KTE54; KTE55; KTE56; KTE57; KTE58; KTE59; KTE6; KTE60; KTE61 ; KTE62; KTE63; KTE64; KTE65; KTE66; KTE67; KTE68; KTE69; KTE7; KTE70; KTE71 ; KTE72; KTE73; KTE74; KTE75; KTE76; KTE77; KTE78; KTE79; KTE8; KTE80; KTE81 ; KTE82; KTE83; KTE84; KTE85; KTE86; KTE87; KTE88; KTE89; KTE9; KTE90; KTE91 ; KTE93; KTE94; KTE95; KTE96; KTE97; KTE98; KTE99; LAU-EC1 ; LAU-EC10; LAU-EC2; LAU-EC3; LAU-EC4; LAU-EC5; LAU-EC6; LAU-EC7; LAU-EC8; LAU-EC9; LB226692; LCT- EC106; LCT-EC52; LCT-EC59; LSU-61 ; LT-68; M056; Ml ; M10; Mi l ; Ml 14; M12; M13; M14; M15; M16; M17; M18; M19; M2; M20; M21; M22; M23; M3; M4; M5; M6; M605; M646; M7; M718; M8; M863; M9; M919; MA6; MC19; MC21; MC23; MC6002; MC6003; MG1655star; MP020940.1; MP020980.1; MP020980.2; MP021017.1; MP021017.10; MP021017.il; MP021017.12; MP021017.2; MP021017.3; MP021017.4; MP021017.5; MP021017.6; MP021017.9; MP021552.il; MP021552.12; MP021552.7; MP021552.8; MP021561.2; MP021561.3; MP021566.1; MS 107-1; MS 110-3; MS 115-1; MS 116-1; MS 117-3; MS 119-7; MS 124-1; MS 145-7; MS 146-1; MS 153-1; MS 16-3; MS 175-1; MS 182-1; MS 185-1; MS 187-1; MS 196-1; MS 198-1; MS 200-1; MS 21-1; MS 45-1; MS 57-2; MS 60-1; MS 69-1; MS 78-1; MS 79-10; MS 84-1; MS 85-1; MT#2; Nl; NC101; NCCP15647; NCCP15657; NCCP15658; NCCP15738; NCCP15739; NE037; NE098; NE1487; NIPH- 11060424; Nissle 1917; O08; 091; OK1114; OK1180; OK1357; ON2010; ON2011; OP50; P0298942.1; P0298942.10; P0298942.ll; P0298942.12; P0298942.14; P0298942.15; P0298942.2; P0298942.3; P0298942.4; P0298942.6; P0298942.7; P0298942.8; P0298942.9; P0299438.10; P0299438.ll; P0299438.2; P0299438.3; P0299438.4; P0299438.5; P0299438.6; P0299438.7; P0299438.8; P0299438.9; P0299483.1; P0299483.2; P0299483.3; P02997067.6; P0299917.1; P0299917.10; P0299917.2; P0299917.3; P0299917.4; P0299917.5; P0299917.6; P0299917.7; P0299917.8; P0299917.9; P0301867.1; P0301867.ll; P0301867.13; P0301867.2; P0301867.3; P0301867.4; P0301867.5; P0301867.7; P0301867.8; P0301904.3; P0302293.10; P0302293.2; P0302293.3; P0302293.4; P0302293.6; P0302293.7; P0302293.8; P0302293.9; P0302308.1; P0302308.10; P0302308.ll; P0302308.12; P0302308.13; P0302308.14; P0302308.2; P0302308.3; P0302308.4; P0302308.5; P0304777.1; P0304777.10; P0304777.ll; P0304777.12; P0304777.13; P0304777.14; P0304777.15; P0304777.2; P0304777.3; P0304777.4; P0304777.5; P0304777.7; P0304777.8; P0304777.9; P0304799.3; P0304816.1; P0304816.10; P0304816.ll; P0304816.12; P0304816.13; P0304816.14; P0304816.15; P0304816.2; P0304816.3; P0304816.4; P0304816.5; P0304816.6; P0304816.7; P0304816.8; P0304816.9; P0305260.1; P0305260.10; P0305260.ll; P0305260.12; P0305260.13; P0305260.15; P0305260.2; P0305260.3; P0305260.4; P0305260.5; P0305260.6; P0305260.7; P0305260.8; P0305260.9; p0305293.1; p0305293.10; p0305293.11; p0305293.12; p0305293.13; p0305293.14; p0305293.15; p0305293.2; p0305293.3; p0305293.4; p0305293.5; p0305293.6; p0305293.7; p0305293.8; p0305293.9; P4; P4-96; P4-NR; PA10; PA11; PA13; PA14; PA15; PA19; PA2; PA22; PA23; PA24; PA25; PA28; PA3; PA31; PA32; PA33; PA34; PA35; PA38; PA39; PA4; PA40; PA41; PA42; PA45; PA47; PA48; PA49; PA5; PA7; PA8; PA9; PCN033; PUTI459; R424; R527; R529; RN587/1; S17; SCD1; SCD2; SCI-07; SEPT362; STEC_7v; STEC_94C; STEC_B2F1; STEC_C165-02; STEC_DG131-3; STEC_EH250; STEC_H.1.8; STEC_MHI813; STEC_031; STEC_S1191; SWW33; T1282_01; T1840_97; T22; T234_00; T408; T426; T924_01; TA004; TA007; TA008; TA014; TA024; TA054; TA103; TA124; TA141; TA143; TA144; TA206; TA249; TA255; TA271; TA280; TA435; TA447; TA464; ThroopD; TOP2386; TOP2396-1; TOP2396-2; TOP2396-3; TOP2515; TOP2522-1; TOP2652; TOP2662-1; TOP2662-2; TOP2662-3; TOP2662-4; TOP291; TOP293-1; TOP293-2; TOP293-3; TOP293-4; TOP379; TOP382-1; TOP382-2; TOP382-3; TOP498; TOP550-1; TOP550-2; TOP550-3; TOP550-4; TT12B; TTSH84137; TW00353; TW06591; TW07509; TW07793; TW07945; TW09098; TW09109; TW09195; TW09231; TW09276; TW10119; TW10246; TW10509; TW10598; TW10722; TW10828; TW11039; TW11681; TW14182; TW14301; TW14313; TW14425; TW14588; TW15838; TW15901; Txl686; TX1999; Tx3800; TY-2482; UMD753; UMEA 3014-1; UMEA 3022-1; UMEA 3033-1; UMEA 3041-1; UMEA 3052-1; UMEA 3053-1; UMEA 3065-1; UMEA 3087-1; UMEA 3088-1; UMEA 3097-1; UMEA 3108-1; UMEA 3113-1; UMEA 3117-1; UMEA 3121-1; UMEA 3122-1; UMEA 3124-1; UMEA 3139-1; UMEA 3140-1; UMEA 3144-1; UMEA 3148-1; UMEA 3150-1; UMEA 3151-1; UMEA 3152-1; UMEA 3155-1; UMEA 3159-1; UMEA 3160-1; UMEA 3161-1; UMEA 3162-1; UMEA 3163-1; UMEA 3172-1; UMEA 3173-1; UMEA 3174-1; UMEA 3175-1; UMEA 3176-1; UMEA 3178-1; UMEA 3180-1; UMEA 3185-1; UMEA 3190-1; UMEA 3193-1; UMEA 3199-1; UMEA 3200-1; UMEA 3201-1; UMEA 3203-1; UMEA 3206-1; UMEA 3208-1; UMEA 3212-1; UMEA 3215-1; UMEA 3216-1; UMEA 3217-1; UMEA 3220-1; UMEA 3221-1; UMEA 3222-1; UMEA 3230-1; UMEA 3233-1; UMEA 3240-1; UMEA 3244-1; UMEA 3257-1; UMEA 3264-1; UMEA 3268-1; UMEA 3271-1; UMEA 3290-1; UMEA 3292-1; UMEA 3298-1; UMEA 3304-1; UMEA 3314-1; UMEA 3317-1; UMEA 3318-1; UMEA 3323-1; UMEA 3329-1; UMEA 3336-1; UMEA 3337-1; UMEA 3341-1; UMEA 3342-1; UMEA 3355-1; UMEA 3391-1; UMEA 3426-1; UMEA 3489-1; UMEA 3490-1; UMEA 3585-1; UMEA 3592-1; UMEA 3609-1; UMEA 3617-1; UMEA 3632-1; UMEA 3652-1; UMEA 3656-1; UMEA 3662-1; UMEA 3671-1; UMEA 3682-1; UMEA 3687-1; UMEA 3693-1; UMEA 3694-1; UMEA 3702-1; UMEA 3703-1; UMEA 3705-1; UMEA 3707-1; UMEA 3718-1; UMEA 3805-1; UMEA 3821-1; UMEA 3834-1; UMEA 3889-1; UMEA 3893-1; UMEA 3899-1; UMEA 3955-1; UMEA 4075-1; UMEA 4076-1; UMEA 4207-1; USDA 5905; W; W26; Wal; Wa2; WC1; WC2; WV_060327; XH001; XH140A; Table 3. 62 completely sequenced E. coli strains used in this study

BL21-Gold; 042; 536; 55989; ABU 83972; APEC 01; APEC 078; ATCC 8739 REL606; BL21; BW2952; c321.deltaA; CFT073; DHl; DHl (ME8569); E24377A ED la; H10407; HS; IAI1; IAI39; IHE3034; JJ1886; K011FL; LF82; LY180; NA114: 12009; 2009EL-2050; 2009EL-2071; 2011C-3493; 11128; E2348/69; EC4115 EDL933; Sakai; TW14359; EC958; 11368; CB9615; RM12579; CE10; NRG857c: P12b; PMV-1; S88; SE11; SE15; SMS-3-5; clone D il4; clone D i2; DH10B; MC4100: MDS42; MG1655; W3110; UM146; UMN026; UMNF18; UMNK88; UTI89: Xuzhou21.

Table 4. 144 E. coli strains missing more than one essential gene

08bkt77219; 09bkt024447; 101-1; 1044; 1125; 174900; 178850; 180050; la; 2722950; 27a; 2862600; 2872800; 3.4880; 541-1; 87a; 908519; 909957; 910096-2; 95.0083; 95jbl; 97.1742; 99.1775; ai27; bl l4; bl71; b40; b7a; b94; bce008_ms-01; cl244_91; cl55_l l; c58_l l; clonea_il; cvml0021; cvm9455; cvm9534; cvm9545; cvm9553; cvm9570; cvm9574; cvm9952; declOf; deci le; decl2a; declb; dec2e; dec3c; dec4a; dec7b; dec9c; dora_a_5_14_21; dora_b_14; el 10019; e22; e482; ec4024; ec4076; ec4113; ec4192; ec4205; ec4401; ec4421; ec4448; ec4486; ec4501; ec508; ec869; eca- 0157; eca-727; ecc-1470; fl l; gsk202b; hOOl; h093800014; hi 12180283; hi 12180540; hi 12180541; h494; h591; hm27; hm69; hvh_221; hvh_222; hvh_225; hvh_29; kl; koege_61; koege_68; koege_71; ktel03; kte204; kte234; kte39; kte70; kte90; lau-ecl; ml3; ml; m21; m22; m23; m5; m8; m919; mp021017.1; ms_124-l; op50; p0298942.7; p0301867.8; p0304816.14; p0304816.1; p0305293.13; pal9; scdl; scd2; sci-07; ta004; ta014; tal41; ta206; top2386; top2396-3; top2515; top2522-l; top291; top293-l; top293-3; top293-4; top382-2; top498; top550-l; ttsh84137; tw09231; tw09276; twl0722; twl l039; twl4182; twl5838; umea_3257-l; umea_3298-l; umea_3899-l; wa2; wv_060327.

Table 5. E. coli strains missing YncE

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