A vaccine for the treatment or prevention of pseudomonas aeruginosa infection in a subject.

Abstract

A vaccine for the treatment or prevention of Pseudomonas aeruginosa infection in a subject. A composition comprising one or more immunogens is provided. The composition is for use in vaccine therapy to treat or prevent P. aeruginosa infection in subject.

Description

[0001] Title of the Invention

[0002] A vaccine for the treatment or prevention of Pseudomonas aeruginosa infection in a subject.

[0003] Field of the Invention

[0004] The invention relates to a vaccine for the treatment or prevention of Pseudomonas aeruginosa infection in a subject. The invention further relates to methods for treating or preventing Pseudomonas aeruginosa infection in a subject.

[0005] Background of the invention

[0006] Pseudomonas aeruginosa is a highly diverse, adaptable, and notorious opportunistic pathogen. P. aeruginosa causes infections in people with damaged epithelial barriers (e.g., patients on ventilators, with burns, intravascular and urinary catheters, or wounds), in people with compromised immune systems (e.g., cancer, human immunodeficiency virus (HIV) infection, diabetes mellitus, neutropenia, or organ transplantation), or in patients that are critically ill, such as those in intensive care units (ICUs). Moreover, it causes persistent infections in the lower respiratory tract of patients with pulmonary disorders that display a damaged airway epithelium, impaired clearance mechanisms and excessive mucus, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (nCFBE), or ventilator-associated pneumonia (VAP).

[0007] P. aeruginosa is one of the top-listed pathogens causing life-threatening infections, which have a deleterious impact on global public health due to the associated morbidity, mortality, and antimicrobial resistance (AMR) rates. P. aeruginosa was associated with more than 500,000 deaths globally in 2019, positioning it among the five bacterial pathogens that were responsible for more than half of all global bacterial deaths (Collaborators, G.B.D.A.R., Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet, 2022. 400(10369): p. 2221-2248). The surveillance carried out by the International Nosocomial Infection Control Consortium between 2010 and 2015 showed that nosocomial infections caused by P. aeruginosa are a healthcare concern, mainly due to its high level of AMR. Between 2015-2017, it was the 4thmost frequent pathogen causing healthcare-associated infections, (Weiner-Lastinger, L.M., et al., Antimicrobialresistant pathogens associated with adult healthcare-associated infections: Summary of data reported to the National Healthcare Safety Network, 2015-2017. Infect Control Hosp Epidemiol, 2020. 41 (1): p. 1-18), and the 3rdin Spain in 2022. It is considered a major nosocomial pathogen with the ability to cause outbreaks, which are associated with a mortality rate of 23%. In 2017, the World Health Organisation (WHO) included carbapenem-resistant P. aeruginosa (CR-Pa) in the list of antibiotic-resistant priority pathogens for which new antibiotics are urgently needed. The Centres for Diseases Control and Prevention (CDC) subsequently estimated that 32,600 hospitalised patients and 2,700 deaths were associated with multidrug resistance P. aeruginosa (MDR-Pa) infections in the United States (USA) in 2019 at a cost of $767,000 and declared MDR-Pa as a serious threat.

[0008] P. aeruginosa bloodstream infections (BSIs) are amongst the most serious infections caused by this pathogen. In 2019, almost 30% of P. aeruginosa-associated deaths were due to BSIs (Shortridge, D., et al., Geographic and Temporal Patterns of Antimicrobial Resistance in Pseudomonas aeruginosa Over 20 Years From the SENTRY Antimicrobial Surveillance Program: 1997-2016. Open Forum Infect Dis, 2019. 6(Suppl 1): p. S63-68). It was the fourth most common aetiological agent of BSIs worldwide between 1997 and 2016, and the sixth of ICU-acquired BSIs in 2019 in Europe. P. aeruginosa BSIs are associated with increased mortality compared to other Gram-negative microorganisms with rates ranging from 15 to 40%.

[0009] Antibiotic therapy is becoming ineffective due to the high percentage of infecting isolates displaying multi- or extensive-drug resistance. Additionally, P. aeruginosa can quickly become resistant to newly developed antibiotics. Therefore, P. aeruginosa infections are associated with high morbidity and mortality rates, reduced quality of life, and increased economic costs. Because therapeutic options for P. aeruginosa infections are increasingly limited, preventive vaccination of susceptible individuals may potentially present the most effective intervention. Despite extensive efforts made by the scientific community to develop an efficacious antiFI aeruginosa vaccine, only three candidates have progressed to phase III clinical trials, and none achieved the expected clinical benefit.

[0010] Dbring, G., et al discuss the alginate LPS- based vaccines for P. aeruginosa in patents with cystic fibrosis. (Dbring, G Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients. Int J Med Microbiol, 2010. 300(8): p. 573-7).

[0011] IC43 recombinant vaccine has been investigated for use in mechanically ventilated intensive care patients. IC43 is a recombinant protein (OprF / l) based vaccine against P. aeruginosa. Unfortunately, the vaccine provided no clinical benefit over placebo in terms of overall mortality. (Adlbrecht, C., et al., Efficacy, immunogenicity and safety of IC43 recombinant Pseudomonas aeruginosa vaccine in mechanically ventilated intensive care patients-a randomized clinical trial. Grit Care, 2020. 24(1): p. 74)

[0012] A bivalent P. aeruginosa flagella vaccine, containing two flagella subtype antigens (a0a1a2 and b) was investigated for its safety and efficacy in cystic fibrosis patients not colonised with P. aeruginosa. The authors conclude that P. aeruginosa strains, exhibiting flagella subtypes included in the vaccine, were significantly less frequently isolated from vaccinates than from placebo controls (Ddring, G., et al., A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Sci USA, 2007. 104(26): p. 11020-1102).

[0013] Unfortunately, there are no approved vaccines against P. aeruginosa infections to date, nor candidates in active clinical development, highlighting the need for searching and studying novel candidates and strategies.

[0014] The current invention serves to address the problems of the prior art.

[0015] Summary of the Invention

[0016] The inventors have identified a series of P. aeruginosa proteins for use in a vaccine that can prevent or treat serious infection of subjects with P. aeruginosa. Furthermore, the use of multiple subunit antigens widens the protective response against bacterial infection amongst a greater range of strains of the bacterium.

[0017] The inventors are the first to discover the use of the proteins of the invention as vaccine antigens. Furthermore, the inventors are the first to discover that these proteins were involved in the attachment of P. aeruginosa to host cells, in particular, to human lung epithelial cells.

[0018] For example, Figure 8 confirms that OpmH, FtsZ and LptH are involved in host cell attachment. In addition, the inventors have shown that FtsZ and LptH are protective in an acute P. aeruginosa pneumonia mouse model (Figure 13), reducing both bacterial colonisation and dissemination to the spleen, which is indicative of invasive disease. The inventors have further shown that the protective effect of immunisation with a multivalent vaccine containing OmpH, FtsZ and LptH against P. aeruginosa challenge in mice (Figure 20).

[0019] The proteins of the invention are listed in Table 1 .

[0020] Table 1 : Proteins of the Invention.

[0021] Accordingly, in an aspect of the invention, there is provided a composition comprising one or more immunogens of the invention (herein “composition of the invention”).

[0022] The immunogens of the invention may be one or more proteins of the invention, or an immunogenic portion of the protein of the invention. In an embodiment, the composition comprises a nucleotide or mRNA encoding the immunogen of the invention.

[0023] In an embodiment, the immunogenic portion is a polypeptide or a peptide.

[0024] In an embodiment, the composition is an immunogenic composition.

[0025] In an embodiment, the composition is a vaccine.

[0026] In an embodiment, the composition is an adenoviral vector vaccine.

[0027] The composition may optionally include one or more adjuvants.

[0028] The composition may optionally include one or more additional antigens.

[0029] The composition may be a pharmaceutical composition and optionally comprising a pharmaceutically acceptable excipient.

[0030] Preferably, the immunogen is present in a therapeutically effective amount.

[0031] It will be appreciated that the one or more immunogens may be any combination of the proteins of the invention, or immunogenic portions thereof.

[0032] In an embodiment, the composition comprises all the proteins of the invention.

[0033] In an embodiment, the immunogens comprise (or consist of) OpmH, FtsZ and LptH or immunogenic portions thereof.

[0034] In an embodiment, immunogens comprise (or consist of) LptH and FtsZ.

[0035] An aspect of the invention provides an immunogen of the invention (or a nucleotide or mRNA encoding the same), for use in a vaccine therapy to prevent or treat infection by P. aeruginosa in a subject.

[0036] An aspect of the invention provides the composition of the invention for use in a vaccine therapy to prevent or treat infection by P. aeruginosa in a subject.

[0037] An aspect of the invention provides an immunogen of the invention (or a nucleotide or mRNA encoding the same), for use as a therapy to induce an immune response against P. aeruginosa in a subject. An aspect of the invention provides the composition of the invention, or an immunogen of the invention (or a nucleotide or mRNA encoding the same) for use as a medicament.

[0038] The invention also provides a method of treating or preventing infection by P. aeruginosa in a subject, the method comprising a step of administering a therapeutically effective amount of one or more immunogens of the invention (or a nucleotide or mRNA encoding the same) to the subject.

[0039] The invention also provides a method of treating or preventing infection by P. aeruginosa in a subject, comprising administering the composition of the invention to the subject.

[0040] The invention also provides a method of treating P. aeruginosa infection in a subject, the method comprising a step of administering an antibody against one or more immunogens of the invention to the subject.

[0041] In an embodiment of any aspect of the invention, the subject is human.

[0042] In an embodiment, the subject is one with a compromised immune system. Examples of this subject include but are not limited to a subject with cancer, or recovering from cancer, a subject with human immunodeficiency virus (HIV) infection, diabetes mellitus, neutropenia, or organ transplantation.

[0043] In an embodiment, the subject is one with a damaged epithelial barrier. The epithelial barrier may be on the skin, surface of organs and / or soft tissue. Examples of this subject include but are not limited to subjects on ventilators, with burns, intravascular and urinary catheters, or wounds.

[0044] In an embodiment, the subject is a subject that is critically ill, such as a subject in an intensive care units (ICUs).

[0045] In an embodiment, the subject is one with a pulmonary disorder. This pulmonary disorder is one that displays a damaged airway epithelium, impaired clearance mechanisms and excessive mucus. The pulmonary disorder may be cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (nCFBE), pneumonia, or ventilator- associated pneumonia (VAP).

[0046] In an embodiment, the P. aeruginosa infection is an infection in the respiratory tract of the subject, for example, including but not limited to the lower respiratory tract..

[0047] In an embodiment, P. aeruginosa infection is in the skin and / or soft tissue.

[0048] In an embodiment, the P. aeruginosa infection is a bloodstream infection (BSI). In an embodiment, the P. aeruginosa infection is an eye infection. In such an embodiment, the subject may be a contact lens user.

[0049] Definitions and general preferences

[0050] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0051] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0052] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0053] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning, or nutritional deficiencies.

[0054] As used herein, the term "treatment" or "treating" refer to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s). In this case, the term is used synonymously with the term “therapy”. It can be manifested by a permanent or temporary improvement in the subject's condition. In this context, it includes limiting and / or reversing disease progression.

[0055] As used herein the terms "prevention" or "preventing" refers to an intervention (e.g., the administration of an agent or vaccine to a subject), which prevents or delays the onset or progression of a disease or infection, e.g., Pseudomonas aeruginosa infection, or the severity of a disease in a subject, or reduces (or eradicates) its incidence within a treated population. When used herein, the term “composition” should be understood to mean something made by the hand of man, and not including naturally occurring compositions. Compositions may be formulated in unit dosage form, i.e. , in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

[0056] When used herein, the term “pharmaceutical composition” may comprise one or more pharmaceutically acceptable diluents, excipients, or carriers. Even though the proteins, peptides and compositions of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine. Examples of such suitable excipients for the different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients”, 2ndEdition, (1994), edited byA Wade and PJ Weller. In particular, formulations for topical delivery are described in “Topical Drug Delivery Formulations” edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

[0057] In this specification, the term “sequence identity” should be understood to mean the amount of nucleic acids, or amino acids, which match between different sequences. For example, a sequence, such as a polypeptide or a polynucleotide sequence, that shares 90% sequence identity with a sequence is one in which any 90% of aligned residues are identical to the corresponding nucleic acids or amino acids in the reference sequence across the entire length of the sequence. Sequence homology can be calculated by a BLAST program (www.ncbi.nlm.nih.gov / cgi-bin Blast). Hereby, gaps are not counted.

[0058] The term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where the context permits) defines any subject, particularly a mammalian subject, for whom treatment or prevention is indicated. Mammal is a human or an animal, typically a human. In many cases, the subject will be a mammal which has, or which is suspected of having, a Pseudomonas aeruginosa infection, or at risk of having a pathogenic Pseudomonas aeruginosa infection.

[0059] In this specification, the term “infection by Pseudomonas aeruginosa", should be understood to mean infections of mammals that are caused by Pseudomonas aeruginosa.

[0060] In this specification, the term “effective variant” or “functional variant” as applied to any of the amino acid sequences discussed herein means an amino acid sequence that shares sequence identity, e.g. at least 90% or more (e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), with the sequence, and that is capable of eliciting a protective immune response in a mammal immunised with the variant against subsequent challenge by Pseudomonas aeruginosa as described below. The term can include peptides or proteins having an amino acid sequence that is substantially identical to the reference, and which is therapeutically effective as defined herein. Thus, for example, the term should be taken to include variants that are altered in respect of one or more amino acid residues. Preferably, such alterations involve the insertion, addition, deletion and / or substitution of 6 or fewer amino acids, preferably 5 or fewer, 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. It should be noted that any variant will have principally the same therapeutic effect, or may have an enhanced effect when tested in in vitro or in vivo models of the disease. This term includes “immunogenic portions” of the protein.

[0061] The term “effective variant” or “functional variant” may also be applied to the immunogenic portions, mRNA and nucleic acid sequences of the invention. For instance, the “effective variant”, of the nucleotide is one that encodes a protein or immunogenic portion thereof that is capable of eliciting a protective immune response in a mammal immunised with the protein or immunogenic portion thereof against subsequent challenge by Pseudomonas aeruginosa species as described below.

[0062] The term “immunogen” is a substance that is capable of generating a B-cell and / or T-cell immune response in a host organism. In this context, the immunogen may be a protein or an immunogenic portion of the protein such as an isolated polypeptide, or peptide. The immunogens may be isolated, purified, recombinant or synthetic or be delivered as nucleic acid such as mRNA or DNA.

[0063] In this specification, the term “immunogenic portion thereof” as applied to any of the proteins of the invention should be understood to mean a portion of the protein that when administered to a mammal using the methods described below elicits an immune response that is specific to the portion that is administered. In this context immune response includes IFN-y and / or IL- 17 responses in CD4+ and CD8+ cells, natural killer cells and humoral responses (B-cells).

[0064] The term “vaccine therapy” should be understood to mean the administration of an immunogen, e.g., an immunogenic protein or immunogenic portion thereof or an mRNA or viral vector comprising a nucleic acid sequence encoding the protein or immunogenic portion, to a subject with a view to eliciting a response by the host immune system. Typically, it results in the immunogen being recognised and process by the host immune system. It is subsequently recognised by the components of the host adaptive immune system to destroy infectious microorganisms.

[0065] The term “immune response” should be understood to mean induced humoral or cellular response in a host subject. In this context immune response includes IFN-y response and / or IL-17 responses in CD4+ and CD8+ cells, natural killer cells and humoral responses (B-cells).

[0066] As used herein, the term “active pharmaceutical ingredient” or “API” refers to an immunogen that may be used in vaccine therapy such as a protein, protein subunit, polypeptide, botanical (e.g., a plant extract) a fraction of a cell such as a bacterial cell, viral cell, or yeast cell (e.g. a cell wall fraction), or a nucleic acid encoding an immunogen. The nucleic acid may be an RNA (e.g. mRNA) or DNA molecule.

[0067] In this specification, the term “vaccine” should be understood to mean a composition comprising at least one immunogen, e.g., a protein of the invention, and optionally a suitable adjuvant and / or carrier. The preparation of vaccines comprising a protein or peptide as an active agent is well known in the art, for example US4599230 and US4601903 as examples. In an embodiment, the vaccine is a subunit vaccine. A “subunit” vaccine contains one or more specific antigens from a pathogen. It is acellular, i.e. it does not contain whole bacteria.

[0068] The term “DNA vaccine” should be understood to mean a composition that comprises a nucleic acid, typically on a nucleic acid construct that is capable of being delivered to a subject and encodes one or more proteins or immunogenic portions of the invention. The nucleic acid construct expresses said one or more protein or immunogenic portions in said subject. The protein or immunogenic portion thereof may be expressed as separate entities or in the form of a fusion protein. The vaccine may include one or more suitable adjuvants and / or one or more suitable pharmaceutically acceptable carriers. The construct may be an expression vector. Expression vectors suitable for this purpose are known in the art, for example a plasmid or a virus. In an embodiment, the vector is an adenovirus vector.

[0069] The term “adjuvant” should be understood to mean an agent that enhances the subject, i.e., the recipient’s, immune response to an immunogenic protein or portion thereof. Details of a suitable adjuvant composition are well known to a person skilled in the art.

[0070] The term “immunogenic composition” refers to a composition comprising one or more proteins of the invention, or an immunogenic portion thereof, (or nucleotide or mRNA encoding same) that is capable of inducing an immune response in an individual.

[0071] Proteins, peptides, and polypeptides (and their variants, including fragments) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).

[0072] In this specification, the term “amount effective” or “therapeutically effective amount” should be taken to mean an amount which results in a clinically significant reduction or prevention of Pseudomonas aeruginosa infection.

[0073] Suitably, the immunogenic component of the vaccine is administered at a dose of from 1 pg to 1OOpg. In one embodiment, it is administered at a dose of from 1Opg to 70pg, or from 1Opg to 50pg, or from 20pg to 40pg, or from 25pg to 30pg.

[0074] Typically, it is given as a bolus dose.

[0075] In the context of the therapeutic aspects of the present invention, the term “individual in need thereof” shall be taken to mean a mammal, preferably a human, who has an infection caused by Pseudomonas aeruginosa.

[0076] The term "symptom" is defined as an indication of disease, illness, injury, or that something is not right in the body.

[0077] The term "excipient" refers to a diluent, adjuvant, carrier or vehicle with which the therapeutic or active is administered. Such pharmaceutical carriers or excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous buffered solution, are the preferred carriers when the pharmaceutical composition is administered intramuscularly. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0078] The vaccine or composition of the invention can be formulated in any suitable means for the desired route of administration. Formulation should suit the mode of administration. Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g. , intra-nasally. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intranasal, intracerebral, and oral routes. Typically, the mode is intramuscularl, subcutaneous, oral or transdermal, such as by microneedle patches. In a typical embodiment, the mode is intramuscular. The vaccine or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). The vaccine or composition may be administered together with adjuvants and or other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the vaccine or composition of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0079] An “mRNA vaccine” is a vaccine that uses a copy of messenger RNA (mRNA) to produce an immune response. The vaccine delivers the immunogen encoding mRNA into the subject’s immune cells. This uses the mRNA to provide the protein, in this case the B. pseudomallei protein BpPA26. It is translated in the cell cytosol. This protein then stimulates the immune response which trains the body to identify, retain a memory for and destroy the pathogen. Typically, the mRNA is delivered by a co-formulation of the RNA encapsulated in a lipid nanoparticle that protects the RNA strand and aids absorption into the cells. Methods to make mRNA and mRNA vaccines are known in the art. mRNA vaccines are described in Kowalski PS, Rudra A, Miao L, Anderson DG (April 2019). "Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery". Mol Ther. 27 (4): 710-28; and Verbeke R, Lentacker I, De Smedt SC, Dewitte H (October 2019). "Three decades of messenger RNA vaccine development". Nano Today. 28: 100766.

[0080] In this specification, the term “sample” or “fluid” is a biological sample. The sample is one obtained from a subject, such as, for example, urine or urine cell-sediment, blood, saliva, or a tissue sample.

[0081] In this specification, the term “one or more” should be understood to mean and encompass that one or more, two or more, three or more, four or more, five or more, and so on. In an embodiment, it includes all 31 proteins of the invention.

[0082] Brief Description of the Figures

[0083] The current invention will now be described with reference to the following Figures in which;

[0084] Figure 1 : Attachment of six P. aeruginosa clinical isolates to HBE cells, assessed by colony counting. Bars represent the mean + SEM, with each dot representing one independent experiment. Statistically significant differences (p-value<0.05) were calculated using one-way ANOVA and are indicated by letter-based representation of pairwise comparison. The strains were assigned a letter from “a” to “f” from left to right. The letters on top of each bar indicate the strains to which they are compared that gave a statistically significant difference.

[0085] Figure 2: Confocal microscopy investigation of the attachment of six P. aeruginosa clinical isolates to HBE cells. These were obtained with the Opera Phenix™ High Content Screening automated confocal microscope. Bacteria are shown in green (anti- / ? aeruginosa Ab + secondary Ab-Alexa Fluor®488), and HBE cells in red (membranes, CellMask™ Deep Red) and blue (nuclei, DAPI).

[0086] Figure 3: Identification of P aeruginosa proteins in CF transmissible strain LES 431 involved in attachment to HBE cells. Two-dimensional gel (left) and corresponding cell blot (right) of two independent experiments performed with the same bacterial protein preparation from CF transmissible strain LES 431.

[0087] Figure 4: Identification of proteins involved in attachment of CF early strain AA2 to HBE cells. Two-dimensional gel (left) and corresponding cell blot (right) of two independent experiments (A and C) performed with the same bacterial protein preparation from CF early strain AA2.

[0088] Figure 5: Identification of proteins involved in attachment of COPD strain 57P31 PA to HBE cells. Two-dimensional gel (left) and corresponding cell blot (right) of two independent experiments (G and H) performed with the same bacterial protein preparation from COPD strain 57P31 PA.

[0089] Figure 6: Identification of proteins involved in attachment of CAP strain A5803 to HBE cells. Two-dimensional gel (left) and corresponding cell blot (right) of three independent experiments performed with the same bacterial protein preparation (L, N) and a different one (E) from CAP strain A5803.

[0090] Figure 7: Representative image of a negative control bot, where the membrane was probed with both primary and secondary antibodies, but not HBE cells.

[0091] Figure 8: Attachment of recombinant E. coli BL21 cells expressing either of the five antigen candidates. A) Number of bacteria attached per 100 HBE cells, counted automatically by the Harmony®4.8 software. Bars represent the mean and SD of the replicates. Raw data are available in appendix B.7. B) Representative images taken with the automated Opera PhenixTM confocal HSC microscope, showing the increased attachment of the induced cultures. Bacteria are shown in green (anti-E. coli Ab-FITC), and HBE cells in red (membranes, CellMaskTM Deep Red) and blue (nuclei, DAPI). C) Increased attachment levels of the induced cultures, compared to their uninduced controls, represented as the log2(FC). D) Confirmation by 12% SDS-PAGE (upper) and Western blot (lower) of protein (pointed with a red arrow) expression only in the induced cultures (+).

[0092] Figure 9: Evaluation of the purity of the recombinant protein batches by 12% SDS-PAGE (left) and Western blot (right) with an anti-6xHis Ab, and densitogram analysis using the Imaged software. The bands and peaks corresponding to the protein in study are pointed with a red arrow. The numbers inside the squares indicate the sample number that was excised from the gel for LC-MS analysis.

[0093] Figure 10: Antigen-specific serum IgG antibodies generated after subcutaneous immunisation of mice with three doses of either Ag+SAS (1 :1, 50 pg / mouse). Each dot represents the mean ± SD of the mice in the group. The grey, dash lines show the cut-off (2SD+mean) for antibody titration. Significant differences between control and immunised groups were analysed via two-way ANOVA Sidak’s multiple comparison test (p-value<0.05).

[0094] Figure 11 : Antigen-specific lgG1 and lgG2c antibodies generated after subcutaneous vaccination of mice with three doses of either Ag+SAS (1 :1, 50 pg / mouse). Each dot represents the average ± SD of the mice in the group. The grey lines show the cut-off (2SD+mean) for antibody titration. Significant differences between control and immunised groups were analysed via two-way ANOVA Sidak’s multiple comparison test (p-value < 0.05).

[0095] Figure 12: Antibody titres raised against each antigen, and the corresponding lgG2 / ig1 ratio. Bars represent the mean ± SD of mice per group. Antibody titres were calculated using 2SD+mean as a cut-off. On top of the bars are indicated the lgG2 / 1 ratios. Each point represents one mouse.

[0096] Figure 13: Bacterial colonisation of lungs (A, B), spleen (C, D) and stomach (E, F) of mice immunised with SAS alone (control group) or either Ag+SAS (rOprM, rFtsZ, rOpmH, rLptH), 24 h after challenge with P. aeruginosa KK1 strain (6.3x106-1.8x107CFU / mouse). Bars indicate the mean ± SEM, and each dot represents one mouse. Graphs A, C, and E show data pooled from two independent experiments. Statistically significant differences were calculated using the non-parametric Kruskal-Wallis test (p-value<0.05).

[0097] Figure 14: Weight change (A, B) and severity score (C, D) of mice in either the control (SAS) or immunised (r-Ag+SAS) group 24 h after challenge with P. aeruginosa KK1. Data are represented as mean ± SEM, and each dot represents one mouse. Statistically significant differences were calculated using the One-way ANOVA test (p-value<0.05).

[0098] Figure 15: Attachment of recombinant E. coli BL21 clones overexpressing either LptH or OprM to HBE cells, a) Representative images taken with the automated Opera Phenix™ confocal HSC microscope, showing the increased attachment of the induced E. coli cultures. Bacterial cells are shown in green (anti-E. coli antibody-FITC), and HBE cells in red (membranes, CellMask™ Deep Red) and blue (nuclei, DAPI). b) Number of bacteria attached per 100 HBE cells, automatically counted using a pipeline on the Harmony®4.8 software in 99 fields. Bars represent the mean and SD of the technical duplicates, c) Confirmation of protein expression (red arrows) only in the induced cultures (+) by 12% SDS-PAGE (upper) and Western blot (lower). Control data have been previously published1and are reproduced here as the experiments were performed simultaneously for comparison purposes.

[0099] Figure 16: Recombinant protein purification. Representative 12% SDS-PAGE (upper / left) and Western blot (bottom / right) examples of the purification of rOprM (a) and rLptH (d) by IMAC using a Ni-NTA resin, and the final batch after dialysis and concentration (b, e). The bands corresponding to each protein are pointed with a red arrow. Proteins were detected by Western blot using an anti-6xHis tag antibody conjugated to HRP. Densitograms showing the purity of the batches (c,f) were obtained with the Imaged software. CS, chaotrope- soluble fraction; FT, flow-through; W-io, (wash with 10 mM imidazole); W20, (wash with 20 mM imidazole); E250 (elution with 250 mM imidazole); E3oo (elution with 300 mM imidazole).

[0100] Figure 17: Protective effect of immunisation with rOprM and rLptH plus SAS against P. aeruginosa challenge in mice, a) Timeline of the immunisation schedule. Mice were immunised three times subcutaneously with 50 pg rOprM or rLptH plus SAS, blood samples were collected one week after the last dose, and one week later they were challenged via oropharyngeal aspiration with P aeruginosa KK1 strain (1.2x107CFU / mouse). P aeruginosa burden in the lungs (b) and spleens (c) and severity scores (d) of immunised mice 24 h after challenge. Data are represented as mean ± SEM, with each point representing one mouse. Statistically significant differences (p<0.05) were calculated using the non-parametric Kruskal- Wallis or One-way ANOVA tests.

[0101] Figure 18: Serological analysis of antigen-specific total IgG (a, d), lgG1 (b, e) and lgG2c (c, f) production one week after the last immunisation. Each point represents the mean ± SD of the )differences between control and immunised groups were analysed via two-way ANOVA Sidak’s multiple comparison test (p<0.05) (*p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001).

[0102] Figure 19: Cytokine recall responses in splenocytes of immunised mice. A) secretion of Interferon -y or IL-17 from splenocytes of mice immunised with rLptH that were restimulated with antigen; B) secretion of Interferon -y from splenocytes of mice immunised with either rFtsZ or rOmpH Splenocytes from mice immunised once with 50 pg antigens plus SAS or SAS-only controls were restimulated ex vivo with 10 pg antigen / 106cells. IFN-y and IL-17 production was analysed after 48 h using ELISpot. Data represent the number of spot-forming units (SFU) per million total cells and are shown as the average ± SD, with each dot representing one mouse. Statistically significant differences (p<0.05) were evaluated using Student’s t-test. (*p<0.05; **p<0.01 ; ***p<0.001).

[0103] Figure 20: Protective effect of immunisation with multivalent vaccines against P. aeruginosa challenge in mice, a) Timeline of the immunisation schedule. Mice (n=6-7) were immunised three times subcutaneously with 25 pg of either antigen (trivalent, rFtsZ+rOpmH+rLptH; Bivalent-O, rFtsZ+rOpmH; Bivalent-L rFtsZ+rLptH) plus SAS, and challenged via oropharyngeal aspiration with P. aeruginosa KK1 strain. P. aeruginosa burden in the lungs (b) and spleens (c) and severity score (d) of immunised mice 24 h after challenge. Data are represented as mean ± SEM, with each point representing one mouse. Statistically significant differences (p<0.05) were calculated using the non-parametric Kruskal-Wallis test. Black asterisks indicate statistically significant differences compared to PBS group, and blue asterisks to SAS-only group.

[0104] Detailed Description of the Invention

[0105] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0106] There is a need to provide a vaccine to protect and / or treat infections of P. aeruginosa in a subject, preferably a human. There are currently no vaccines available. Patients that are immunocompromised, critically ill, or that have a pulmonary disorder, such as cystic fibrosis, are especially vulnerable.

[0107] The invention provides compositions, such as a vaccine, comprising one or more immunogens of the invention. The one or more immunogens are generally one or more proteins of the invention. The proteins of the invention are selected from the group comprising ALAD, HfIK, ArcA, ArgA, FtsZ, AguA, GlnA, OpmH, Hslll, Gimli, FumC2, PchG, LpdG, GuaB, MmsA, CycH, BamB, OprQ, LptH, TssK1 , GltA, HGDO, MetK, PrpD, llvC, PsIB, HemX, ApeB, Uncharacterised protein (FixH), YjiA and peptidase M16.

[0108] In an embodiment, the immunogen is an immunogenic portion of the protein.

[0109] The proteins of the invention or immunogenic portions thereof are for use as vaccine antigens.

[0110] In an embodiment, the composition of the invention comprises two or more immunogens, three or more immunogens, four or more immunogens, five or more immunogens, six or more immunogens, seven or more immunogens, eight or more immunogens, nine or more immunogens, ten or more immunogens, eleven or more immunogens, twelve or more immunogens, thirteen or more immunogens, fourteen or more immunogens, fifteen or more immunogens, sixteen or more immunogens, seventeen or more immunogens, eighteen or more immunogens, nineteen or more immunogens, twenty or more immunogens, twenty one or more immunogens, twenty two or more immunogens, twenty three or more immunogens, twenty-four or more immunogens, twenty-five or more immunogens, twenty-six or more immunogens, twenty-seven or more immunogens, twenty-eight or more immunogens, twenty- nine or more immunogens, thirty or more immunogens, thirty-one or more immunogens, or thirty-two immunogens of the invention.

[0111] In an embodiment, the composition comprises at least FtsZ immunogen.

[0112] In an embodiment, the composition comprises at least OpmH immunogen.

[0113] In an embodiment, the composition comprises at least LptH immunogen.

[0114] In these embodiments, the composition may have one or more additional immunogens of the invention. It will be appreciated that any number or combination of the immunogens of the invention may be present.

[0115] In an embodiment, the composition comprises at least two immunogens, which are LptH and FtsZ. In an embodiment, the only immunogens present in the composition are FtsZ and LptH. In other words, in an embodiment, the composition comprises immunogens which consist of two immunogens namely FtsZ and LptH. Typically, the immunogen is a protein.

[0116] In an embodiment, the composition comprises at least two immunogens, which are LptH and OpmH. In an embodiment, the only immunogens present in the composition are LptH and OmpH. In other words, in an embodiment, the composition comprises immunogens which consist of two immunogens namely LptH and OmpH. Typically, the immunogen is a protein.

[0117] In an embodiment, the composition comprises at least two immunogens, which are FtsZ and OpmH. In an embodiment, the only immunogens present in the composition are FtsZ and OmpH. In other words, in an embodiment, the composition comprises immunogens which consist of two immunogens namely FtsZ and OmpH. Typically, the immunogen is a protein.

[0118] In an embodiment, the composition comprises at least three immunogens, which are FtsZ, OpmH and LptH. In an embodiment, the only immunogens present in the composition are FtsZ, OpmH and LptH. In other words, in an embodiment, the composition comprises immunogens which consist of FtsZ, OpmH and LptH. Typically, the immunogen is a protein. In an embodiment, the composition comprises all thirty-two proteins of the invention or immunogenic portions thereof.

[0119] The composition of the invention may comprise a combination of immunogenic portions. This may be any combination of the immunogenic portions of the invention.

[0120] It will be appreciated that in the embodiments disclosed above, the composition may comprise a nucleotide encoding the one or more immunogen, or an mRNA encoding the one or more immunogen instead of the immunogen itself. In such an embodiment, the vaccine may be a DNA vaccine. It may be an adenoviral vector vaccine which comprise one or more nucleotide sequences encoding one or more immunogen of the invention.

[0121] In such an embodiment, the vaccine may be an mRNA vaccine.

[0122] In an embodiment of the invention the composition is a vaccine composition. The vaccine may have one or more adjuvants. Typically, the adjuvant is one that induces a T cell and B cell response, e.g., the CAF series, such as CAF-01 or CAF-09 or CpG ODNS (ODN 2006). It may be manno-heptose capsular polysaccharide (CPS) (Scott et al., Infect Immun. 2014 Aug;82(8):3206-13). It will be appreciated that such adjuvants are known in the art.

[0123] The invention further provides a DNA vaccine comprising an active agent, optionally a pharmaceutically acceptable carrier and optionally an adjuvant. In an embodiment, the active agent is a nucleic acid sequence encoding one or more immunogens of the invention. In an embodiment, the nucleotide acid sequence comprises (or consists of) a sequence selected from SEQUENCE ID NO .1 to SEQUENCE ID NO. 31 or a therapeutically effective variant thereof, typically having at least 90% sequence identity with the sequence.

[0124] In an embodiment, the vaccine of the invention is an mRNA vaccine. Thus, in an aspect, a vaccine is provided that comprises an mRNA that encodes one or more immunogens of the invention.

[0125] The vaccine or composition of the invention may comprise a combination of immunogenic portions.

[0126] Notably, the composition is for use as a vaccine to prevent or treat infection by P. aeruginosa in a subject. A vaccine for use to treat an infection can be referred to as “post exposure vaccines”.

[0127] The composition can be used to induce an immune response against P. aeruginosa in a subject. An aspect of the invention includes one or more immunogens of the invention or a composition comprising said one or more immunogens, for use in immunotherapy.

[0128] An embodiment of the treatment aspect of the current invention includes the use of an antibody directed or raised against, to one or more immunogens of the invention, which are suitable for immunotherapy and administered to the subject. The antibody is typically a monoclonal antibody specific to the immunogen of the invention. This type of therapy is particularly effective as a treatment but also lends itself to passive immunisation. Passive immunisation is used when there is a high risk of infection but insufficient time or ability to develop an immune response. Additionally, it can mean administering antibodies to treat an infection. This can be an effective route for subjects with a compromised immune system and who are in an environment or circumstances that place them at increased risk of infection.

[0129] Thus also provided by the invention is an antibody (or a composition comprising the same) directed to one or more immunogens of the invention and its use in immunotherapy. The composition may contain multiple antibodies directed to different immunogens of the invention. Antibodies to said immunogens or antigens can be obtained with routine methods known in the art, for example hybridoma technology. The use of monoclonal antibodies and generation of such antibodies, to provide long term protection has been investigated for Pseudomonas aeruginosa infection, e.g. see Pitiot et al., Mucosal Immunology, (2023) 16: 312-325 and Mousavi, et al., Microbial Pathogenesis, vol 101 , December 2016, pages 83-88.

[0130] This use or method of treatment would be especially beneficial to subjects on ventilators who develop ventilator acquired P. aeruginosa pneumonia and need a fast-acting treatment. However, it will be appreciated that any subject with a P. aeruginosa infection may benefit.

[0131] The nucleotide sequence and amino acid sequence of the proteins of the invention are as follows:

[0132] 1 . Delta-aminolevulinic acid dehydratase (ALAD)

[0133] The term ALAD encompasses this protein and effective variants, e.g. that share at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO. 32.

[0134] The nucleic acid sequence of ALAD is provided in SEQUENCE ID NO. 1. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0135] >PA5243 |hemB

[0136] SEQUENCE ID NO. 1 : gtgagcttcactcccgccaatcgcgcctatccctacacccgcctgcggcgcaatcgccgcgatgacttctcccgccgcctggtccgcgagaatgtcc tcaccgtcgacgacctgatcctgccggtgttcgtcctcgatggggtcaaccagcgtgaaagcattccgtcgatgcccggcgtcgagcgcctgtccat cgaccagttgctgatcgaggcggaagagtgggtggccctgggcattccggcgctggcgctgttccctgtgaccccggtggagaagaagtccctgg acgccgccgaggcatacaacccggaaggcatcgcccagcgcgccacccgtgccctgcgcgagcgctttccggagctgggcatcatcaccgacgt ggcgctcgacccgttcaccacccatggccaggacggcatcctggatgacgatggctacgtcctcaacgacgtcagcatcgacgtgctggtacggc aggcgctgtcccacgccgaggccggcgcccaggtggtagcgccctcggacatgatggacggtcgcatcggtgcgatccgcgaggccctggagtc tgccggccacaccaatgtgcggatcatggcctactcggccaagtacgccagcgcctactacggtccgttccgcgatgcggtcggctcggcttcgaa cctcggcaagggcaacaaggccacctaccagatggatccggcgaacagcgacgaggcacttcacgaagtggccgccgacctggccgaaggcg ccgacatggtgatggtcaagccgggcatgccctacctcgacatcgtgcgtcgggtgaaggatgaattccgcgccccgacctttgtctaccaggtca gcggcgagtatgcgatgcacatgggcgccatccagaacggctggctggccgaatcggtgatcctcgaatcccttaccgccttcaaacgtgccggc gccgatggcattctgacctacttcgccaagcaggccgcagaacaattaagacgggggcgttga

[0137] SEQUENCE ID NO. 32

[0138] MSFTPANRAYPYTRLRRNRRDDFSRRLVRENVLTVDDLILPVFVLDGVNQRESIPSMPGVERLSIDQLLIEAEEWVAL GIPALALFPVTPVEKKSLDAAEAYNPEGIAQRATRALRERFPELGIITDVALDPFTTHGQDGILDDDGYVLNDVSIDVL VRQALSHAEAGAQVVAPSDMMDGRIGAIREALESAGHTNVRIMAYSAKYASAYYGPFRDAVGSASNLGKGNKATY QMDPANSDEALHEVAADLAEGADMVMVKPGMPYLDIVRRVKDEFRAPTFVYQVSGEYAMHMGAIQNGWLAES VILESLTAFKRAGADGILTYFAKQAAEQLRRGR

[0139] 2. Protein HfIK

[0140] The term HfIK encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.33.

[0141] The nucleic acid sequence of HfIK is provided in SEQUENCE ID NO. 2. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0142] >PA4942 |hflK

[0143] SEQUENCE ID NO.2 atggcttggaacgagccgggtgacaactcgaacaacaacgatcgagacccgtggggaggccgccgcggtggtggtggcggcggtggtgaccgc aaggggccgcccgacctggatgaggctttccgcaagcttcaggacagcctgaacgggctgttcggtggaaaaaaacgcagtggcaacggttccg gctccggttccggcggcaagggcggtggcctgggcctgttcggcatcggcctggcgatcctcgccgtgctctggctgtacaacgccatctatgtggt ggacgagcaggagcaggcggtcatcctgcgcttcggcaagtattacgagacggtcggtcccggcctgaacttctacttcccgccgatcgacaagc gcttccaggagaacgtcacccgcgagcgtgcgtacagcaagcaggggcagatgctcaccgaggacgagaacatcgtcgaagtgccgctgaccgt gcagtacaagatcagcaacctgcaggacttcgtgctcaacgtcgaccagcccgaggtcagcctgcagcaggcgaccgagagcgcgctgcggcat gtcgccggctccaccaccatggaccggatcctcaccgaaggccgcgagcagatggcgaccgaggtgcgcgagcgcctgcaacgcttcctggata cctacaggaccggtatcaccgtgacccaggtgaacatccagagcgcccaggcaccgcgtgaggtgcaggaagcgttcgacgacgtgatccgggc ccgcgaggacgagcagcgcgagaagaaccaggccgaagcctatgccaacggcgtggtgccggaggcacgtggccaggcccagcgcatcatcg aagaagccaacggttaccgcgacgaggtgatttcgcgcgcccagggcgaggcggatcgcttctccaagctgctggtggaatatcgcaaggctccg gaagtgacgcgcgagcgtctgtacctggacaccatgcaggaagtcttcagccagaccagcaaggtgctggtgaccggtcagcaggggcagaac aacctgctctacctgccgctggacaagatgatcgatggtcgtaacgccgcgcccgctacgggggccggtgcttccgccggtggtggcagcgccag cgcttccgacctggggtcgcggatcgtcaacgatctgcgccagcaagacgcgcgtacgagggagagccgctga

[0144] SEQUENCE ID NO. 33

[0145] MAWNEPGDNSNNNDRDPWGGRRGGGGGGGDRKGPPDLDEAFRKLQDSLNGLFGGKKRSGNGSGSGSGGKG GGLGLFGIGLAILAVLWLYNAIYVVDEQEQAVILRFGKYYETVGPGLNFYFPPIDKRFQENVTRERAYSKQGQMLTED ENIVEVPLTVQYKISNLQDFVLNVDQPEVSLQQATESALRHVAGSTTMDRILTEGREQMATEVRERLQRFLDTYRTGI TVTQVNIQSAQAPREVQEAFDDVIRAREDEQREKNQAEAYANGVVPEARGQAQRIIEEANGYRDEVISRAQGEAD RFSKLLVEYRKAPEVTRERLYLDTMQEVFSQTSKVLVTGQQGQNNLLYLPLDKMIDGRNAAPATGAGASAGGGSAS ASDLGSRIVNDLRQQDARTRESR

[0146] 3. Arginine deiminase (ArcA)

[0147] The term ArcA encompasses this protein and effective variants, e.g. that share at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.34.

[0148] The nucleic acid sequence of ArcA is provided in SEQUENCE ID NO. 3. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0149] >PA5171 |arcA

[0150] SEQUENCE ID NO. 3 atgagcacggaaaaaaccaaacttggcgtccactccgaagccggcaaactgcgcaaagtgatggtctgctcgcccggactcgcccaccagcgcc tgaccccgagcaactgcgacgagttgctgttcgacgacgtgatctgggtgaaccaggccaagcgcgaccacttcgacttcgtcaccaagatgcgc gagcgcggcatcgacgtcctcgagatgcacaatctgctgaccgagaccatccagaacccggaagcgctgaagtggatcctcgatcgcaagatca ccgccgacagcgtcggcctgggcctgaccagcgagctgcgctcctggctggagagcctggagccgcgcaagctggccgagtacctgatcggcgg cgtcgccgctgacgacctgcccgccagcgaaggcgccaacatcctcaagatgtaccgcgagtacctgggccattccagcttcctgctgccgccgtt gccgaacacccagttcacccgcgacaccacttgctggatctacggcggcgtgaccctgaacccgatgtactggccggcgcgacgacaggaaacc ctgctgaccaccgccatctacaagttccaccccgagttcgccaacgccgagttcgagatctggtacggcgacccggacaaggaccacggctcctc gaccctggaaggcggcgacgtgatgccgatcggcaacggcgtggtcctgatcggcatgggcgagcgctcctcgcgccaggccatcggtcaggtc gcccagtcgctgttcgccaagggcgccgccgagcgggtgatcgtcgccggcctgccgaagtcccgcgccgcgatgcacctggacaccgtgttcag cttctgcgaccgcgacctggtcacggtcttcccggaagtggtcaaggaaatcgtgcccttcagcctgcgccccgatccgagcagcccctacggcat gaacatccgccgcgaggagaaaaccttcctcgaagtggtcgccgaatccctcggcctgaagaaactgcgcgtggtcgagaccggcggcaacagc ttcgccgccgagcgcgagcaatgggacgacggtaacaacgtggtctgcctggagccgggcgtggtggtcggctacgaccgcaacacctacacca acaccctgctgcgcaaggccggcgtcgaggtcatcaccatcagcgccagcgaactgggtcgcggtcgcggcggcggccactgcatgacctgccc gatcgtccgcgacccgatcgactactga SEQUENCE ID NO.34

[0151] MSTEKTKLGVHSEAGKLRKVMVCSPGLAHQRLTPSNCDELLFDDVIWVNQAKRDHFDFVTKMRERGIDVLEMHN LLTETIQNPEALKWILDRKITADSVGLGLTSELRSWLESLEPRKLAEYLIGGVAADDLPASEGANILKMYREYLGHSSFLL PPLPNTQFTRDTTCWIYGGVTLNPMYWPARRQETLLTTAIYKFHPEFANAEFEIWYGDPDKDHGSSTLEGGDVMPI GNGVVLIGMGERSSRQAIGQVAQSLFAKGAAERVIVAGLPKSRAAMHLDTVFSFCDRDLVTVFPEVVKEIVPFSLRP DPSSPYGMNIRREEKTFLEVVAESLGLKKLRVVETGGNSFAAEREQWDDGNNVVCLEPGVVVGYDRNTYTNTLLRK AGVEVITISASELGRGRGGGHCMTCPIVRDPIDY

[0152] 4. Amino-acid N-acetyltransferase (ArgA)

[0153] The term ArgA encompasses this protein and effective variants, e.g. that share at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO. 35.

[0154] The nucleic acid sequence of ArgA is provided in SEQUENCE ID NO. 4. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0155] >PA5204 |argA

[0156] SEQUENCE ID NO. 4 atgcccgactacgtcaactggttacgtcacgcttcgccctacatcaactcgcaccgggaccgcaccttcgtggtcatgctccccggcgaaggggtg gagcatcccaatttcggcaatatcgtccacgacctagtccttctccacagcctcggtgcgcgcctggtgctggtgcacggttcgcgtccgcagatcg aggcgcgcctggccgcgcgcggcctggctccgcgctatcaccgcgacctgcgagtgaccgatgcaccgaccctggagtgcgtcatcgacgccgtg ggtagcctgcgcatcgccatcgaggcgcggctgtcgatggacatggccgcctcgccgatgcagggcgcgcgcctgcgggtcgctggcggcaacc tggtcaccgcgcggccgatcggcgtggtcgagggtgtcgactatcaccataccggtgaggtccgtcgcatcgaccgcaagggtattggccgccttc tcgacgagcgcagcatcgttctgctctcgccgctgggctactcgccgaccggggaaatcttcaacctggcctgtgaagacgtggccatgcgcgccg ccatcgacctggaagcggaaaagctgattctctacggcgccgaacagggcttgctggacgcatccggcaaactggtccgcgaactgcgcccgca gcaggtgcccgcgcacctgcaacggctgggcaacagctaccaggccgagctgctggacgccgcggcgcaggcctgccgggccggggtcaagc gcagccatatcgtcagctacaccgaggacggcgcgctgctcagcgagctattcacccgcaccggcaacggcaccctggtcgcccaggagcagttc gagcagttgcgcgaggcgggcatcgaggacgttggcgggctgatcgagctgatccgcccgctggaagagcagggcatcctggtacgccgttccc gcgaggtgctggaacgcgagatcgaacagttcagcatcgtcgagcgcgaagggctgatcatcgcctgcgccgcgctctacccgatcgccgattcc gaggcgggcgagctggcctgtctggcggtcaacccggagtaccgccacggcgggcgtggcgacgaactgctggagcggatcgaggagcgcgcg cgcggactcggcctgaagaccctgttcgtgctcaccacgcggaccgcgcactggttccgcgagcgcggcttccagcctagcagcgtcgaacggct accggcggcgcgggcctcgctgtacaacttccagcgcaattcgcaggtattcgagaagagcctgtga

[0157] SEQUENCE ID NO. 35

[0158] MPDYVNWLRHASPYINSHRDRTFVVMLPGEGVEHPNFGNIVHDLVLLHSLGARLVLVHGSRPQIEARLAARGLAPR YHRDLRVTDAPTLECVIDAVGSLRIAIEARLSMDMAASPMQGARLRVAGGNLVTARPIGVVEGVDYHHTGEVRRID RKGIGRLLDERSIVLLSPLGYSPTGEIFNLACEDVAMRAAIDLEAEKLILYGAEQGLLDASGKLVRELRPQQVPAHLQRL GNSYQAELLDAAAQACRAGVKRSHIVSYTEDGALLSELFTRTGNGTLVAQEQFEQLREAGIEDVGGLIELIRPLEEQGI LVRRSREVLEREIEQFSIVEREGLIIACAALYPIADSEAGELACLAVNPEYRHGGRGDELLERIEERARGLGLKTLFVLTTR TAHWFRERGFQPSSVERLPAARASLYNFQRNSQVFEKSL 5. Cell division protein FtsZ

[0159] The term FtsZ encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.36.

[0160] The nucleic acid sequence of FtsZ is provided in SEQUENCE ID NO. 5. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0161] >PA4407 |ftsZ

[0162] SEQUENCE ID NO. 5 atgtttgaactggtcgataacatcgcacaaaccgccgtcataaaagtgatcggtgtaggtggtggtggcggcaacgccgtcaatcacatggccaa gaacaacgtcgagggcgtcgagttcatctgcgccaacaccgacgcacaagcgctgaagaacatcgcggcgcgcaccgttctgcaactcggcccg ggcgtcaccaaggggctgggtgccggcgccaatccggaagtcggtcgtcaggcggctctggaagatcgcgagcgcatttccgaagtgctggaag gcgccgacatggtcttcatcaccaccggcatgggtggcggcaccggtaccggcgccgcgccgatcatcgccgaagtggcgaaggaaatgggcat cctcaccgtcgcggtggtgacccgcccgttcccgttcgaaggtcgcaagcgcatgcagatcgccgacgagggcatccgcgcgctggccgagagcg tcgattcgctgatcaccatcccgaacgagaagctgctgaccatcctcggcaaggacgccagcctgctggccgccttcgccaaggccgatgacgtg ctggccggtgccgtgcgcggtatctccgacatcatcaagcgtccgggcatgatcaacgtcgacttcgccgacgtgaagaccgtcatgagcgaaatg ggcatggcgatgatgggtaccggctgcgccagcggtccgaaccgtgcccgcgaggccaccgaggcggcaatccgcaacccgctgctggaagac gtcaacctgcagggcgcgcgcggcatcctggtgaacatcaccgcgggtccggacctgtccctgggcgagtactccgatgtcggcaacatcatcga acagttcgcttccgagcacgccactgtgaaggtgggcaccgtgatcgacgcggacatgcgcgatgagctgcacgtcaccgtagtcgccaccggcc tgggcgcgcgcctggagaaaccggtgaaggtcgtcgacaacaccgtgcagggcagtgcagcccaggcagccgctccggcccagcgcgagcagc agtcggtgaactaccgcgacctcgaccgtcctaccgtgatgcgcaaccagtctcacggcagcgcggcgaccgcggccaagctgaacccgcagga tgacctggattacctggatatcccggcgttcctgcgtcgtcaggccgattga

[0163] SEQUENCE ID NO. 36

[0164] MFELVDNIAQTAVIKVIGVGGGGGNAVNHMAKNNVEGVEFICANTDAQALKNIAARTVLQLGPGVTKGLGAGAN PEVGRQAALEDRERISEVLEGADMVFITTGMGGGTGTGAAPIIAEVAKEMGILTVAVVTRPFPFEGRKRMQIADEGI RALAESVDSLITIPNEKLLTILGKDASLLAAFAKADDVLAGAVRGISDIIKRPGMINVDFADVKTVMSEMGMAMMGT GCASGPNRAREATEAAIRNPLLEDVNLQGARGILVNITAGPDLSLGEYSDVGNIIEQFASEHATVKVGTVIDADMRDE LHVTWATGLGARLEKPVKVVDNTVQGSAAQAAAPAQREQQSVNYRDLDRPTVMRNQSHGSAATAAKLNPQDD LDYLDIPAFLRRQAD

[0165] 6. Mannose-1 -phosphate guanylyltransferase (PsIB)

[0166] The term PsIB encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.37.

[0167] The nucleic acid sequence of PsIB is provided in SEQUENCE ID NO. 6. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0168] >PA2232 |pslB

[0169] SEQUENCE ID NO. 6 atgaacgccgtcgccccgctgatcccctgcatcgtttccggcggttccgggacccgcctgtggccggtttcccgggagagcatgccgaaacccttca tgcgcctggccgatgaccagagcctgttgcagaagaccttcctgcgtatcgccgggctgccggacgtcgcccgcctgctcacggtgaccaaccgcg acctgctgttccgcaccctggacgactaccgcgcggtcaaccgcagcggcctggcccaggacctgctgctggagccggtgggacgcaataccgc gccggccatcgccgccgccgcgctgcatgtgcaggagcacttcggcgaccaggcgcagttgctgatccttcccgccgaccacctgatccgcgacg agcaggccttcgccgcggcggtggccgaggctcgcggactcgcggcccaaggctacctggtgaccttcggcatcaccccggagcgcgccgagac cggcttcggctatatcgaacagggcgccccgctgggcaacggcttccgggtcgcgcgcttcgtcgagaaacccgaccaggccaccgcccagtcct acctggacagcggcaagtacctgtggaacgccggcatgttctgcttccaggccgccaccgtgttgcaggaactggagcgccacgcgccggaggtg ctgatcgccgcccgcgccgccctggccgacggcagcagcctggagaacggccagtgccgccagcgcgagctggccgccggggccttcgccgag gcgccggacatctccgtcgactacgcgctgatggagcgctcggacaaggtcgccgtggtgccctgctccatcggctggagcgacatcggctcctg gcaggcgctgcgcgaactcagcgcggcggacgagaacggcaaccaggtacgcggcgaaagcgtgctgcacgacgtcagcaactgctacatcga ttcgccgaagcgcctggtcggcgccgtcggcgtgcacgacctgatcatcgtcgacacccccgacgccctgctggtggccgacgcggcgcgcagcc aggacgtcaagttcgtcgcccaggaactcaagcgtcgcggccacgacgccttccgcctgcaccgcacggtcagccggccctggggcacctacacc gtgctcgaggaaggccgccgcttcaagatcaagcgcatcgtggtgcgccccaaggcttcgctgtcgttgcagatgcaccaccatcgcagcgagca ctggatcgtggtcagcggcatggcgctggtggagaacggcgagcgcgagtttctcctcaacaccaacgaatccaccttcatccccgccgggcata gccaccgcctgagcaatccggggatcatcgacctggtgatgatcgaggtacagagcggcgagtacctcggcgaggacgacatcgtccgcttcaac gacatctacggtcgcgcccccgccagcgacgagaagaaagcctga

[0170] SEQUENCE ID NO. 37

[0171] MNAVAPLIPCIVSGGSGTRLWPVSRESMPKPFM RLADDQSLLQKTFLRIAGLPDVARLLTVTNRDLLFRTLDDYRAV NRSGLAQDLLLEPVGRNTAPAIAAAALHVQEHFGDQAQLLILPADHLIRDEQAFAAAVAEARGLAAQGYLVTFGITP ERAETGFGYIEQGAPLGNGFRVARFVEKPDQATAQSYLDSGKYLWNAGMFCFQAATVLQELERHAPEVLIAARAAL ADGSSLENGQCRQRELAAGAFAEAPDISVDYALM ERSDKVAVVPCSIGWSDIGSWQALRELSAADENGNQVRGES VLHDVSNCYIDSPKRLVGAVGVHDLIIVDTPDALLVADAARSQDVKFVAQELKRRGHDAFRLHRTVSRPWGTYTVLE EGRRFKIKRIVVRPKASLSLQMHHHRSEHWIVVSGMALVENGEREFLLNTNESTFIPAGHSHRLSNPGIIDLVMIEVQ SGEYLGEDDIVRFNDIYGRAPASDEKKA

[0172] 7. Agmatine deiminase AguA

[0173] The term AguA encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.38.

[0174] The nucleic acid sequence of AguA is provided in SEQUENCE ID NO. 7. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0175] >PA0292 |aguA

[0176] SEQUENCE ID NO. 7 atgagcaacccgaccagcaccccacgcgccgacggcttccgcatgcccgccgaatgggaaccccacgagcagacctggatggtctggccggaa cgcccggacaactggcgcaacggcggcaagccggcccaggcggccttcgccgcggtggccaaggccatcgcgcgcttcgagccggtcaccgtct gcgccagcgccgggcagtacgagaacgcgcgcgcacgcctcgacgacggcaacatccgcgtcgtggaaatcagcagcgacgatgcctgggtcc gcgacaccggcccgaccttcgtcatcgacgacaagggcgatgtacgcggcgtcgactggggcttcaacgcctggggcggcttcgaaggcggcct gtacttcccctggcagcgcgacgaccaggtggcacgcaagatcctcgagatcgaacggcgcgcccgctaccgcaccgacgacttcgtcctcgagg gcggctcgatccacgtcgacggcgaaggcacgctgatcaccaccgaggaatgcctgctcaaccacaaccgcaacccgcacctgagccaggcgg agatcgagcggaccctgcgcgactaccttgcggtggagagcatcatctggctgccgaacggcctctacaacgacgagaccgacggccacgtcga caacttctgctgctacgcgcgtcccggcgaggtgctgctggcctggaccgacgaccaggacgacccgaactacctgcgctgccaggccgccctcc gcgtgctggaagaaagccgcgacgccaagggacgcaagctggtggtacacaagatgccgatccccggcccgctgtacgcgacccaggaagagt gcgacggcgtggatatcgtcgagggcagccagccgcgcgatccctccattcgcctggccggctcctacgtgaacttcctgatcgtcaacggcggca tcatcgcgccgagcttcgacgatcccaaggacgccgaggccagggcgatactccagcgcgtgttccccgagcacgaggtggtgatggtcccgggg cgcgagatcctcctcggcggcggcaacatccattgcatcacccagcagcaaccggcgccgcgcaaggcctga SEQUENCE ID NO. 38

[0177] MSNPTSTPRADGFRMPAEWEPHEQTWMVWPERPDNWRNGGKPAQAAFAAVAKAIARFEPVTVCASAGQYEN ARARLDDGNIRVVEISSDDAWVRDTGPTFVIDDKGDVRGVDWGFNAWGGFEGGLYFPWQRDDQVARKILEIERR ARYRTDDFVLEGGSIHVDGEGTLITTEECLLNHNRNPHLSQAEIERTLRDYLAVESIIWLPNGLYNDETDGHVDNFCC YARPGEVLLAWTDDQDDPNYLRCQAALRVLEESRDAKGRKLVVHKMPIPGPLYATQEECDGVDIVEGSQPRDPSIRL AGSYVNFLIVNGGIIAPSFDDPKDAEARAILQRVFPEHEVVMVPGREILLGGGNIHCITQQQPAPRKA

[0178] 8. Glutamine synthetase (GlnA)

[0179] The term GlnA encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.39.

[0180] The nucleic acid sequence of GlnA is provided in SEQUENCE ID NO. 8. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0181] >PA5119 |glnA

[0182] SEQUENCE ID NO. 8 atgtcgtacaagtcgcaccaactgatcaaagaccatgacgtgaagtgggtagacctgcgcttcaccgataccaagggcaagcagcaacacgtca ccatgccggctcgcgacgcgctggacgatgagttcttcgaagccggcaagatgttcgacggctcctccatcgccggctggaaaggcatcgaagcc tccgacatgatcctgatgccggacgacagcaccgccgtcctcgatccgttcaccgaagagccgaccctgatcctggtctgcgacatcatcgagccg agcaccatgcaaggctacgagcgcgacccgcgcaacatcgccaagcgcgccgaggagtacctgaagtccaccggcatcggcgacaccgtgttcg tcggcccggagccggagttcttcatcttcgacgaagtgaagttcaagtccgacatctccggctcgatgttcaagatcttctccgagcaggcttcctgg aacaccgacgccgacatcgagtccggcaacaagggccatcgcccgggcgtgaagggcggctacttcccggtaccgccggtcgaccacgaccat gaaatccgcaccgccatgtgcaacgccctggaagaaatgggcctggtggtcgaagtccaccaccacgaagtggcaaccgccggccagaacgag atcggcgtgaagttcaacaccctggtcgccaaggccgacgaagtgcagaccctgaagtactgcgtgcacaacgtcgccgacgcctacggcaaga ccgtgaccttcatgccgaagccgctgtacggcgacaacggttcgggcatgcacgtgcacatgtcgatctccaaggacggcaagaacaccttcgcc ggcgaaggctatgccggcctgtccgagaccgccctgtacttcatcggcggcatcatcaagcacggcaaggccctgaacggcttcaccaacccctc gaccaactcctacaagcgcctggtcccgggcttcgaagctccggtgatgctggcctactcggcgcgcaaccgttccgcctcgatccgtatcccctac gtatccagcccgaaggcccgccgtatcgaagcgcgcttcccggacccggcagccaacccctacctggccttcgccgcgctgctgatggccggcct ggacggcatccagaacaagatccaccccggcgatgccgccgacaagaacctgtacgacctgccgccggaagaggcgaaggaaatcccgcagg tttgcggcagcctgaaagaggcgctggaagaactcgacaagggccgcgcgttcctgaccaagggcggcgtgttcaccgacgagttcatcgatgcc tacatcgagctgaagagcgaagaagagatcaaggtgcgcaccttcgtgcacccgctggaatacgacctgtactacagcgtctga

[0183] SEQUENCE ID NO. 39

[0184] MSYKSHQLIKDHDVKWVDLRFTDTKGKQQHVTMPARDALDDEFFEAGKM FDGSSIAGWKGIEASDMILMPDDS TAVLDPFTEEPTLILVCDIIEPSTMQGYERDPRNIAKRAEEYLKSTGIGDTVFVGPEPEFFIFDEVKFKSDISGSMFKIFSE QASWNTDADIESGNKGHRPGVKGGYFPVPPVDHDHEIRTAMCNALEEMGLVVEVHHHEVATAGQNEIGVKFNTL VAKADEVQTLKYCVHNVADAYGKTVTFMPKPLYGDNGSGMHVHMSISKDGKNTFAGEGYAGLSETALYFIGGIIKH GKALNGFTNPSTNSYKRLVPGFEAPVMLAYSARNRSASIRIPYVSSPKARRIEARFPDPAANPYLAFAALLMAGLDGI QNKIHPGDAADKNLYDLPPEEAKEIPQVCGSLKEALEELDKGRAFLTKGGVFTDEFIDAYIELKSEEEIKVRTFVHPLEY DLYYSV 9. Outer membrane efflux protein (OpmH) ( Channel protein TolC);

[0185] The term OpmH encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.40.

[0186] The nucleic acid sequence of OpmH is provided in SEQUENCE ID NO. 9. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0187] >PA4974

[0188] SEQUENCE ID NO. 9 atgctgcgcagactctccctggcggccgccgtggccgctgctaccggtgtcgcctgggccgcccagccgaccccgctgccgaccaagaccgacct gatcagcgtctacaaggaagccgtcgacaacaacgccgacctcgccgccgcccaggccgactacctggcgcgcaaggaagtggtgccccaggc ccgcgccggcctgctgccgcaactcggcgccggcgcccgcgtcggagacacccggatcgcattcgacgagcgtccggcgaccgtcaagcgcaac agccaggtcgtccaggccaccctcagccagccgttgttccgcgccgaccgctggttccagtggcaggccgccaaggaaaccagcgaccaggccc ggctggaattctccgcgacccagcaggacctgatcctgcgcagcgccgagacctacttcacggtgctccgcgcccaggacaacctggccaccagc aaggccgaggaagccgccttcaagcgccagctggaccaggccaacgagcgcttcgacgtgggcctttccgacaagaccgacgtgctcgaggccc aggccagctacgacaccgcccgcgccaaccggttgatcgccgaacagcgcgtggacgatgccttccaggccctggtgaccctgaccaaccgcga ctacagcgccatcgagggcatgcgccacaccctgccggtggtgccgccggcgccgaacgacgccaaggcctgggtcgacaccgcggtgcagca gaacctgcgcctgctggccagcaactacgcggtcaacgccgccgaggaaaccctccgccagcgcaaggccgggcacctgccgaccctcgatgcc gtggcccagtaccagaagggcgacaacgacgccctcggcttcgccaacagcgccgccaatccgctggtgcactatggcaagtatgtcgacgagc gcagcattggcctggaactgaacatcccgatctacagcggcggcctgacctcctcccaggtccgcgagtcctaccagcgcctcaaccagagcgag caatcccgcgaaggccagcgccgccaggtggtgcaggatacccgcaacctgcaccgcgcggtgaataccgacgtcgagcaggtccaggcgcgg cgccaggcgatcatctccaaccagagttcgctggaagccaccgagatcggctaccaggtcggcacccgcaacatcgtcgacgtgctcaacgccca gcgccagctgtacgccgccgtgcgcgactacaacaacagccgctacgactacatcctcgataccctgcgcctgaagcaggcggccggcaccctc agcccggccgacctggaggcgctcagcgcctacctgaagcaggactacgatccggacaaggacttcctcccgccggacctggccaaggccgccg ccgagcagttacagagcaagccgcgccagcagtactag

[0189] SEQUENCE ID NO. 40

[0190] MLRRLSLAAAVAAATGVAWAAQPTPLPTKTDLISVYKEAVDNNADLAAAQADYLARKEVVPQARAGLLPQLGAGA RVGDTRIAFDERPATVKRNSQVVQATLSQPLFRADRWFQWQAAKETSDQARLEFSATQQDLILRSAETYFTVLRAQ DNLATSKAEEAAFKRQLDQANERFDVGLSDKTDVLEAQASYDTARANRLIAEQRVDDAFQALVTLTNRDYSAIEGM RHTLPVVPPAPNDAKAWVDTAVQQNLRLLASNYAVNAAEETLRQRKAGHLPTLDAVAQYQKGDNDALGFANSAA NPLVHYGKYVDERSIGLELNIPIYSGGLTSSQVRESYQRLNQSEQSREGQRRQVVQDTRNLHRAVNTDVEQVQARR QAIISNQSSLEATEIGYQVGTRNIVDVLNAQRQLYAAVRDYNNSRYDYILDTLRLKQAAGTLSPADLEALSAYLKQDYD PDKDFLPPDLAKAAAEQLQSKPRQQY

[0191] 10. ATP-dependent protease ATPase subunit (HsIU)

[0192] The term HsIU encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.41. The nucleic acid sequence of Hslll is provided in SEQUENCE ID NO. 10. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0193] >PA5054 |hslU

[0194] SEQUENCE ID NO. 10 atgtccatgacgccccgcgagatcgtccacgaactcaaccgccacatcatcggccaggacgacgccaagcgcgccgtcgccatcgccctgcgca accgctggcgacgcatgcaactgccggccgagctgcgcgccgaggtaacaccgaagaacatcctcatgatcggccccaccggcgtcggcaaga ccgagatcgcccgccgcctggcgcgcctggcgaacgcgccgttcatcaaggtcgaggcgaccaagttcaccgaggtcggctatgtcggacgcgac gtcgaatcgatcatccgcgatctcgccgacgccgcggtgaagatgctccgcgaacaggagatccagaaggtcaagtatcgcgccgaggacgccg ccgaagagcgcatcctcgatgccctgctgccggccgcgcgtcccgccatgggcttcggcgacgagccggcacgcgaggacagcaacacccgcca gctgttccgcaagcgcctgcgcgaaggccagctggacgacaaggaaatcgacatcgaggtggccgacaacccggccggcgtggagatcatggc cccgcccggcatggaggagatgaccaaccagttgcagaacctgttctccggcatgagcaagggcaagaagaagacccgcaagctgaaggtcgc cgaggccctgaagctgatccgcgacgaagaggcggtgcgcctggtcaacgaggaagagctcaaggcacgcgccctggaggcggtcgagcagc acggcatcgtcttcatcgacgagatcgacaagatcgccaagcgcgccaacgccggcggcgccgacgtctcccgcgagggcgtacagcgcgacct gctgccgctgatcgagggctgcacggtgaacaccaagctgggcatggtcaagaccgaccacatcctgttcatcgcctccggcgccttccacctgag caagccgagcgacctggttcccgagctgcagggccgcctgccgatccgcgtggagctcaaggccctcagtccgaacgatttcgagcgcatcctca ccgagccgcatgcctcgctcaccgagcagtaccgcgagctgctgaagaccgaggggctggccatcgagttcgccgaggacggcatcaagcgcct tgccgagatcgcctggcaggtcaacgagaagaccgagaacatcggtgcccgccgcctgcatacgctgctcgagcggctgctggaagaggtctcg ttcagcgccgccgacctggccagcgagcatagcgacaagccgatcctgatcgatgccggctacgtcaacagccacctcggcgagctggccgagg acgaggacctgtcccgctacatcctttga

[0195] SEQUENCE ID NO. 41

[0196] MSMTPREIVHELNRHIIGQDDAKRAVAIALRNRWRRMQLPAELRAEVTPKNILMIGPTGVGKTEIARRLARLANAP FIKVEATKFTEVGYVGRDVESIIRDLADAAVKMLREQEIQKVKYRAEDAAEERILDALLPAARPAMGFGDEPAREDSN TRQLFRKRLREGQLDDKEIDIEVADNPAGVEIMAPPGMEEMTNQLQNLFSGMSKGKKKTRKLKVAEALKLIRDEEA VRLVNEEELKARALEAVEQHGIVFIDEIDKIAKRANAGGADVSREGVQRDLLPLIEGCTVNTKLGMVKTDHILFIASG AFHLSKPSDLVPELQGRLPIRVELKALSPNDFERILTEPHASLTEQYRELLKTEGLAIEFAEDGIKRLAEIAWQVNEKTEN IGARRLHTLLERLLEEVSFSAADLASEHSDKPILIDAGYVNSHLGELAEDEDLSRYIL

[0197] 11. Bifunctional protein (GlmU)

[0198] The term GlmU encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.42.

[0199] The nucleic acid sequence of GlmU is provided in SEQUENCE ID NO. 11. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0200] >PA5552 |glmU

[0201] SEQUENCE ID NO. 11 atgtcccttgaaatcgtgattctcgccgccggccagggcacccgcatgcgttcggccctgcccaaggtcctccatcccattgccggcaagccgatgc tcggccacgtcatcgactgcgcgcgccagttgcagccggaacggatccacgtggtgatcggccacggcgcggatctggtgcgtgagcggatggcc gccgacgacctgaatttcgtgctccaggccgaacagctcggtacgggccatgcggtggcccaggccctgccgttcctcagtgcagaccaggtgctg atcctctacggcgacgtgccgctgatccagctggacaccttgcagcgcctgctggcgcaagtcaccccggaccagttgtcgttgctcaccgtcgaca tgctcgaccccaccggctacggccgcatcgtccgtgacgaccagggcgcggtgcaggccatcgtcgagcacaaggacgcgaccccggcgcagc gccagatcggcgagatcaacaccggcatcctcgcggtgccgggcaagcgccttgccgactggctcggccgcctgtccaacgacaacgcccaggg cgagtactacctgaccgacgtgatcgccatggcggtcggcgacggcctggtggtggccagcgcccaaccgctggacgcgatggaagtgcagggc gtcaatgaccgcatgcaacaggcccagctggagcgtcactaccagcgcctgcgcgcagaggagctgatgcgccagggcgtgaccctgctcgatc cgcagcgcctggatgtccgtggcgaaatctcggtcggccgcgacgtgctgatcgacgtcaatgtggtgctggaaggccgcgtggtgatcgaggac gacgtgcgcatcggaccgaactgcgtgattcgcgacagcgtcctgcggcgtggcgcggtgatcaaggccaacagccacctggaaggcgcggagc tgggcgagggtagcgatgccggtccgttcgcccgcctgcgtccgggcagtgtgctcggcgctcgggcccatgtcggcaacttcgtcgaactgaaga atgcccgcctgggcgaggggagcaaggccggccacctgagctacctgggcgatgccgagctgggcgccaactgcaacatcggcgccggcacca tcacctgcaactacgatggcgcgaacaagttccgcaccgagctgggcgatgatgtgttcatcggctcgaacaactcgctggtggcgccgctgaag atcggcgatggcgcgacgaccgcggccggctccaccatcacccatgaggttccggcgaagaacctggccttcggccgcgcccggcagaagaacc tggaaaactggaagcgcccggaaaaaatcaagaagtaa

[0202] >glucosamine-1 -phosphate acetyltransferase / N-acetylglucosamine-1 -phosphate uridyltransferase

[0203] SEQUENCE ID NO. 42

[0204] MSLEIVILAAGQGTRM RSALPKVLHPIAGKPMLGHVIDCARQLQPERIHVVIGHGADLVRERMAADDLNFVLQAE QLGTGHAVAQALPFLSADQVLILYGDVPLIQLDTLQRLLAQVTPDQLSLLTVDMLDPTGYGRIVRDDQGAVQAIVEH KDATPAQRQIGEINTGILAVPGKRLADWLGRLSNDNAQGEYYLTDVIAMAVGDGLVVASAQPLDAMEVQGVNDR MQQAQLERHYQRLRAEELM RQGVTLLDPQRLDVRGEISVGRDVLIDVNVVLEGRVVIEDDVRIGPNCVIRDSVLRR GAVIKANSHLEGAELGEGSDAGPFARLRPGSVLGARAHVGNFVELKNARLGEGSKAGHLSYLGDAELGANCNIGAG TITCNYDGANKFRTELGDDVFIGSNNSLVAPLKIGDGATTAAGSTITHEVPAKNLAFGRARQKNLENWKRPEKIKK

[0205] 12. Fumarate hydratase class II 2 (FumC2)

[0206] The term FumC2 encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO. 43.

[0207] The nucleic acid sequence of FumC2 is provided in SEQUENCE ID NO. 12. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0208] >PA4470 |fumC1

[0209] SEQUENCE ID NO. 12 atgactgacacccgcatcgaacgcgacagcatgggcgaactggcggtcccggcgaccgccctctatggcgcccagacccagcgcgcggtgaac aacttcccggtcagcggccaacgcatgccccaggccttcgtccgcgccctgctgctggccaaggccgcggcggcgcgggcgaacgtctccctgca gcaactggatgcaccgatgggcgaggcgatcgccgacacctgcctgcaactgctgcaggaggacttcatgcagcatttcccggtggatgtgttcca gaccggctccggcaccagttccaacatgaacgccaacgaagtggtggcgaccctggccagtcgtcgcctgggcggcaaggtcaatccgaacgac catgtgaactgcgggcagagcagcaacgacatcattccctcgaccatccacatcagtgccgccctcgagatcagcgaacgcctgctgccggcgct gcgccacctggagcagactatccagagcaaggccggcgaagtgcatgcctatgtgaagaccggccgcacccacctgatggatgcgatgccggta cgcatgagccaggtgctcggtggctgggcgcagcaggtgcgacaggccggcgtgcacatcgagagcgtacttcctgccttgcagcagttggcaca gggcggcaccgcggtgggcaccgggatcaacgcccatccgcggttcgccgaacgtttcagccaggagctgaacgacctgaccgggctggcgttc cgccccggcgacgacttcttcgccctgatcggttcgcaggacaccgcggtcgctgcgtccggccagttgaagaccctggccgtgaccctgatgaag ctggccaacgacctgcgctggatgaactccgggccgcttgccgggctcggcgagatcgagctggaagcgctgcaaccaggctcctcgatcatgcc cggcaaggtcaacccggtgatcccggaggccaccgcgatggtcgccgcccaggtgatcggcaatgacgccgccattgccgtcgctggccaatcg ggcaacttcgaactgaacgtgatgctgccgctggtggcggacaacctgttgcacagcatccagttgctggccaacgtcagccggctgctggcgga caaggccatcgccagcttcaaggtcaaccagggcaagctcagcgaggcgttggcgcgcaacccgatcctggtcactgcgctgaacccgatcatcg gctaccagaaggctgcggaaatcgccaagcaggcctatcgcgagggacggccgatcatcgatgtggcgctggaaaacaccgatctggaccgtgc gcgcctggaggtgttgctggatccggaaaaactcacggctggcggcctctga

[0210] SEQUENCE ID NO. 43

[0211] MTDTRIERDSMGELAVPATALYGAQTQRAVNNFPVSGQRMPQAFVRALLLAKAAAARANVSLQQLDAPMGEAIA DTCLQLLQEDFMQHFPVDVFQTGSGTSSNMNANEVVATLASRRLGGKVNPNDHVNCGQSSNDIIPSTIHISAALEI SERLLPALRHLEQTIQSKAGEVHAYVKTGRTHLMDAMPVRMSQVLGGWAQQVRQAGVHIESVLPALQQLAQGGT AVGTGINAHPRFAERFSQELNDLTGLAFRPGDDFFALIGSQDTAVAASGQLKTLAVTLMKLANDLRWMNSGPLAGL GEIELEALQPGSSIMPGKVNPVIPEATAMVAAQVIGNDAAIAVAGQSGNFELNVMLPLVADNLLHSIQLLANVSRLL ADKAIASFKVNQGKLSEALARNPILVTALNPIIGYQKAAEIAKQAYREGRPIIDVALENTDLDRARLEVLLDPEKLTAGG L

[0212] 13. Pyochelin biosynthetic protein (PchG)

[0213] The term PchG encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.46.

[0214] The nucleic acid sequence of PchG is provided in SEQUENCE ID NO. 13. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0215] >PA4224 |pchG

[0216] SEQUENCE ID NO. 13 atgagcgacgtccgttccgtggtggtcgccggttcccggttcggccagttctatgccgccggcgtcgccgccgatccgcgcttcgtcctgcgcggcat ccttggccagggcagccggcgttcgcgggcgttggccgaacgcctcggggtggagacctggtgcgaggtcgaggcgttgcccgacgatgtccgcc tggcctgcgtcgcggtcggcggcgcggcacgcggcgagcagggcccggcgctggccgaggcgctgatggcgcgtggcatcgacgtgctgatcga gcatccgttgctgccgcgcgaatggcaggacctgctgcgcagcgccgagcgcctgggccggcgctgcctgctcaacaccttctatccgcagttgcc ggcggtggcgcgtttcatcgagctgggccgccagttgcatcgccggcgcggcatccgccacctggacgcggcctgcggggtgcaggtcggtttcg ccaccctggacatcctcgccgcgttgctggagggcgttggcccctggtcgctggagtcgccctcgaacgacctgtcggcgatgcgcgggctgtccc tggtgttggcggaagtgccattgagcctgctcgtgctcaacgaactggcggccgccgacgacgggcgcatgaccttgttgcagcgcgtcagcctga ccaccgatcgcggcacgctgagcctgctcgcccccatggcccgttgttgtggacgcctgcggtggcggtaccggcagaggatgacgacggcctgtt cgcgctgttcgacgagatagccggcgaaccgctgcccagcgcccagctctggtatgccgaaccgtgcagctgggcccaggtgcaccagcgcctgt ggccggcggcggcggccgaggcgctggcgctgctcgccgacggcgacgaggtgcgccggcgcaaccagcgcagcctggaggtcgccgccctgt ggcagcggatcggcgagcgccttggcttccccgaggcgccgccggcgtcgctggcgccggcgagcctggaacaggtgctggagcaagcctcgtg a

[0217] SEQUENCE ID NO. 44

[0218] MSDVRSVVVAGSRFGQFYAAGVAADPRFVLRGILGQGSRRSRALAERLGVETWCEVEALPDDVRLACVAVGGAAR GEQGPALAEALMARGIDVLIEHPLLPREWQDLLRSAERLGRRCLLNTFYPQLPAVARFIELGRQLHRRRGIRHLDAAC GVQVGFATLDILAALLEGVGPWSLESPSNDLSAMRGLSLVLAEVPLSLLVLNELAAADDGRMTLLQRVSLTTDRGTLS LLSPHGPLLWTPAVAVPAEDDDGLFALFDEIAGEPLPSAQLWYAEPCSWAQVHQRLWPAAAAEALALLADGDEVRR RNQRSLEVAALWQRIGERLGFPEAPPASLAPASLEQVLEQAS 14. Dihydrolipoyl dehydrogenase (LpdG)

[0219] The term LpdG encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.45.

[0220] The nucleic acid sequence of LpdG is provided in SEQUENCE ID NO. 14. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0221] >PA1587 |lpd

[0222] SEQUENCE ID NO. 14 atgagccagaaattcgacgtggtagtgattggtgccggccccggcggttacgttgccgccatccgcgctgcccagctgggcctgaagaccgcctgc atcgagaagtacatcggcaaggaaggcaaggtggcgctcggcggtacctgcctgaacgttggctgcatcccgtccaaggcgctgctggacagct cctacaagtaccatgaggccaaagaggccttcaaagtccatggtatcgaagccaagggcgtcaccatcgacgttccggcgatggttgcgcgcaag gccaacatcgtcaagaacctgaccggcggcatcgctaccctgttcaaggccaacggcgtgacctccttcgaaggccacggcaagctgctggccaa caagcaggtggaagtgaccggcctggacggcaagacccaggtcctggaagccgagaacgtgatcatcgcttccggctcccgcccggtggagatc ccgccggccccgctgaccgacgacatcatcgtcgactccaccggcgccctggaattccaggccgtgccgaagaagctgggtgtgatcggcgccgg cgtcatcggcctggagctgggttcggtatgggcccgcctgggtgccgaggtgaccgtgctggaagccctggacaagttcctcccggctgccgacg agcagatcgccaaggaagccctgaaggtcctgaccaagcaaggcctgaacattcgcctgggcgcccgcgtgaccgcttcggaagtgaagaaga agcaggtcaccgtgaccttcaccgatgccaacggcgagcagaaggaaaccttcgacaagctgatcgtcgcggtcggccgtcgcccggtgaccac cgacctgctggccgccgacagcggcgtgaccctggacgagcgtggcttcatctacgtcgacgaccactgcaagaccagcgttccgggcgtcttcgc catcggtgacgtggttcgcggcgcgatgctcgcgcacaaggcctcggaagagggcgtgatggtcgccgagcgcatcgccggccacaaggcgcag atgaactacgacctgattccgtcggtcatctacacccacccggaaatcgcctgggtcggcaagaccgagcagaccctcaaggccgagggcgtcg aagtcaacgtcggcaccttcccgttcgccgccagcggtcgcgcgatggctgccaatgacaccaccggcctggtcaaggtcatcgccgatgcgaag accgaccgcgtcctgggcgtccacgtgatcggcccgagcgccgccgagctggtacagcaaggcgcgatcggcatggaattcggcaccagcgccg aagacctgggcatgatggtcttctctcacccgacgctgtccgaagccctgcacgaagctgccctggcagtgaatggccatgccatccacatcgcca accgcaagaagcgctga

[0223] SEQUENCE ID NO. 45

[0224] MSQKFDVVVIGAGPGGYVAAIRAAQLGLKTACIEKYIGKEGKVALGGTCLNVGCIPSKALLDSSYKYHEAKEAFKVHG IEAKGVTIDVPAMVARKANIVKNLTGGIATLFKANGVTSFEGHGKLLANKQVEVTGLDGKTQVLEAENVIIASGSRPV EIPPAPLTDDIIVDSTGALEFQAVPKKLGVIGAGVIGLELGSVWARLGAEVTVLEALDKFLPAADEQIAKEALKVLTKQG LNIRLGARVTASEVKKKQVTVTFTDANGEQKETFDKLIVAVGRRPVTTDLLAADSGVTLDERGFIYVDDHCKTSVPGV FAIGDVVRGAMLAHKASEEGVMVAERIAGHKAQMNYDLIPSVIYTHPEIAWVGKTEQTLKAEGVEVNVGTFPFAA SGRAMAANDTTGLVKVIADAKTDRVLGVHVIGPSAAELVQQGAIGMEFGTSAEDLGMMVFSHPTLSEALHEAALA VNGHAIHIANRKKR

[0225] 15. lnosine-5-monophosphate dehydrogenase (GuaB)

[0226] The term GuaB encompasses this protein and effective variants., e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.46.

[0227] The nucleic acid sequence of GuaB is provided in SEQUENCE ID NO. 15. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence. >PA3770 |guaB

[0228] SEQUENCE ID NO. 15 atgctgcgaatcagtcaagaagccctgactttcgacgacgtccttctgatccccggttattccgaagtcctgcccaaggacgtgagtttgaaaactc gcctgacccgcggcatcgaactgaacatcccgctggtatccgccgcgatggataccgtgaccgaagcccgcctggccatcgccatggcccaggaa ggcggcatcggcatcatccacaagaacatgggcatcgagcagcaggccgcggaagtccgcaaggtcaagaagcacgaaacggccatcgtccgc gacccggtcaccgtgaccccctcgaccaagatcatcgaactgctgcagatggcccgcgagtacggcttctccggtttcccggtggtggagcagggc gagctggtcggtatcgtcaccggtcgcgacctgcgggtgaagccgaacgccggcgataccgtcgccgcgatcatgaccccgaaggacaagctgg tcaccgcccgcgaaggcaccccgctggaagagatgaaggccaagctctacgagaaccgcatcgagaagatgctggtggtcgacgagaacttcta cctgcgtggcctggtgaccttccgtgacatcgagaaggccaagacctacccgctggcgtccaaggacgagcagggccgcctgcgcgtcggcgcc gccgtcggcaccggcgccgataccggcgagcgcgttgccgcgctggtcgccgccggggtcgacgtggtggtggtggacaccgcccacggccact ccaagggcgtgatcgagcgtgtgcgctgggtcaagcagaccttcccggacgtccaggtgatcggcggcaacatcgccaccgccgaggccgccaa ggccctcgccgaggctggcgccgacgcggtcaaggtcggcatcggcccgggctcgatctgcaccacccgcatcgtcgccggtgtcggcgtgccgc agatctccgccatcgccaacgttgccgccgccctcgaaggcaccggcgtaccgctgatcgccgacggcggcatccgcttctccggcgacctggcc aaggccatggtcgccggcgcctactgtgtgatgatgggctcgatgttcgccggtaccgaggaagcgccgggcgagatcgagctgttccagggccg ttcctacaagtcctaccgcggcatgggttcgctgggcgccatgtccggctcccagggctcgtccgaccgctacttccaggacgcctccgccggcgcc gagaagctggtgccggaaggcatcgagggtcgcgtgccgtacaagggtgccctgtccgccatcgtccaccagctgatgggcggcctgcgcgccg ccatgggctacaccggcagcgccgacatccagcagatgcgcacgcagccgcagttcgtccggatcaccggcgcgggcatggccgagtcccatgt ccacgacgtccagatcaccaaggaagcccccaactaccgggttggttga

[0229] SEQUENCE ID NO. 46

[0230] MLRISQEALTFDDVLLIPGYSEVLPKDVSLKTRLTRGIELNIPLVSAAMDTVTEARLAIAMAQEGGIGIIHKNMGIEQQ AAEVRKVKKHETAIVRDPVTVTPSTKIIELLQMAREYGFSGFPVVEQGELVGIVTGRDLRVKPNAGDTVAAIMTPKD KLVTAREGTPLEEMKAKLYENRIEKMLVVDENFYLRGLVTFRDIEKAKTYPLASKDEQGRLRVGAAVGTGADTGERVA ALVAAGVDVWVDTAHGHSKGVIERVRWVKQ.TFPDVQ.VIGGNIATAEAAKALAEAGADAVKVGIGPGSICTTRIVA GVGVPQISAIANVAAALEGTGVPLIADGGIRFSGDLAKAMVAGAYCVMMGSMFAGTEEAPGEIELFQGRSYKSYRG MGSLGAMSGSQGSSDRYFQDASAGAEKLVPEGIEGRVPYKGALSAIVHQLMGGLRAAMGYTGSADIQQMRTQP QFVRITGAGMAESHVHDVQITKEAPNYRVG

[0231] 16. Methylmalonate-semialdehyde dehydrogenase [acylating] (MmsA)

[0232] The term MmsA encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.47.

[0233] The nucleic acid sequence of MmsA is provided in SEQUENCE ID NO. 16. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0234] >PA3570 |mmsA

[0235] SEQUENCE ID NO. 16 atgtcagtcccagtccgccacctcatcgcgggagccttcgtcgaaggtctcggcgcccagcgcatcccggtcagcaatcccctcgacaacagcacc ctggcggagatcgcctgcgccagcgcggagcaggtcgagcaggcggtggccagtgcccgcgaaaccttcgccagctggaaggaaaccccggttt ccgagcgcgcgcgggtgatgctgcgttaccaggcgttgctcaaggaacaccacgacgaactggcgaagatcgtctccagcgagctgggcaagac tttcgaggacgccaagggcgacgtctggcgcggtatcgaggtggtcgaacacgcctgcaacgtgccctcgctgctgatgggcgagacggtggag aacgtcgcccgcaacatcgacacctacagcatcacccagccgctgggcgtgtgcgttggcatcaccccgttcaacttcccggcgatgatcccgctg tggatgttcccgctggccatcgcctgcggcaacgccttcatcctcaagccttccgagcaggtgccgctgaccagcgtgcggctcgccgagctgttcc tcgaggccggtgcgccgaagggcgtgctgcaggtggtgcatggcggcaaggaacaggtcgaccagttgctcaagcacccgcaggtgaaggcgg tgtccttcgtcggctcggtcgccgtcggccagtacgtctaccacaccggcaccgcgcacaataagcgcgtgcagagcttcgccggggcgaagaac cacatggtgatcatgcccgacgcggacaaggcacaggtcatcagcaacctggtcggtgcctcggtgggcgcggccgggcagcgctgcatggcga tctcggtggcggtgctggtgggggccgcccgcgagtggattccggaaatccgcgacgcgctggccaaggtccgccccggcccatgggacgacag cggcgccagctacggcccggtgatcaacccgcaggccaaggcgcgcatcgagcgcctgatcggccagggcgtggaggagggcgcgcaactgct gctggatggccgcggctacaaggtcgagggatacccggacggcaactgggtcggcccgacgctgttcgccggggtgcgcccggacatggcgatc taccgcgaagaggtgttcggcccggtgctctgcctggccgaggtcgacagcctggagcaggcgatccgcctgatcaacgaaagcccctacggca acggcacctcgatcttcaccagctccggcgccgcggcgcggaccttccagcaccacatcgaggtcggccaggtgggcatcaacatcccgattccg gtaccgctgccgttcttctccttcaccggttggaagggctcgttctacggcgacctgcacgcctacggcaagcagggcgtgcgcttctacaccgaga ccaagaccgtgaccgcgcgctggttcgacagcgacagcgtggccgggaccaacttctcgatccagatgcgctga

[0236] SEQUENCE ID NO. 47

[0237] MSVPVRHLIAGAFVEGLGAQRIPVSNPLDNSTLAEIACASAEQVEQAVASARETFASWKETPVSERARVMLRYQALL KEHHDELAKIVSSELGKTFEDAKGDVWRGIEVVEHACNVPSLLMGETVENVARNIDTYSITQPLGVCVGITPFNFPA

[0238] MIPLWMFPLAIACGNAFILKPSEQVPLTSVRLAELFLEAGAPKGVLQWHGGKEQVDQLLKHPQVKAVSFVGSVAV

[0239] GQYVYHTGTAHNKRVQSFAGAKNHMVIMPDADKAQVISNLVGASVGAAGQRCMAISVAVLVGAAREWIPEIRDA LAKVRPGPWDDSGASYGPVINPQAKARIERLIGQGVEEGAQLLLDGRGYKVEGYPDGNWVGPTLFAGVRPDMAIY REEVFGPVLCLAEVDSLEQAIRLINESPYGNGTSIFTSSGAAARTFQHHIEVGQ.VGINIPIPVPLPFFSFTGWKGSFYGD LHAYGKQGVRFYTETKTVTARWFDSDSVAGTNFSIQMR

[0240] 17. Cytochrome c-type biogenesis protein (CycH)

[0241] The term CycH encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.48.

[0242] The nucleic acid sequence of CycH is provided in SEQUENCE ID NO. 17. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0243] > PA 1483 |cycH

[0244] SEQUENCE ID NO. 17 atgatcgatttctggctcgctgccggcctgctgctgctggcggccctggcattcctgctgattccgctgctgcgtggccgtcgcgcccaggccgagg aagaccgtaccgccctgaacgtcgcgctttatcaggagcggatcgccgagctggccggccagcaggccgccggcacgctgaccgccgagcaact ggagaatggccgcgccgaggctgcccgcgaactgctcgccgacaccgagggcagcggcgaagagcgtagctcgcgcctgggccgggcggtccc gctggtcgcggcgctgctggtgccgttggtcgcgctcggcctgtacctgcattggggcgccagcgacaaggtcgagctggcccgcgaattcgccca ggcgccgcactccatggaagagatgactgcgcgcctggagcgcgcggtacaggctcagccggattccgcccagggctggtacttcctcggtcgca cctacatgacccaggaacgcggcgccgatgcagcgcgggccttcgaacgcgccgccgaactcagtgggcggcagccggaagtgctcggccagt gggcgcaggcactgtatttctccggtggcaagaaaatgaccgcgcagatcaaggccctggccgacgaggcgctgaagggcgacccggccgaag tgaccacccttggcctgctcggcatcgccgccttcgaagagcagcgctacgccgacgccatcggcttctgggagcgcctggtgtcggtgttgccga acgaggacccggcacgctcggcgatccagggcggcatccagcgcgcccgcgagcgcatgaccgaggccgggcagactccgccggcgccggcc gcgcccgctgccgccggggtgaccctgacggtgaaggtggatatcagcgacgcggtgaagggccaggtcaaggccgacgacagcgtgttcgtct tcgcccgcgccgtcggcggcccgccgatgccgttggcagtgaagcgcctgaccgtcgccgacctgccggccgaggtcagcctgagcgatgccgac gccatgatgccgcagctcaagctcagcggtttcccgcaggtcgagctggtggcacgggtatcccgcgccggcaacgccatctccggcgagtggat cggacgcggcaaaccgttgtctactgcccagtcgtcggcgcaagcgctgaccatcgacggcccggaccagccccaggcggcccggccataa SEQUENCE ID NO.48

[0245] MIDFWLAAGLLLLAALAFLLIPLLRGRRAQAEEDRTALNVALYQERIAELAGQQAAGTLTAEQLENGRAEAARELLAD TEGSGEERSSRLGRAVPLVAALLVPLVALGLYLHWGASDKVELAREFAQAPHSMEEMTARLERAVQAQPDSAQGW YFLGRTYMTQERGADAARAFERAAELSGRQPEVLGQWAQALYFSGGKKMTAQIKALADEALKGDPAEVTTLGLLGI AAFEEQRYADAIGFWERLVSVLPNEDPARSAIQGGIQRARERMTEAGQTPPAPAAPAAAGVTLTVKVDISDAVKGQ VKADDSVFVFARAVGGPPMPLAVKRLTVADLPAEVSLSDADAMMPQLKLSGFPQVELVARVSRAGNAISGEWIGR GKPLSTAQSSAQALTIDGPDQPQAARP

[0246] 18. Probable zinc protease (peptidase M16)

[0247] The term peptidase M16 encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.49.

[0248] The nucleic acid sequence of peptidase M16 is provided in SEQUENCE ID NO. 18. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0249] >PA0372

[0250] SEQUENCE ID NO. 18 gtggcggcacgaaaggtccagatcaccatgaaaaccccagctcgccgccgcgtcggcctgctcctagccagcctttgtctcccgcttttcgcccag gcggcggaaacccagcccacccacgagttcagcctcgacaacggcctgaaggtcatagtgcgtgaagaccaccgcgccccggtggtggtttccc agctctggtacaggatcggctccagctacgagacacccgggctcaccggcctgtcccacgccctcgaacacatgatgttcaagggcagccgcaag ctcggcccgggcgaagcctcgcgggtgctgcgcgacctgggtgcggaagagaacgccttcaccaccgacgactacaccgcctattaccaggtcct ggcccgcgaccgcctgccggtagcgctggagatggaagccgaccgcatggcccacctgagcctgccggtcgaccagttcaagagcgagatcgag gtgatcaaggaggagcgccgcctgcgcaccgacgacaatcccaacgccctcgccttcgagcgcttcaaggccgccgcctacccggccagcggct accacaccccgaccatcggctggatggccgacctgcagcgcatgaccatcgacgacctgcgccactggtacgaatcctggtacgcgccgaacaa cgccaccctggtagtggtcggcgacgtcaccgccgacgaggtcaagaccctcgccaagcgctacttcggcgagatcccctggcgccagctgccgc cggcgcgcaagccgctggaactggccgagcccggcgagcgccggctcaagctgtacgtacgcacccagctgccgaacctgatcatgggcttcaa cgtgcccagcctgggcagcagcgagaaccctcgcgaagtcaacgccctgcgcctgatcggcgcgctgctcgatggcggctacagcgcccgcctg gcctcgcgcctggagcgcggcgaggagctggtcgccggcgcctccacctactatgacgcgttcaaccgcggcgacagcctgttcgtcctctcggcc acgccgaacgtgcagaagggcaagaccctcgagcaggtcgaggccggtctgtggaagcagctcgatgacctcaagcagaacccgcccagcgcc gcggagatcgagcgcgtgcgcgcgcagatgatcgccgggatggtctacgagaaggactccatcgccgcccaggccagcagcatcggccagttgg aaagcgtcggcctgtcctggaagctgatcgaccaggacctggaggccctcaaggcagtgactccggacgatatccagaaagccgcccgcaccta tttcaccccgtcgcgcctgaccctggcgcaagtgctgcctgtgaaggccgaggaaaaggaggcccgtcatgagtga

[0251] SEQUENCE ID NO.49

[0252] MAARKVQITMKTPARRRVGLLLASLCLPLFAQAAETQPTHEFSLDNGLKVIVREDHRAPVVVSQLWYRIGSSYETPG LTGLSHALEHMM FKGSRKLGPGEASRVLRDLGAEENAFTTDDYTAYYQVLARDRLPVALEMEADRMAHLSLPVDQ FKSEIEVIKEERRLRTDDNPNALAFERFKAAAYPASGYHTPTIGWMADLQRMTIDDLRHWYESWYAPNNATLVVVG DVTADEVKTLAKRYFGEIPWRQLPPARKPLELAEPGERRLKLYVRTQLPNLIMGFNVPSLGSSENPREVNALRLIGALL DGGYSARLASRLERGEELVAGASTYYDAFNRGDSLFVLSATPNVQKGKTLEQVEAGLWKQLDDLKQNPPSAAEIERV RAQMIAGMVYEKDSIAAQASSIGQLESVGLSWKLIDQDLEALKAVTPDDIQKAARTYFTPSRLTLAQVLPVKAEEKE ARHE 19. Outer membrane protein assembly factor B (BamB)

[0253] The term BamB encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.50.

[0254] The nucleic acid sequence of BamB is provided in SEQUENCE ID NO. 19. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0255] >PA3800 |bamB

[0256] SEQUENCE ID NO. 19

[0257] ATGGTGCAATGGAAACACGCGGCGCTGCTCGCCCTGGCCCTGGCGGTCGTGGGTTGCAGCAGCAACAGCAAG AAGGAACTCCCGCCCGCCGAACTGACCGACTTCAAAGAGGAAGTCGTGTTGAGCAAGCAGTGGAGCCGCTCG GTCGGTGATGGTCAGGGCGACCTGTACAACCTGCTCGAACCGGCCGTCGATGGTTCCACCATCTACGCCGCGTC CGCCGAAGGGCGGGTGATGGCGATCCAGCGCGAGACCGGCGACGTGCTCTGGAAGAAGGACCTGGAACGTCC GGTTTCCGGCGGTGTCGGCGTTGGCTACGGCCTGGTGCTGGTGGGTACCCTGCGCGGTGACGTGATCGCCCTC GACGAAGCCACCGGCAAGAAGAAGTGGACCAAGCGAGTCAACAGCGAAGTGCTGTCGGCGCCGGCCACCAAT GGCGACGTGGTGGTGGTGCAGACCCAGGACGACAAGCTGATCGGCCTCGATGCGGCCAGCGGCGACCAGCGC TGGATCTACGAAAGCACCGTGCCGGTGCTGACCCTGCGCGGCACCGGCGCGCCGCTGATTGCCGGCAACATGG CCCTGGCTGGCCTGGCCAGCGGCAAGGTAGTGGCGGTCGACGTACAGCGCGGCCTGCCGATCTGGGAGCAGC GGGTAGCGATTCCCCAGGGGCGTTCCGAACTGGATCGCGTGGTGGACATCGACGGCGGCCTCCTGCTGTCCGG CGACACCCTCTACGTGGTCAGCTACCAGGGCCGTGCCGCGGCGCTGGACGTGAACAGCGGCCGCCTGCTCTGG CAGCGCGAAGCGTCGAGCTACGTCGGCGTCGCCGAAGGCTTCGGCAATATCTACGTCAGCCAGGCCAGCGGTT CGGTGGAAGGCCTGGACTCGCGCGGCGCTTCTTCGCTGTGGAACAACGACGCCCTGGCGCGTCGCCAACTGTC GGCTCCGGCGGTGTTCTCCAGCAACGTGGTGGTCGGCGACCTGGAAGGCTACGTGCACCTGCTGAGCCAGGTG GACGGTCGCTTCGTCGGTCGCGAGCGGGTCGACAGCGATGGCGTGCGGGTTCGTCCGCTGGTGGTCGGGAGC TGGATGTACGTGTTCGGCAACGGTGGCAAGCTCGTCGCCTACACCATCCGCTAG

[0258] SEQUENCE ID NO. 50

[0259] MVQWKHAALLALALAVVGCSSNSKKELPPAELTDFKEEVVLSKQWSRSVGDGQGDLYNLLEPAVDGSTIYAASAEG RVMAIQRETGDVLWKKDLERPVSGGVGVGYGLVLVGTLRGDVIALDEATGKKKWTKRVNSEVLSAPATNGDVVVV

[0260] QTQDDKLIGLDAASGDQRWIYESTVPVLTLRGTGAPLIAGNMALAGLASGKWAVDVQRGLPIWEQRVAIPQGRSE LDRVVDIDGGLLLSGDTLYVVSYQGRAAALDVNSGRLLWQREASSYVGVAEGFGNIYVSQASGSVEGLDSRGASSL WNNDALARRQLSAPAVFSSNVVVGDLEGYVHLLSQVDGRFVGRERVDSDGVRVRPLVVGSWMYVFGNGGKLVA YTIR

[0261] 20. OprD family porin (OprQ)

[0262] The term OprQ encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.51.

[0263] The nucleic acid sequence of OprQ is provided in SEQUENCE ID NO. 20. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence. >PA2760 |oprQ

[0264] SEQUENCE ID NO. 20 atgttgaagaaaaggatttgcctgctggcactgggcatcgcgactgcaagccaggcgatggccaatgaccaagaagcagcaaaggggtttgtcg aggacagccacctcgatctgttcttccgcaatggctacatcagccgtgactacaaacatggccggcaggacaaggccgaatggggccaggccgc cactgccaccttcacctccggcttcacccaggggacggtgggcgtcggtgtcgacgcgttcggcttgtacgcggtacgcctggacggcggcaagg gtcgtagcggcgccgccggtatcgacttcttcaagcagggcgacagcggctccgcggcggacgatctgtccaagggcggcgcggcggtgaagttc cgcatctccaacaccgtgctcaagtacggcgaccagatgcccagcctgccggtgctcagctacgacaactcgcgcctgctcccggaaagctacag cggcaccctgatcacctccaaggagatcgagggcctggagctgaacgccggtcgcttcaccgcggaatcccgcaagagcgccgaagggcgcga cagcggcggcctgaagagcatcaacgtgttcggcggcaagtacgccttcaccgatcacttcaacgcctcgctgtacgcttccgacgtcgaggacgt actgaagaagcagtacatcaacctgaactacaccatcccgctgcaggccgaccagtcgctgaacttcgacttcaacggctatcgcaccaagctgg acagcgacttcgccgaccagaacttcaacggtaaccgcgacaacaagatctggagcctggcggcgtcctacaccatcgacgcccacaccttcatg atcgcccaccagcgcaacacgggcgacaccggctacaactacggctggtaccagaacgccggcggcatcggcgacggcggcaccaccatctgg ctggccaactcctactggtcggacttcaacgccgaggacgagcgttcctggcaggtcagctacgccctggacttcgccaagtacggcgtgcccggc ctgacctaccgcgtagcctacgtgcgcggcgacaacatcaagaccgccgagaccagcaacggcaaggagcgcgagatcttcaaccaggtccagt acgtggtgcagagcggtccggccaaggacctcaccctgcgtctgcgcagctcgttcctgcgtgtctccaacgacgctcgctcgtacaacgacgacg gcaacgaaatccgtgccttcgtcgagtacccgttcagcgtgttctga

[0265] SEQUENCE ID NO. 51

[0266] MLKKRICLLALGIATASQAMANDQEAAKGFVEDSHLDLFFRNGYISRDYKHGRQDKAEWGQAATATFTSGFTQGTV GVGVDAFGLYAVRLDGGKGRSGAAGIDFFKQGDSGSAADDLSKGGAAVKFRISNTVLKYGDQMPSLPVLSYDNSRL LPESYSGTLITSKEIEGLELNAGRFTAESRKSAEGRDSGGLKSINVFGGKYAFTDHFNASLYASDVEDVLKKQYINLNYTI PLQADQSLNFDFNGYRTKLDSDFADQNFNGNRDNKIWSLAASYTIDAHTFMIAHQRNTGDTGYNYGWYQNAGG IGDGGTTIWLANSYWSDFNAEDERSWQVSYALDFAKYGVPGLTYRVAYVRGDNIKTAETSNGKEREIFNQVQYVVQ SGPAKDLTLRLRSSFLRVSNDARSYNDDGNEIRAFVEYPFSVF

[0267] 21. Lipopolysaccharide export system protein A (LptH)

[0268] The term LptH encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.52.

[0269] The nucleic acid sequence of LptH is provided in SEQUENCE ID NO. 21. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0270] >PA4460 |lptH

[0271] SEQUENCE ID NO. 21 atgaggttcgttaataccctcccccttattttcggtctgactgccgccctcggcagctccatggccttggccctgccttcggaccgcgaacaaccgatc cgcgtccaggccgacagcgccgaactggacgacaagcagggcgtcgcggtctaccgcggcgacgtcgtggtgacccagggcagcaccaagctg accggcaacaccgtgaccctgaagcaggacaagaacggcgacatcgaggtcgtgacctcagtcggcaagcctgcctactacgagcagaaaccc gctccggacaaggacgtgaccaaggcctatggcctgaccatccagtacttcgtcacgcagaaccgggtcgtgctgatcgaccaggccaaggtgat ccaggaaggcaacaccttcgaaggcgagaagatcgtctacgacacccagcgccagatcgtcaacgcgggtcgtgccaccggctcccaggtgacc agcccgcgcccacgcatcgacatggtcatccagccgaagaaaaaggcccagtaa SEQUENCE ID NO. 52

[0272] MRFVNTLPLIFGLTAALGSSMALALPSDREQPIRVQADSAELDDKQGVAVYRGDVVVTQGSTKLTGNTVTLKQDKN GDIEVVTSVGKPAYYEQKPAPDKDVTKAYGLTIQYFVTQNRVVLIDQAKVIQEGNTFEGEKIVYDTQRQIVNAGRATG SQVTSPRPRIDMVIQPKKKAQ

[0273] 22. Type VI secretion system baseplate component (TssK1)

[0274] The term TssK1 encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO. 53.

[0275] The nucleic acid sequence of Tsskl is provided in SEQUENCE ID NO. 22. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0276] >PA0079 |tssK1

[0277] SEQUENCE ID NO. 22 atgtcctggaataaccgcgtggtgtggtccgaaggcatgttcctgcggccacagcatttccagcagcacgaccgctacctggaaaccctggtggat ggccgctgccgctcgctcctggcgggcggctggggcttttccgagctgaagctggacgatgccctgctgacccagggcaagctggccatcgtcag cgcccgcggcgtgctgccggatggtacgccgttcaacatccccgccgacgatccggcgccggcgccgctgaatgtcgaggaaagcctgcgtgatg gcatcgtctacctcggcctgccgctgaagcgggtcggcacccgcgacacggtagaggagggcgaggccctcggcggcgcccgctacgtcagcca ggtgcaggaagtgcgcgacgacaatgccgccttcgagagccgcgcgccagtggccctgggcagccaggcgttccgcctgctgaccgagcgcgac ggcctcggcgaatacgccgcggtgggcgtcgcgcgggtccgcgagaagcgcgcggaccaggccctgagcctggacgaggactacctgccgccg gtactcgacattgccgccgcgccgcccctggcgtccttcgccaaggagctgctgggcctgctccaccagcgcggcgaagccctcgccgggcgggt ggtggcctccagcgccgggggcgcctcggaaatcgccgacttcctgctgctgcaactggtcaaccgcgccgaggccctcaccggtcacctgtccc gcgtgcgcccgctgcacccgcaggagctgtaccgcgaactggtggccctggccggcgagttctgcaccttcaccgccagccagcggcgtcccgag gagtacccggtgtacaaccacgacgacctggccgccagcttcgccccggtgatgctcgccctgcgccaggcgctggcgacggtgatcgacgccaa ggcgatcgccatcccgatcgtcgagaaggcctacggcgtgcacgtcgcgatgctcagcgaccgcagcctgatcgacaacgccagcttcgtcctggt ggtgcgcgccgacgtccccggcgagagcctgcgcgggcacttcccgcagcaggccaaggtcggctcggtggagcacatccgcgacctggtcaac ctgcaactgccgggtatcggcctgctgccgatgccggtggcgccgcggcagatcccctaccacgccggctccacctatttcgaactggaccgcggc agcgcgcattggaagcagctgacgcactccggcggcttcgccttccacatcgccggccagttccccgggctgaacctggccttctgggccattcga ggataa

[0278] SEQUENCE ID NO. 53

[0279] MSWNNRWWSEGMFLRPQHFQQHDRYLETLVDGRCRSLLAGGWGFSELKLDDALLTQGKLAIVSARGVLPDGTP FNIPADDPAPAPLNVEESLRDGIVYLGLPLKRVGTRDTVEEGEALGGARYVSQVQEVRDDNAAFESRAPVALGSQAF RLLTERDGLGEYAAVGVARVREKRADQALSLDEDYLPPVLDIAAAPPLASFAKELLGLLHQRGEALAGRVVASSAGGA SEIADFLLLQLVNRAEALTGHLSRVRPLHPQELYRELVALAGEFCTFTASQRRPEEYPVYNHDDLAASFAPVMLALRQ ALATVIDAKAIAIPIVEKAYGVHVAMLSDRSLIDNASFVLVVRADVPGESLRGHFPQQAKVGSVEHIRDLVNLQLPGI GLLPMPVAPRQIPYHAGSTYFELDRGSAHWKQLTHSGGFAFHIAGQFPGLNLAFWAIRG 23. Uroporphyrinogen-Ill synthase O (HemX)

[0280] The term HemX encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO. 54.

[0281] The nucleic acid sequence of HemX is provided in SEQUENCE ID NO. 23. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0282] >PA5258

[0283] SEQUENCE ID NO. 23 gtgagcgaagcagtcactccccaagacgacgcaagaacttccacttccaccccttctcccgttgcacccgatgcatccaggcgttccggtaccgcc accggcctggcgttgctcgccctgctggttggcgtggcgggactcggcgcgggtggctggagcttctggcaggtccagcagttgcagggcaccga gcgtgaccagctggcgcagctggaagatgcccgtggcaagaccgaatccctggcccagcgtaaccagcaactggctgcccgcctggacgctttg cctaccgcggacgaactggaggcccggcgacgccttgtggtggagctgcagggcgaccagcagcatctcagcgagcaactgaagaaggtcctc ggccagagccgccagcaatggcgactggccgaggccgaacacctgctgcgcctggccagcctgcgcctgtccgccttgcaggacgtgggcagcg ccaccgaactggtgcagggcgccgatgacatcctgcgcgcgcagaacgatcccggcgcctatgccgcccgcgccaagctggcggccagcctcga ggccctgcgtgcgttgccggagccggaccgtaccgggctgttcgtgcaactggcggcgttgcgcgagcaggtcaaccagttgcaaccgctcacgc cgaccttcaaggaaggcgaaagcggtgccagcaatgtcgcctggggcgacggcaacagcacctgggagctgtggtgggagaagatctcgcgct acatccgcatcgatttcaatgccggccaggacattcgtccgctgctggccggcgagcaattggcgcaggtgcgcctgaccatcggcctggccctgg agcaagcccagtgggcggcgctgaatggcaagcaggaagtctacgagcaggcgctgaagcaggcccaggacgtgctggacggctatttcaacg tagagctggccgacagccgcgcgttgaagttgcgtatcgatgagttggcgaagcagccggtcaccgtgcaggtccctgacctcgcaccggccctg agcgccttgcaggcctacgtgtcgcgccgcgaggccgctggcaacctggtgcccgaggcgcaggcggctccggccacggagggccgtccatga

[0284] SEQUENCE ID NO. 54

[0285] MSEAVTPQDDARTSTSTPSPVAPDASRRSGTATGLALLALLVGVAGLGAGGWSFWQVQQLQGTERDQLAQLEDA RGKTESLAQRNQQLAARLDALPTADELEARRRLVVELQGDQQHLSEQLKKVLGQSRQQWRLAEAEHLLRLASLRLS ALQDVGSATELVQGADDILRAQNDPGAYAARAKLAASLEALRALPEPDRTGLFVQLAALREQVNQLQPLTPTFKEGE SGASNVAWGDGNSTWELWWEKISRYIRIDFNAGQDIRPLLAGEQLAQVRLTIGLALEQAQWAALNGKQEVYEQAL KQAQDVLDGYFNVELADSRALKLRIDELAKQPVTVQVPDLAPALSALQAYVSRREAAGNLVPEAQAAPATEGRP

[0286] 24. Citrate synthase (GltA)

[0287] The term GltA encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.55.

[0288] The nucleic acid sequence of GltA is provided in SEQUENCE ID NO. 24. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence. > PA 1580 |gltA

[0289] SEQUENCE ID NO.24 atggctgacaaaaaagcgcagttgatcatcgagggctcagcccccgtcgaactgcccgtcctatccggtaccatgggtcccgatgtagtggatgta cggggcctcaccgccacgggccacttcaccttcgatcctggcttcatgtcgaccgcctcctgcgagtcgaagatcacctatatcgacggcgacaaa ggcgtcctcctccatcgcggctaccccatcgagcaactggcagagaaatccgactacctggaaacctgctacctgctgctgaacggcgagctgcc caccgccgcgcagaaggaacagttcgtcggcaccatcaagaaccacaccatggttcacgagcagttgaagaccttcttcaacggcttccgccgcg acgcccacccgatggccgtgatgtgcggcgtgatcggcgccctctcggccttctaccacgactccctggacatcaataacccgaagcatcgcgaag tctccgcgcatcgcctgatcgccaagatgccgaccatcgccgccatggtgtacaagtactccaagggcgagccgatgatgtatccgcgtaacgacc tgaactacgcggaaaacttcctgcacatgatgttcaacaccccctgcgagaccaagccgatcagccccgtgctggccaaggccatggaccgcatc ttcattctccacgccgaccacgagcagaacgcctccacctccacggtgcgtctggccggctcctccggcgccaatccgttcgcctgcatcgcctccg gcatcgccgccctgtggggaccggcccatggcggcgcgaacgaagcggtgctgcgcatgctcgacgagatcggcgacgtgtccaacatcgacaa gttcgtcgagaaggccaaggacaagaacgatccgttcaagctgatgggcttcggccatcgtgtctacaagaacttcgacccgcgcgccaaggtca tgaagcagacctgcgacgaggtcctccaggagctgggcatcaacgacccgcaactggaactggcgatgaagctagaagaaatcgcccgccacg acccctacttcgtggaacgcaacctgtacccgaacgtcgacttctactcggggatcatcctcaaggcgatcggcattccgaccagcatgttcaccgt gatcttcgccctggcgcgtaccgtcggctggatctcgcactggcaggaaatgctctccggcccctacaagatcggccgcccgcgccagctctatac cggccacacccagcgcgacttcaccgccctcaaggatcgcggctga

[0290] SEQUENCE ID NO. 55

[0291] MADKKAQLIIEGSAPVELPVLSGTMGPDVVDVRGLTATGHFTFDPGFMSTASCESKITYIDGDKGVLLHRGYPIEQLA EKSDYLETCYLLLNGELPTAAQKEQFVGTIKNHTMVHEQLKTFFNGFRRDAHPMAVMCGVIGALSAFYHDSLDINN PKHREVSAHRLIAKMPTIAAMVYKYSKGEPMMYPRNDLNYAENFLHMMFNTPCETKPISPVLAKAMDRIFILHAD HEQNASTSTVRLAGSSGANPFACIASGIAALWGPAHGGANEAVLRMLDEIGDVSNIDKFVEKAKDKNDPFKLMGF GHRVYKNFDPRAKVM KQTCDEVLQELGINDPQLELAMKLEEIARHDPYFVERNLYPNVDFYSGIILKAIGIPTSMFTV IFALARTVGWISHWQEMLSGPYKIGRPRQLYTGHTQRDFTALKDRG

[0292] 25. Probable M18 family aminopeptidase 2 (ApeB)

[0293] The term ApeB encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.56.

[0294] The nucleic acid sequence of ApeB is provided in SEQUENCE ID NO. 25. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0295] > PA 3247

[0296] SEQUENCE ID NO. 25 atgcgcgcagaactcaaccagggcctgatcgatttcctcaaggcctcgcccacgcctttccatgccaccgccagcctcgcccgccgcctggaagcc gccggctaccgccgcctcgacgagcgcgacgcctggcacaccgaaaccggcggccgctactacgtgacccgtaacgactcgtcgctgatcgccat ccgcctgggccgtcgctcgcccctggaaagcggcttccgcctggtcggcgcgcacaccgacagcccctgcctgcgggtcaagccgaacccggag atcgctcgcaacggcttcctccagctcggcgtcgaagtctatggcggcgccctcttcgccccctggttcgaccgtgacctgtcactggccgggcgcg tcaccttccgcgccaatggcaagctggaaagccgcctggtcgacttccgcaaggccatcgcggtaatccccaacctggccatccatctcaaccgcg ccgccaacgagggttggccgatcaacgcgcagaacgaactgccgccgatcatcgcccagctggcgccgggcgaggccgccgacttccgcctgct gctcgacgaacagctgctgcgcgagcacggcatcaccgccgacgtggtactggactacgagctgtcgttctacgacacccagtccgccgcggtgg taggtctcaacgacgagttcatcgccggggcgcgcctggacaacctgctgtcctgccacgccggcctggaagccctgctcaacgccgaaggcgac gagaactgcatcctggtctgcaccgaccacgaggaagtcggttcctgttcgcattgcggcgccgacggtccgttcctcgaacaggtactgcgccgc ctgctgccggaaggcgacgccttcagccgggcgatccagcgctcgctgctggtctcggccgacaacgcccatggcgtacacccgaactacgccga caggcacgacgccaaccatggcccggcgctgaacggcggtccggtgatcaagatcaacagcaaccagcgctatgccaccaacagcgaaaccgc cggcttcttccgccacctctgccaggacagcgaagtgccggtgcagagcttcgtgacccgcagcgacatgggatgcggctcgaccatcggcccga tcaccgccagccaggtcggcgtgcgcaccgtcgacataggcctgccgaccttcgccatgcactcgattcgcgagctggccggtagccatgacctgg cgcacctggtcaaggtgctcggcgccttctacgccagcagcgagctgccctga

[0297] SEQUENCE ID NO. 56

[0298] MRAELNQGLIDFLKASPTPFHATASLARRLEAAGYRRLDERDAWHTETGGRYYVTRNDSSLIAIRLGRRSPLESGFRL VGAHTDSPCLRVKPNPEIARNGFLQLGVEVYGGALFAPWFDRDLSLAGRVTFRANGKLESRLVDFRKAIAVIPNLAIH LNRAANEGWPINAQNELPPIIAQLAPGEAADFRLLLDEQLLREHGITADVVLDYELSFYDTQSAAVVGLNDEFIAGAR LDNLLSCHAGLEALLNAEGDENCILVCTDHEEVGSCSHCGADGPFLEQVLRRLLPEGDAFSRAIQRSLLVSADNAHG

[0299] VHPNYADRHDANHGPALNGGPVIKINSNQRYATNSETAGFFRHLCQDSEVPVQSFVTRSDMGCGSTIGPITASQVG VRTVDIGLPTFAMHSIRELAGSHDLAHLVKVLGAFYASSELP

[0300] 26. S-adenosylmethionine synthase (MetK)

[0301] The term MetK encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.57.

[0302] The nucleic acid sequence of MetK is provided in SEQUENCE ID NO. 26. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0303] >PA0546 |metK

[0304] SEQUENCE ID NO. 26 atgagcgaatattccgttttcacctccgagtccgtgtccgaaggccatccggacaagatcgcggaccagatctccgacgcggtgctggacgccatc atcgccaaggacaagtacgctcgcgtggcatgcgaaaccctggtcaagaccggcgtggcgatcatcgccggtgaagtcaccacctccgcctgggt cgacctggaagagctggtgcgcaaggtcatcatcgacatcggctacgacagctccgacgtcggcttcgatggtgccacctgcggcgtgctgaaca tcatcggcaagcagtcggtggacatcaaccagggcgtcgaccgggccaagccggaagaccagggcgccggcgaccagggcctgatgttcggct acgccagcaacgagaccgacgtgctgatgccggcgccgatctgtttctcccaccgcctggtcgagcgccaggccgaggcgcgcaagtccggcctg ctgccatggctgcgcccggacgccaagtcccaggtgacctgccgctacgaaggcggcaaggtggtcggcatcgatgcggtggtgctgtccaccca gcacaacccggaagtctcctataacgacctgcgcgacggcgtgatggagctgatcatcaagcaggtgctgccggccgagctgctgcacaaggac acccagttccacatcaacccgaccggcaacttcgtgatcggcggcccggtcggcgactgcggcctgaccggacgcaagatcatcgtcgactccta cggcggcatggcccgccacggcggcggcgcgttctccggcaaggacccgtccaaggtcgaccgctccgccgcctacgccggccgctacgtggcg aagaacatcgtcgccgccggcctcgccgagcgttgcgagatccaggtttcctacgccatcggcgtggcccagccgacctcgatctcgatcaacacc ttcggcaccggcaaggtcagcgacgagaagatcgtgcaactggtccgcgagcacttcgacctgcgcccgtacgcgatcaccaagatgctcgacct gctccacccgatgtaccagccgaccgcggcctacggccacttcggtcgtcatccgttcgagctgaccgtggacggcgataccttcaccgcgttcacc tgggagaagaccgacaaggccgctctcctgcgcgacgccgccggcctctga

[0305] SEQUENCE ID NO. 57

[0306] MSEYSVFTSESVSEGHPDKIADQISDAVLDAIIAKDKYARVACETLVKTGVAIIAGEVTTSAWVDLEELVRKVIIDIGYDS SDVGFDGATCGVLNIIGKQSVDINQGVDRAKPEDQGAGDQGLMFGYASNETDVLMPAPICFSHRLVERQAEARKS GLLPWLRPDAKSQVTCRYEGGKWGIDAVVLSTQHNPEVSYNDLRDGVMELIIKQ.VLPAELLHKDTQFHINPTGNFV IGGPVGDCGLTGRKIIVDSYGGMARHGGGAFSGKDPSKVDRSAAYAGRYVAKNIVAAGLAERCEIQVSYAIGVAQPT SISINTFGTGKVSDEKIVQLVREHFDLRPYAITKMLDLLHPMYQPTAAYGHFGRHPFELTVDGDTFTAFTWEKTDKAA LLRDAAGL

[0307] 27. Uncharacterised protein (FixH)

[0308] The term FixH encompasses this protein and effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.58.

[0309] The nucleic acid sequence of FixH is provided in SEQUENCE ID NO. 27. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0310] > PA 1550

[0311] SEQUENCE ID NO. 27 atgcagtccgagacccaccctcaaccctggtacaaacacttctgggcctggttcatcgtggccctcctggcgacctcggtggtgctcgggctgagc ctggtcaccatcgcggtgcgcaaccaggacaccctggtcaccgacaactactacgaggccggcaagggcatcaaccgctccctggaacgcgaga acctggccgtacgcctgcaggtgcacgccaaggtcaaggtcgacgacctgaccggcgaagtcgaagtgcgcctgaccggcaacagccgtccgg atcggctcaccctcaacctgatctcgccgacccagccggagaaggaccgccgcgtggagctgctgcgcagcaactccgaccgcgagcgctacgt cgggcaactgaccgacgccgtcgagggccgtcgcttcgtcgaactgctcggccaggaaggcagcggccagtggcgcctgttcgaggaagaggtg gtatcgccgaccgtggtcatggagctggg tgacgaaccgctccagggagcagaagcgaagcgcccatga

[0312] SEQUENCE ID NO. 58

[0313] MQSETHPQPWYKHFWAWFIVALLATSVVLGLSLVTIAVRNQDTLVTDNYYEAGKGINRSLERENLAVRLQVHAKVK VDDLTGEVEVRLTGNSRPDRLTLNLISPTQPEKDRRVELLRSNSDRERYVGQLTDAVEGRRFVELLGQEGSGQWRLF EEEVVSPTVVMELGDEPLQGAEAKRP

[0314] 28. 2-methylcitrate dehydratase (PrpD)

[0315] The term PrpD encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.59.

[0316] The nucleic acid sequence of PrpD is provided in SEQUENCE ID NO. 28. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0317] >PA0792 |prpD

[0318] SEQUENCE ID NO. 28 atgagtgccaacgtcgatctcaaccaacgtcccgactacgacgccgtcctgcaggacatcgccgactacgtgctggactaccgcatcgattccacg gaggccctggacaccgcccgcaactgcctgatggataccctcggctgcggcctgctggcgctgcgcttcccggaatgcaccaagcacctcggccc gctggtcgagggcaccctggtcccgcacggcgcgcgggtgccgggcacctccttccggctcgacccggtcaaggccgcctgggacatcggctgc atcgtccgctggctggactacaacgacacctggctggccgccgagtggggccatccctcggacaacctcggcggcatcctcgccgtcgccgacca cctctcgcagaaacgcctggccaacggcgaagcgccgctgagcatgcgccaggtactggaagcgatgatcatggcccacgagatccagggcgtg atcgccctggagaactcgttcaatcgcgtcggcctcgaccatgtgctgctggtcaaggtcgcgtccaccgccgtctgcgccaagctgatgggggcc gaccgcgagcaactgctggccgccctctcccacgccttcgtcgacggccaggccctgcgcacctaccgtcacgcaccgaacgccggctcgcgcaa gtcctgggccgctggcgacgcgacgagccgcggcgtgcgcctggcggacatcgccttgcgcggcgagatgggcattcccggcgtgctcagcgcg ccgcagtggggtttctacgatgtcctgttcagccacaccagcaaggacctggcgaccaagcccgaggacaagcggcgcttcagcttcccccaggg ctacggcagctacgtgatggaaaacgtgctgttcaagatcagcttccccgccgaattccacgcgcagaccgccgccgaggccgccgtgcgcctgc atccgctggtgaaggaccgcctgcaacggatcagccgcatcgtcatcaccacccacgagtcggcgatccgcatcatttccaaggtcggcccgctg gccaaccccgcagaccgcgaccattgcctgcagtacatgaccgccgtgccgctgatcttcggcgacctggtggccgagcactacgaggacgcctt ccacgccgcccacccgctgatcgaccgcttgcgcgagaaaatggagatcgtcgaggagccgcgctacagccgtgaatacctggaggcggacaa gcgctccatcgccaacgccgtggaagtgttcttcgacgacggcagcagcaccggccaggtcgccgtggagtacccgctcggccatcgccgccggc gcgccgaaggcatcccgctgctgcaggagaagttcaaggccaacctggccacgcgcttcccgccgcagcgctgccagaggatcttcgacctgtgc agccaccaggcatcgctggaagccacgccggtgaaccgcttcatggacctcctggcgatctga

[0319] SEQUENCE ID NO. 59

[0320] MSANVDLNQRPDYDAVLQDIADYVLDYRIDSTEALDTARNCLMDTLGCGLLALRFPECTKHLGPLVEGTLVPHGAR VPGTSFRLDPVKAAWDIGCIVRWLDYNDTWLAAEWGHPSDNLGGILAVADHLSQKRLANGEAPLSMRQVLEAMI MAHEIQGVIALENSFNRVGLDHVLLVKVASTAVCAKLMGADREQLLAALSHAFVDGQALRTYRHAPNAGSRKSWA AGDATSRGVRLADIALRGEMGIPGVLSAPQWGFYDVLFSHTSKDLATKPEDKRRFSFPQGYGSYVMENVLFKISFPA EFHAQTAAEAAVRLHPLVKDRLQRISRIVITTHESAIRIISKVGPLANPADRDHCLQYMTAVPLIFGDLVAEHYEDAFHA AHPLIDRLREKM EIVEEPRYSREYLEADKRSIANAVEVFFDDGSSTGQVAVEYPLGHRRRRAEGIPLLQEKFKANLATR FPPQRCQRIFDLCSHQASLEATPVNRFMDLLAI

[0321] 29. Ketol-acid reductoisomerase (NADP(+)) (HvC)

[0322] The term II vC encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NQ.60.

[0323] The nucleic acid sequence of HvC is provided in SEQUENCE ID NO. 29. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0324] >PA4694 |ilvC

[0325] SEQUENCE ID NO. 29 atgcgcgttttctacgataaagactgtgacctctcgatcatccagggcaagaaagttgccatcatcggctacggctcccagggccacgcccatgcct gcaacctgaaggactccggcgtcgacgtcaccgtgggcctgcgtagcggctccgccaccgtggccaaggccgaagcgcacggtctgaaggttgc cgacgtgaagaccgccgtcgccgcagccgacgtggtcatgatcctcaccccggacgagttccagggccgcctgtacaaggaagagatcgagccg aacctgaagaagggcgccaccctggccttcgctcacggcttctccatccactacaaccaggtcgtcccgcgcgccgacctcgacgtgatcatgatc gcgccgaaggcaccgggtcacaccgtgcgttccgagttcgtcaagggcggtggcatccctgacctgatcgccatctaccaggacgcttccggcaa cgccaagaacgtcgccctgtcctacgcctgcggcgtcggcggcggtcgtaccggtatcatcgaaaccaccttcaaggacgagaccgaaaccgac ctgttcggtgagcaggccgttctctgcggtggttgcgtcgagctggtcaaggccggtttcgaaaccctggtcgaagccggttacgcgccggaaatg gcctacttcgagtgcctgcacgagctgaagctgatcgtcgacctgatgtacgaaggcggcatcgccaacatgaactactccatctccaacaatgcc gaatacggtgagtacgtaaccggtccggaggtgatcaacgccgagtcccgtgctgccatgcgcaacgccctgaagcgcatccaggacggcgagt acgcgaaaatgttcattaccgaaggtgcggccaactacccgtcgatgactgcctaccgccgcaacaacgccgctcacccgatcgagcagatcggc gagaagctgcgcgcgatgatgccgtggatcgcagccaacaagatcgtcgacaagagcaagaactaa

[0326] SEQUENCE ID NO. 60

[0327] MRVFYDKDCDLSIIQGKKVAIIGYGSQGHAHACNLKDSGVDVTVGLRSGSATVAKAEAHGLKVADVKTAVAAADVV MILTPDEFQGRLYKEEIEPNLKKGATLAFAHGFSIHYNQVVPRADLDVIMIAPKAPGHTVRSEFVKGGGIPDLIAIYQD ASGNAKNVALSYACGVGGGRTGIIETTFKDETETDLFGEQAVLCGGCVELVKAGFETLVEAGYAPEMAYFECLHELKLI VDLMYEGGIANMNYSISNNAEYGEYVTGPEVINAESRAAMRNALKRIQDGEYAKM FITEGAANYPSMTAYRRNNA AHPIEQIGEKLRAMM PWIAANKIVDKSKN 30. CobW C-terminal domain-containing protein; GTP-binding protein (YjiA)

[0328] The term YjiA encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.61.

[0329] The nucleic acid sequence of YjiA is provided in SEQUENCE ID NO. 30. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0330] >PA4604

[0331] SEQUENCE ID NO. 30 atgtccgaagtagccaacgacaaccacgccccgatccccgtcaccgtgctcaccggcttcctcggcgccggcaagaccaccctgctcaagtacct gctgcaggccgagcacggcatgaagatcgcggtgatcgagaacgagtacagcgagacgccgatcgacggccagttgctcggcgtcgagccggt gcaggtgatgacgctgtccaatggctgcgtctgctgctcgatcaacaccgacctggaaaaggccctgttcctcctgctggagcgccgcgacaacg gcgaaatcgacttcgaccgcctggtgatcgagtgcaccggcctggccgatccggcgccggtggcgcagaccttcttcgccgacgaggaactctgc cagcgctacgtgctggacggcatcatcaccctggtggacgccgccaacgccgagcgtcacctgcaggaaaccatcgcccaggcccaggtcggct tcgccgaccgcatcctggtgagcaagaccgacctggtcgacgccgccaccttcgaggccctgggccagcgcctgcaacggatcaaccggagggc gctggtgcatgtggtcgagcacggccgcatcgacctggcgcacctgctcgacgtacgtggcttcaacctcaacgccgacctcggtcctggcatcgg cctgcgcccgctgcgagcggtggcagccaaggacagccgcgatcgcatcggcaccctggtactgcgcagcgacacgccgctcgatctcgaaagg ctgagcgagttcatggacgacctgttgcagtggcatggcaattccctgctgcgctacaagggcgtgctgaacatcgccgacgagccgcgtcgcctg gtgttccagggcgtgctgcgactctacggcttcgactgggatagcgagtggcgggacgacgaggcgcgcgagagcgtgatcgtgttcatcggcga caacctgccggaggacagtatccgcgagggcttcgaacgcatcgtcgaaggccgctga

[0332] SEQUENCE ID NO. 61

[0333] MSEVANDNHAPIPVTVLTGFLGAGKTTLLKYLLQAEHGMKIAVIENEYSETPIDGQLLGVEPVQVMTLSNGCVCCSI NTDLEKALFLLLERRDNGEIDFDRLVIECTGLADPAPVAQTFFADEELCQRYVLDGIITLVDAANAERHLQETIAQAQV GFADRILVSKTDLVDAATFEALGQRLQRINRRALVHVVEHGRIDLAHLLDVRGFNLNADLGPGIGLRPLRAVAAKDSR DRIGTLVLRSDTPLDLERLSEFMDDLLQWHGNSLLRYKGVLNIADEPRRLVFQGVLRLYGFDWDSEWRDDEARESVI VFIGDNLPEDSIREGFERIVEGR

[0334] 31. HGDO

[0335] The term HGDO encompasses this protein and effective variants, e.g. that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO.62.

[0336] The nucleic acid sequence of HGDO is provided in SEQUENCE ID NO. 31. It will be appreciated that the nucleic acid sequence may be an effective variant of this sequence.

[0337] SEQUENCE ID NO. 31

[0338] >PA2009 | hmgA atgaacctcgactccactgccctcgcctatcaatcgggcttcggcaacgaattcagcagcgaagcgctccccggcgccctgccggtcggccagaa ctccccgcagaaagcgccctacggcctgtacgccgaactgctctccggcaccgccttcaccatggctcgcagcgaggcccggcgcacctggctgt accgcatcacgccgtcggccaagcatccgccgttccgccgcctggaacgacagatcgccggtgccgaactggatgcgccgactcccaaccgcctg cgctgggacccgctggcactgcccgagcagcccaccgacttcctcgacggcctgctgcgcatggccgccaacgcgcccggcgacaagcccgccg gcgtgagcatctaccagtacctggccaaccgctcgatggagcgttgcttctacgacgccgatggcgaactgctgctggtcccgcaattgggccgtct gcgcctgtgcaccgaactcggcgcgctgcaagtcgaaccgctggagatcgcggtgatcccgcgcgggatgaagttccgcgtcgagctgctcgacg gcgaggcacgcggctatatcgccgagaaccacggcgcgccgctgcgcctgcccgacctcggcccgatcggcagcaatggcctggccaatccgcg cgacttcctgaccccggtggcgcgctacgaggacagccgccagccgctgcaactggtgcagaaatacctcggcgagctgtgggcctgcgagcttg accactcgccgctggacgtggtcgcctggcacggcaacaacgtgccctacaagtacgacctgcgccgcttcaacaccatcggcacggtcagcttc gaccacccggacccgtcgatcttcaccgtgctgacctcccccaccagcgtccccggcctggccaacatcgacttcgtgatcttcccgccgcgctgga tggtggccgagaacaccttccgtccgccatggttccaccgcaacctgatgaacgaattcatgggcctgatccagggcgcctatgacgccaaggcc ggcggcttcgtgcccggcggcgcctcgctgcacagttgcatgagcgcccacggcccggacgcggaaagctgcgacaaggccatcgccgccgacc tcaagccgcacaggatcgaccagaccatggccttcatgttcgagaccagccaggtcctccggccgagccgtgccgccctcgagacgccggccctg cagaatgactacgatgcctgctgggcgtcgctcgtatccaccttcaacccgcaacggagataa

[0339] SEQUENCE ID NO. 62

[0340] MNLDSTALAYQSGFGNEFSSEALPGALPVGQNSPQKAPYGLYAELLSGTAFTMARSEARRTWLYRITPSAKHPPFRR LERQIAGAELDAPTPNRLRWDPLALPEQPTDFLDGLLRMAANAPGDKPAGVSIYQYLANRSMERCFYDADGELLLV PQLGRLRLCTELGALQVEPLEIAVIPRGMKFRVELLDGEARGYIAENHGAPLRLPDLGPIGSNGLANPRDFLTPVARYE

[0341] DSRQPLQLVQKYLGELWACELDHSPLDWAWHGNNVPYKYDLRRFNTIGTVSFDHPDPSIFTVLTSPTSVPGLANID

[0342] FVIFPPRWMVAENTFRPPWFHRNLMNEFMGLIQGAYDAKAGGFVPGGASLHSCMSAHGPDAESCDKAIAADLK PHRIDQTMAFM FETSQVLRPSRAALETPALQNDYDACWASLVSTFNPQRR

[0343] Table 2: Protein Characterisation

[0344] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention. EXAMPLES

[0345] MATERIALAND METHODS

[0346] Assessment of attachment of arrange of different P. aeruginosa strains from different sources of infection to human lung epithelial cells.

[0347] To assess the attachment of the selected P. aeruginosa strains to human lung epithelial cells, 16HBE14o- (HBE) cells were seeded at a density of 4x105cells / mL in Cellstar® 24-well culture plates (3 wells / strain), and routinely incubated overnight in complete MEM (1 mL / well). The following day, bacterial cultures were grown to ODeoo ~0.6 in LB broth and resuspended in non-supplemented MEM at a concentration of 2x106CFU / well, which corresponds to a multiplicity of infection (MOI) of 5:1. Cells were equilibrated once with MEM to remove antibiotics before applying the bacterial inocula, and the plates were then centrifuged at 700 x g for 5 min and incubated for 30 min at 37°C and 5% CO2 to allow for bacterial attachment. Unattached bacteria were removed by washing twice with sterile PBS, and remaining bacteria and cells were detached by 10 min incubation at room temperature with lysis buffer (0.5% Triton X-100 in PBS) and pipette tip-based scraping. The lysates were serially diluted in PBS, each dilution was plated onto LB agar plates in duplicate, and plates were incubated at 37°C for 24 h, after which the resulting CFU were counted. At least three independent experiments were performed per strain. To test whether there were statistically significant differences in the attachment levels among the six clinical isolates, normal distribution of the data was assessed using the Shapiro-Wilk normality test, and then, a one-way ANOVA (p-value<0.05) with Turkey’s post-hoc test analysis was performed on GraphPad Prism v9.4.1.

[0348] Determination of P. aeruginosa attachment to HBE cells by confocal microscopy.

[0349] Bacterial attachment was also evaluated and visualised using a complementary confocal microscopy method. Cells were seeded on imaging 24-well CG 1.0 plates (4x105CFU / well) and bacteria were applied at two MOIs, 5:1 and 10:1 , after which cells and adhered bacteria were fixed with 3% paraformaldehyde (pH = 7.2) in PBS for 10 min at RT. The wells were then washed with PBS twice and blocked with 5% BSA in PBS for 2 h at RT. Then, the wells were washed as before and incubated overnight at 4°C with a rabbit anti-P. aeruginosa polyclonal Ab (Invitrogen, USA) at a 1 :8,000 dilution in PBS + 1 % BSA. The following day, the wells were washed and incubated for 1 h, in the dark and at RT with an Alexa Fluor®488-conjugated secondary goat anti-rabbit Ab at a 1 :1 ,000 dilution in PBS plus 1% BSA. Finally, HBE plasma membranes and nuclei were counterstained with 0.5X CellMask™ Deep Red for 10 min, at 37°C in the dark, and with 0.25 pg / mL 4',6-Diamidine-2'-phenylindole dihydrochloride for 15 min, at RT in the dark, in two separate steps with two PBS washes in between. Finally, wells were covered with 1 mL PBS and images were taken on an automated Opera Phenix™ High Content Screening confocal microscope. The number of cells and bacteria attached was automatically counted using a specific pipeline with the Harmony®4.8 High Content Imaging and Analysis software. Briefly, cell nuclei and membranes were detected, which was interpreted by the software as cell area; then, green spots ranging from 2 to 8 pm were identified as bacteria, and finally, the software automatically counts the number of bacteria that are within the cell area. Consequently, bacterial attachment was represented as number of bacteria attached per 100 cells. At least three independent experiments were performed per strain.

[0350] Preparation and quantification of bacterial membrane-enriched proteins

[0351] Enriched membrane protein preparations from four P. aeruginosa clinical isolates selected based on their attachment ability (LES 431 , AA2, 57P31 PA, and A5803) were obtained as follows: Overnight bacterial cultures were inoculated into 1 L LB broth (1 :10) and grown overnight at 37°C with agitation. Bacteria were pelleted by centrifugation at 5,000 x g for 10 min at 4°C, and pellets were resuspended and incubated for 20 min with vigorous vortexing in buffer containing 5% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1 -propanesulfonate hydrate) in PBS with protease inhibitor cocktail to allow for membrane proteins solubilisation. Cell debris and any remaining whole cells were eliminated through centrifugation for 15 min at 7,000 x g and 4°C, and supernatants were ultracentrifuged for 30 min at 30,000 x g and 4°C. Then, the supernatants were discarded and the pellets containing the proteins were resuspended in 20 mL 50mM Tris, pH 8 containing 2mM MgCh, with protease inhibitor cocktail and ultracentrifuged using the same conditions. The supernatants were removed, and the pellets were resuspended in 20 ml of 50mM Tris, pH8 containing 2% Triton X-100 and protease inhibitor cocktail, incubated for 30 min at 40°C with gentle shaking, and ultracentrifuged for 1 h at 30,000 x g and 4°C. The supernatants were discarded, and pellets resuspended in 1 mL 50mM Tris, pH8. A final ultracentrifugation step was carried out in the same conditions, after which the supernatants were discarded and the final pellets containing bacterial proteins were solubilised in 1 mL rehydration buffer for the isoelectric focusing (IEF).

[0352] The membrane protein-enriched preparations obtained from the four selected strains (LES 431 , AA2, 57P31 PA, and A5803) were as previously described

[0023] to identify bacterial proteins involved in host attachment during infection. Proteins were separated using 7 cm Inmobiline™ DryStrips, pH 3-11 or pH 4-7. These were rehydrated overnight with 120 pL of protein samples (60-180 pg) resuspended in rehydration buffer with a trace of bromophenol blue. The following day, IEF was carried out on the Ettan™ IPGphor™ 3 IEF System, following a three-step protocol (30 min at 300 V, 30 min at 1 ,000 V, and 4 h at 5,000 V). Then, the strips were equilibrated in reducing buffer (30% glycerol, 2% SDS, 6 M urea, 50 mM Tris base, 2% DTT; 5 mL / strip) for 20 min at RT, and then alkylated with the alkylation buffer (30% glycerol, 2% SDS, 6 M urea, 50 mM Tris base, 2.5% iodoacetamide 5 mL / strip) under the same conditions. The proteins were then separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The strips were placed on top of the polymerised gels, sealed with 0.1 % agarose containing a trace of bromophenol blue as a tracking dye, and a small well was created on the left side of the gel to apply the appropriate protein marker. Protein separation by electrophoresis was carried out at 120 V for ~2,5 h.

[0353] For each individual experiment, two strips and two gels were prepared in parallel, so that one gel was stained overnight with PageBlue™ Protein Staining solution, and the other one was used to transfer the proteins onto an Immobilon-P® polyvinylidene difluoride (PVDF) membrane at 4°C and 30 mA in wet conditions for 16-18 h.

[0354] Membrane probing with HBE cells and detection of bound cells

[0355] After transfer, the membranes were blocked overnight at 4°C with 20 mL 5% BSA, 3% Milk powder in PBS and the membranes were then probed with 4x105HBE cells / mL in PBS for 4 h at 4°C. After incubation, unattached HBE cells were removed by washing the membranes four times with washing solution and attached cells were fixed with 3% paraformaldehyde for 8 min at RT. To detect the spots where the cells adhered, the membranes were probed with specific antibodies. Firstly, the membranes were incubated overnight at 4°C with a mouse anti- epithelial cell-specific antigen Ab (clone VD-1 D9, Sigma-Aldrich) in 5 mL of PBST (PBS / 0.04% tween 20) with 5% BSA. The following day, the membranes were in PBS with 0.5% Tween 20 and incubated for 1 h with a rabbit polyclonal anti-mouse Ab conjugated to horseradish peroxidase in 5 ml PBST with 5% BSA. The membranes were washed as before and chemiluminescence detection performed. The spots revealed by chemiluminescence were matched with the corresponding protein spots observed after destaining the relevant PageBlue-stained gel, based on position (pl and molecular weight) and shape.

[0356] In-gel trypsin digestion and sample purification

[0357] Protein spots corresponding to positive spots on the blots were individually excised from the corresponding 2D gels with a clean scalpel and destained with 100 mM ammonium bicarbonate (AB) / acetonitrile (ACN) (1 :1) for 30 min. Trypsin digestion was performed as described previously

[0024] and the peptides were extracted by incubating the gel pieces with 5% formic acid in acetonitrile (1 :2) at 37°C for 15 min. The supernatants containing the peptides were transferred to new tubes and dried in a vacuum before purification by ziptipping. The peptide samples were then resuspended in 0.1 % FA and analysed in the Mass Spectrometry Core service at the Conway Institute in University College Dublin (UCD). Protein spectra were obtained by LC-MS analysis and matched with a P. aeruginosa reference proteome from strains ATCC 15692, DSM 22644, CIP 104116, JCM 14847, LMG 12228, 1C, PRS 101 , and PAO1 (UniProt accession number: UP000002438). To identify the proteins present in each spot, those with more than 15% of sequence coverage and whose molecular weight and pl (obtained from Compute pl / Mw tool from Expasy, htps: / / web.expasy.org / compute pi / ) matched that of the corresponding spot in the stained gel were selected.

[0358] The antigens were individually cloned into E. coli Top10 cells using a pET-28a cloning vector using standard cloning procedures and subsequently transformed into E. coli BL21 DE3 cells for induction of expression and subsequent antigen purification using standard methods.

[0359] Determination of recombinant E. coli clones attachment to HBE cells by confocal microscopy

[0360] The attachment of the recombinant BL21 clones to lung epithelial cells was analysed by confocal microscopy. Overnight cultures of the recombinant E. coli BL21 clones were inoculated (10:1) into fresh LB+kanamycin and incubated for 2 h before addition of 1 mM IPTG to half of the cultures, and both cultures (induced and uninduced) were grown for 2 h. The inocula were adjusted to ODeoo = 0.1 , resuspended in MEM, and applied to HBE cells. Inocula were also serially diluted, plated onto selective LB agar, and incubated for 24 h at 37°C to count the CFU and calculate the actual inoculum used in each case. Aliquots (1 mL) from the inocula were also taken to confirm recombinant protein expression by SDS-PAGE and Western blot analysis using an anti-6xHis tag-HRP Ab as before. To evaluate the attachment by confocal microscopy, bacterial cells were labelled by a single overnight incubation at 4°C with an anti-E. coli serotype O / K polyclonal Ab conjugated to FITC (fluorescein isothiocyanate; ThermoFisher). HBE cells were stained with DAPI and CellMask™ Deep Red, as before. Bacterial attachment (number of bacteria / 100 cells) of the induced recombinant E. coli BL21 clones was compared to their uninduced counterparts by calculating the fold change (FC; induced / uninduced), and then the log2(FC) was calculated to compare between clones.

[0361] Antigen expression and purification.

[0362] Overnight E. coli BL21 bacterial cultures were inoculated into 1 L LB broth (1 :10) and cultured for 2 h at 37°C and 200 rpm before addition of 1 mM IPTG for 2 to 5 hours at 30 or 37C depending on the optimal expression conditions for each antigen. Bacterial cells were harvested by centrifugation as before and stored at -20°C until further use. To extract protein, the bacterial cell pellets were resuspended in native basic buffer (50 mL / 1 L culture) supplemented with lysozyme and protease inhibitor cocktail as before, incubated on ice for 30 min with gentle shaking, and sonicated on ice 10 times for 30 s at 20% amplitude, with 30 s rest intervals. Separation of soluble and insoluble fractions was performed by centrifugation. Proteins found to be in the soluble fraction were purified from the supernatants after centrifugation. For proteins in the insoluble fraction, centrifugation pellets were resuspended in 20 mL denaturing buffer (basic buffer with 8 M urea), and then incubated, sonicated, and centrifuged to remove insoluble debris. The chaotrope-soluble fractions were purified by affinity chromatography as follows. HisPur™ Ni-NTA (nickel-nitrilotriacetic acid) Superflow Agarose (Thermo Fisher) resin was used to purify the His-tagged recombinant proteins in a gravity flow column. All the buffers used for the IMAC purification are listed in table 3.16. When denaturing conditions were necessary, 8 M urea was added to the buffers.

[0363] Protein purity was determined by SDS PAGE while protein concentrations were determined using BCA protein assays. Protein identity was confirmed by LC-MS. Endotoxin levels were quantified using the Pierce™ Chromogenic Endotoxin Quant Kit.

[0364] Ethics

[0365] All work involving animals in this project was approved by the UCD Animal Research Ethics Committee (AREC-21-19), and mice were maintained according to the regulations of the Health Products Regulatory Authority (Directive 2010 / 63 / EU and Irish Statutory Instrument 543 of 2012), under authorisation number AE18982 / P209.

[0366] Immunisation of mice by subcutaneous injection

[0367] Female 6-8 week old C57BL / 6J mice (n=7) were used to evaluate the protective efficacy of the novel vaccine antigen candidates against an acute P. aeruginosa lung infection. The mice were injected subcutaneously with 100 pL of a 1 :1 mix of SAS adjuvant with 50 pg antigen using a 25-gauge (25G) needle. Mice were immunised on day 0 with two boosters two weeks apart, and they were weighed and monitored daily for three days after each injection, and every second day between boosters.

[0368] Detection of antigen-specific antibodies after immunisation by indirect ELISA

[0369] Seven days after the third immunisation, blood samples were collected from individual mice via submandibular vein bleed and the blood samples centrifuged at 16,000 * g at 4°C for 30 min to isolate and collect sera. The sera were analysed by indirect ELISA (enzyme-linked immunosorbent assay) to evaluate the production of antigen-specific antibodies using Nunc Immuno 96-well Maxisorp plates that had been coated overnight at 4°C with 100 pL / well coating buffer containing 1 pg / mL of individual purified antigen (rOprM, rFtsZ, rOpmH, or rLptH). The wells were washed three times before blocking with 100 pL blocking buffer for 1 h at RT. The sera were diluted in triplicate and incubated for 2 hours with the relevant coated antigen, at which time the wells were washed. The plates were then incubated with 100 uL / well of secondary HRP conjugated goat antibody specific for mouse total IgG, IgG 1 or lgG2c Ab (1 :5,000 in blocking buffer; Abeam) for 1 h at RT and the wells washed again and dried as previously described plus one additional wash with 300 pL / well PBS to avoid potential interference of Tween 20 in the colorimetric reaction. Then, the plates were incubated with 100 pL / well of the 1-stepTMslow TMB (3,3',5,5'-Tetramethylbenzidine) substrate solution (Thermo Scientific), and after 30 min, reactions were stopped using 100 pL / well stop solution. Colour changes were read at an absorbance of 450 nm within 15 min using the Synergy H1 microplate reader.

[0370] P. aeruginosa challenge by intratracheal instillation.

[0371] Seven days after blood collection, mice were challenged by oropharyngeal aspiration with a theoretical dose of 4x106CFU / mouse and culled after 24 h. The actual bacterial CFU administered to mice was calculated by dilution and plating of the inoculum. After 24 hours the mice were sacrificed and both lungs and spleens were collected to enumerate the CFU in the organs. Statistically significant differences between control and immunised mice were analysed using the unpaired non-parametric Kruskal-Wallis test (p-value<0.05).

[0372] Mice immunisation multivalent vaccine candidates and P. aeruginosa challenge.

[0373] Female C57BL / 6J mice (6-8 weeks old) from Charles River (UK) were randomly housed (3-4 mice / cage) with food and water available ad libitum. Mice (n=7) were immunised subcutaneously on day 0 with 25 pg antigen (for multivalent candidates, combined as follows: rFtsZ+rOpmH, rFtsZ+LptH, rFtsZ+rOpmH+rLptH) per mouse in a final volume of 100 pL with Sigma Adjuvant System (SAS, Sigma, UK; 1 :1 v / v), SAS alone or vehicle buffer alone as controls, and boosted on day 14 and day 28 (Figure 3a, 6a). Mice were challenged on day 42 with ~1x107CFU / mouse of the heterologous P. aeruginosa KK1 strain by oropharyngeal instillation, and on day 43 lungs and spleens were aseptically harvested and homogenised to calculate bacterial burden (CFU / mg organ) as described. Animal welfare was evaluated using an experiment-specifically designed scoresheet.

[0374] Evaluation of cytokine production via ELISpot.

[0375] Female C57BL / 6J mice (6-8 weeks old, n=4) were immunised once subcutaneously with 50 pg antigens (individually) plus SAS (1 :1 v / v) or SAS alone as a control. On day 14, mice were humanely killed and splenocytes were harvested as before (Tomas-Cortazar et al. 2021 , 'BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis', Front Immunol, 12, 767359). Polyvinylidene fluoride membranes on ELISpot plates were activated for 1 min with sterile 35% ethanol and washed with deionised water. Plates were coated overnight at 4°C with either anti-mouse IFN-y or anti-mouse IL-17 monoclonal antibodies in PBS as per manufacturer’s instructions (Mabtech, Sweeden). Plates were then washed with PBS and blocked with RPMI + 10% FBS for 30 min at room temperature, and 106cells / well were added in triplicate, stimulated with 10 ug / well of rLptH (50ug / mL), and incubated at 37°C and 5% CO2 for 48 h. Plates were washed and incubated for 2 h at room temperature with either anti-mouse IFN-y or anti-mouse IL-17 monoclonal antibodies in PBS+0.5% FBS as per manufacturer’s instruction. Then plates were washed and incubated for 1 h at room temperature with streptavidin-alkaline phosphatase (1 :1000) in PBS+0.5% FBS. Finally, the BCIP / NBT-plus substrate (Mabtech) was added, and colorimetric reaction was developed and stopped after 5-30 min with water. Plates were air-dried and analysed on a Mabtech Astor 2 reader using the Mabtech Apex 2.0 software.

[0376] RESULTS

[0377] Attachment to host epithelial cells.

[0378] After infection of 16HBE14o_monolayers with the individual P. aeruginosa strains for 30 min at an MOI 5:1 , the adhesion patterns showed statistically significant differences (p-value<0.05) between most of the strains (Figure 1). The CF transmissible strain (LES 431) showed the lowest host cell attachment (0.58%), followed by the burn Mi 162 isolate (6.87%). Two strains isolated from CF patients in early colonisation (AA2 and ATM 0060-3) and one from a COPD patient (57P31 PA) showed 20-30% of adhesion, and the CAP isolate (A5803) showed the highest attachment (54.9% ). The attachment to human lung cells was visualised by confocal microscopy. Overall, the levels of attachment were comparable to those observed by CFU counting although slight differences among strains were observed in the images. The CF early strains (AA2 and ATM 0060-3), 57P31 PA and A5803 tended to bind to the cells on the edges of the cells (Figure 2), while LES 431 and Mi 162 distributed across the cell surface part of the cell surface.

[0379] Identification of proteins involved in adhesion to HBE cells using the Cell Blot platform

[0380] Four P. aeruginosa clinical isolates with different host cell attachment levels were selected for the identification of novel potential adhesins: LES 431 as a low binder, ATM 0060-3 as an intermediate binder, and AA2 and A5803 as the highest binders. Nine cell blot experiments (A to I) were performed on these strains, and 6-11 positive spots were successfully matched in the corresponding gels, except for experiment A, in which only two spots were matched, probably because 1x105instead of 4x105cells / mL were used in the cell probing step (Table 3). The results obtained from the nine cell blot experiments performed are shown in Figures 3 to 6, showing the high reproducibility between replicates. These spots corresponded to cell binding proteins and did not occur due to non-specific Ab binding, as demonstrated by the negative controls (Figure 7). Most of the spots identified in all four strains were in the pH 4-7 range, and 40-75 kDa in size. One large spot (approximately 40 kDa and pH ~4) appeared consistently in 6 / 9 experiments and 3 / 4 strains (F1 , 11 , Figure 3; G1 , H1 , Figure 5; and E1 , N10, Figure 6). Similarly, a smaller spot (15-20 kDa and pH ~8) was consistently detected in 5 / 9 experiments, and 3 / 4 strains (F4, I5, Figure 3; A1 , C1 , Figure 4; and G5, Figure 5).

[0381] Table 3: Number and ID of cell blot experiments performed, bacterial protein samples strain used, and number of spots identified in each of them.

[0382] LES 431

[0383] (CF, transmissible)

[0384] AA2

[0385] (CF, early)

[0386] 57P31PA

[0387] (COPD)

[0388] A5803

[0389] (CAP)

[0390] ID, Identification.

[0391] After trypsin digestion of the matched spots in the nine cell blot experiments and LC-MS analysis, a total of 238 proteins were identified, of which 32 were pre-selected based on frequency of identification, as these proteins were identified in, at least, three out of experiments and two out of four strains. A thorough literature review yielded evidence that six proteins are already known to be involved in P. aeruginosa host attachment (OprF, EF-Tu, FliC, LpdG, OprQ and DnaK), and two of these had already been tested as vaccine antigens (OprF, FliC). UniProt and MoonProt databases predicted 8 proteins as having a membrane location in P. aeruginosa (OprF, AtpD, OpmH, FliC, BamB, PepM16, OprQ, HemX, Pa1550) and six as moonlighting proteins in different bacteria (GroEL, FtsZ, EF-Tu, GlnA, LpdG and DnaK), meaning that they can be found either in the cytoplasm or the membrane and perform more than one function. According to a BLASTp-based search, 34 proteins showed little or no homology with human, mouse, and / or E. coli proteins.

[0392] Confirming the role of antigens in attachment to lung epithelial

[0393] To confirm that HfIK, OprM, FtsZ, OpmH and LptH were involved in host cell attachment, the levels of host cell attachment of IPTG-induced BL21 E. coli cells recombinantly expressing one of the proteins were compared to their uninduced BL21 counterparts, using a confocal microscopy-based method. An increase in attachment was observed in BL21 cells overexpressing either OprM, FtsZ, OpmH or LptH (Figure 8, A, B, C). The involvement of HfIK in attachment to HBE cells could not be confirmed, as no increased attachment was observed in the induced culture, which may suggest it requires other cofactors or components in the process. The expression of the recombinant proteins in induced cultures was confirmed by SDS-PAGE visualisation and Western blot detection (Figure 8, D). As expected, no increase in the level of attachment of the BL21 control strain was observed.

[0394] Production and purification of recombinant proteins batches

[0395] Expression conditions were optimised and the recombinant proteins OprM, FtsZ, OpmH, and LptH were expressed and purified. High levels of expression were achieved for all proteins (Figure 9). The subsequent steps (dialysis and concentration with Amicon® filters) increased the purity of the final batches to over 90% in all the cases, as showed by the densitograms generated with the Image J software (Figure 9). The identity of each protein was confirmed by mass spectrometry (Table 4). Endotoxin levels were determined to be below 1.1 EU / dose in all cases (Table 5).

[0396] Table 4. Confirmation of purified recombinant proteins’ identity by LC-MS. The sample numbers correspond to the numbers pointed in figure 3.17.

[0397] A0A0H2ZKJ8 PSEAB Major intrinsic multiple

[0398] 1 1.02E+09 52 AMR efflux outer membrane protein OprM

[0399] 2 9.01E+08 41 94.90% P47204 PSEAE Cell division protein FtsZ

[0400] _ O-7T- , no m on i / ui / Q9HUJ 1 PSEAE Probable outer membrane

[0401] 3 7.37E+08 37 83.40% . -TTprotein OpmH

[0402] . < / ir- , no oo m -z o / A0A0H2ZGK2 PSEAB Lipopolysaccharide

[0403] 4 5.4E+08 38 97.70%x- . export system proteinTLptA

[0404] Table 5. Endotoxin levels per mouse (50 pL) of the final batches of the four recombinant proteins. rOprM 0.24 rFtsZ 0.18 rOpmH 1.11 rLptH 0.13

[0405] Serological analysis of immune response to P. aeruginosa antigen immunisation in mice

[0406] Immunisation of mice elicited humoral responses as indicated by statistically significant increases in the production of total antigen-specific serum IgG, compared to mice immunised with SAS alone (Figure 10). These results indicate that rOprM, rFtsZ, rOpmH and rLptH are immunogenic antigens. The reciprocal endpoint antibody titres in all cases were 7,812,500. When the presence of different IgG subtypes in sera from immunised mice was assessed, it was observed that vaccination with any of the four antigens significantly raised the production of both lgG1 and lgG2c, compared to mice immunised with SAS alone (Figure 11), indicating that they have stimulated a mixed Th1 / Th2 immune responses (Figure 12).

[0407] Protection from P. aeruginosa infection in a murine acute pneumonia model

[0408] Two weeks after the third immunisation, mice were challenged with 6.3x106- 1.8x107CFU / mouse via oropharyngeal aspiration, and the bacterial burden in the lungs, spleens, and stomachs was determined 24 h post-infection. Immunisation with any of the four antigens reduced bacterial colonisation of the lungs (Figure 13). More precisely, immunisation with rFtsZ and rLptH significantly reduced bacterial counts in the lungs (p-value = 0.0226 and 0.0059, respectively), achieving a 1.9- and 1.1 -log reduction, respectively (Figure 13). Immunisation with rOprM and rOpmH also reduced bacterial burden by 0.7 and 0.8 log, respectively (figure 13). Vaccination with rFtsZ, rOpmH or rLptH, but not rOprM, also reduced bacterial dissemination, as lower bacterial loads were observed in spleens (Figure 13 C, D). These were reduced by 1.8, 0.8, and 0.7 log, respectively (Figure 13, B), and only rFtsZ resulted in a significant bacterial reduction in this organ (p-value = 0.0026, 0.6283 and 0.1052, respectively). Finally, immunisation with either antigen did not reduce bacterial loads in the stomach (Figure 13 E, F). Overall, rFtsZ was the most protective antigen, as it significantly reduced bacterial loads in both lungs and spleens, followed by rLptH. These reductions led to a statistically significant improvement in mice severity scores (p-value = 0.0245 and 0.0493, respectively) (Figure 14 C, D), but no differences were observed in weight loss between groups (Figure 14 A, B). The severity scores of OpmH immunised mice also improved relative to adjuvant control (Figure 14, C) highlighting that rOpmH has potential as a protective antigen in a multivalent vaccine.

[0409] LptH and OprM are involved in P. aeruginosa attachment to lung epithelial cells.

[0410] To confirm that LptH and OprM were involved in host attachment, expression of rLptH or rOprM was induced in E. coli BL21 cells and attachment was examined by confocal microscopy. The IPTG-induced BL21_OprM and BL21_LptH clones showed 8.8- and 3.5-fold increases in attachment to HBE cells in vitro, respectively, compared with the uninduced BL21 counterparts which was also confirmed visually (Figure 15 a, b). Protein expression under these conditions was confirmed by SDS-PAGE and Western blot (Figure 15c). This increase in attachment is not due to non-specific effects of IPTG, as no increased attachment was observed (0.83-fold; Figure 15a, 1b) in the BL21_control strain holding the empty plasmid.

[0411] LptH significantly reduced bacterial burden in an acute pneumonia challenge model.

[0412] Both rOprM and rLptH antigens were purified using Ni-NTA affinity columns followed by dialysis to remove urea (Figure 16). Purities of 92 and 96%, respectively, were obtained based on the densitometry of SDS-PAGE gels (Figure 16 b-c, 2e-f). Prior to immunisation the endotoxin levels were determined to be 0.13 and 0.24 EU / dose for rOprM and rLptH, respectively. Immunisation of C57BL / 6J mice with three doses of 50 ug rLptH plus SAS as adjuvant (Figure 17 a) significantly reduced lung bacterial burden by 1.12 log™ CFU relative to the SAS-only controls (p=0.0059, Figure 17b), which represents a 13-fold reduction in bacterial burden. Of note, a reduction in spleen dissemination of 0.74 log™ CFU (5.5-fold) was observed following immunisation with rLptH, although this was not statistically significant (p=0.1052, Figure 17c). The mice were routinely assessed for severity over the course of the experiment, and it is interesting to note that the severity scores were also significantly reduced in the rLptH-immunised mice post-challenge (p=0.0493, Figure 17d).

[0413] LptH generated high antigen-specific antibody titres and induced a mixed Th1 / Th17 response.

[0414] Serological analysis of immunised mice revealed that both LptH and OprM were also very immunogenic and stimulated the production of total IgG, lgG1 and lgG2c (Figure 18 a-f). Reciprocal endpoint total IgG titres for both antigens were 7.8x106. In addition, isotype analysis showed that titres for lgG1 and lgG2c were 7.8x106and 3.85x106for rLptH, and 7.8x106and 5.3x106for rOprM, respectively. The lgG2c / lgG1 ratios were 0.5 and 0.7, respectively, suggesting that both antigens stimulated a mixed Th1 / Th2 response. Overall, these data demonstrate that while both antigens stimulated strong antibody responses, only rLptH was protective, and other factors apart from antibody production may influence the protective efficacy of the vaccine antigens.

[0415] To explore the protective mechanisms of LptH further, we evaluated recall responses in splenocytes of immunised mice. Enumeration of cytokine-secreting cells via ELISpot after ex vivo restimulation with the antigen showed that rLptH significantly stimulated the proliferation of IFN-y and IL-17-producing cells by 4.83- and 9.87-fold (p=0.0004, p=0.0001 , respectively) in splenocytes from the mice immunised with a single dose of the antigen, in comparison to adjuvant-immunised mice (Figure 19a-b). These suggest that the pro-inflammatory Th1 / Th17 environment may be important to protect against P. aeruginosa infection. In similar ELISpot analyses, but FtsZ and OpmH also stimulated IFN-y cytokine responses in splenocytes from immunised mice that were re-exposed to antigens, confirming antigen-specific recall responses (Figure 19c).

[0416] A trivalent vaccine combining rLptH, rFtsZ and rOpmH confers protection against P. aeruginosa acute infection.

[0417] Immunisation with rLptH and FtsZ showed protection against acute P. aeruginosa pneumonia in mice. Moreover, rOpmH immunisation reduced bacterial burden and stimulated potent IFN- Y responses in splenocytes from immunised mice. This led the inventors to examine whether combining rLptH and / or rOpmH with rFtsZ in bivalent or trivalent formulations (25 pg per antigen) could enhance protection against P. aeruginosa lung infection. The total endotoxin content of the antigens used in the trivalent immunisations was 0.0043 EU / dose. The trivalent vaccine was the most protective vaccine candidate following the same vaccination schedule (Figure 20a), as it significantly reduced the bacterial burden in the lungs by 2.33 log™ CFU relative to SAS-only group (p=0.0091 , Figure 20b) and by 2.05 log™ CFU relative to vehicle- immunised group (p=0.046; Figure 20b). Bacterial dissemination was also reduced by 1.85 log CFU (p=0.0271 ; Figure 20c), which translated into a significant improvement in animal welfare (p=0.0022, p=0.0003, Figure 20d). Immunisation with a bivalent vaccine containing rFtsZ and rOpmH significantly decreased bacterial loads in the lungs by 1.3 log™ CFU (p=0.0357, Figure 20b). All three combinations improved animal welfare scores relative to mice that received PBS or Adjuvant (SAS) alone (Figure 20d), highlighting that all multivalent combinations stimulated protective responses.

[0418] DISCUSSION

[0419] Recombinant E. coli clones overexpressing OprM, FtsZ, OpmH and LptH, antigen candidates showed increased attachment to HBE cells. These results confirm their involvement in host cell attachment. It is the first time that these proteins are reported to participate in host attachment, so the specific mechanism used to bind to lung cells remains unknown. OprM and OpmH are surface located porins that are part of two distinct efflux pump complexes, facilitating direct interaction with host cell receptors (Mima, T., et al., Identification and characterization of TriABC-OpmH, a triclosan efflux pump of Pseudomonas aeruginosa requiring two membrane fusion proteins. J Bacteriol, 2007. 189(21): p. 7600-7609; Pesingi, P.V., et al., MexAB-OprM Efflux Pump of Pseudomonas aeruginosa Offers Resistance to Carvacrol: A Herbal Antimicrobial Agent. Front Microbiol, 2019. 10: p. 2664).

[0420] All four antigens were shown to be immunogenic, as high antigen-specific serum IgG titres were produced in response to vaccination, compared to mice immunised with SAS alone. Despite the strong serological responses observed, not all antigens tested, e.g. OprM were able to protect mice against P. aeruginosa acute lung infection. Recombinant FtsZ proved to be the most efficacious antigen, as it significantly reduced bacterial colonisation in the lungs and spleens, improving animals’ welfare. Mice immunised with FtsZ from S. suis serotype 2 in white oil adjuvant, either as a recombinant protein or DNA vaccine, were also protected against lethal challenge (60 and 70% survival over 10 days, respectively). Immunisation with rLptH also showed promising results, as it reduced bacterial burden the lungs.

[0421] Mice immunised with rOpmH had lower bacterial loads in both organs despite not being statistically significant. Small reductions in bacterial loads can lead to substantial improvements in animal survival. Indeed, the results from the model development studies indicated that small differences in CFU dosage and recovery had a high impact on mice severity. Moreover, other studies have reported that vaccines leading to <1.5 log CFU reduction, including P. aeruginosa ghosts (Sheweita, S.A., et al., Bacterial Ghosts of Pseudomonas aeruginosa as a Promising Candidate Vaccine and Its Application in Diabetic Rats. Vaccines (Basel), 2022. 10(6): p. 910), alginate (Farjah, A., et al., Immunological evaluation of an alginate-based conjugate as a vaccine candidate against Pseudomonas aeruginosa. APMIS, 2015. 123(2): p. 175-183), or LasB peptides (Sokol P.A., et al., Immunization with a Pseudomonas aeruginosa elastase peptide reduces severity of experimental lung infections due to P. aeruginosa or Burkholderia cepacia. J Infect Dis, 2000. 81 (5): p. 1682-1692), translated into lung pathology improvement or 60-100% survival over 6- 7 days. Bioinformatic analysis showed that OpmH was the least conserved protein in P. aeruginosa strain KK1 , with eight conservative and five non-conservative mutations, which may have compromised rOpmH efficacy when tested against this strain. The least protective antigen was rOprM as it reduced bacterial loads in the lungs (almost 1 -log), and only a slight increase in the spleens was observed, neither of which were statistically significant.

[0422] Prophylactic vaccination may not be feasible in all susceptible individuals, such as critically ill patients, immunocompromised hosts, or people who suffer burns or are admitted to ICUs unexpectedly, as there often is insufficient time or capacity to mount a strong immune response, especially if booters are needed. Indeed, a study with 69 patients showed that the number of days admitted to the ICU before P. aeruginosa infection occurred was 31 , on average. Under this scenario, passive immunisation that provides short-lasting but immediate action irrespective of the immune state of the recipient could be useful. To date, five monoclonal antibodies and one polyclonal antibody have been evaluated in clinical trials, and none of these is currently approved for the market as no clinical benefit over the placebo group was observed (Lopes, J. A., et al., Overcoming Barriers to Preventing and Treating P. aeruginosa Infections Using AAV Vectored Immunoprophylaxis. Biomedicines, 2022. 10(12): p. 3162). Although there may be other factors behind their lack of efficacy, the inadequate selection of the antigen may be one of them; actually, the abovementioned antibodies were directed to single serotypes of LPS / alginate, flagellin, or PcrV which are antigenically variable virulence factors and / or are modified over the course of infection. Therefore, the protective antigens identified in this invention, FtsZ and LptH, which seem to be conserved, may also be suitable for immunotherapies. This would be done by retrieving serum from immunised mice or rabbits to prepare antigen-specific IgG-enriched fractions, and then administering these to mice that would be subsequently challenged using relevant mouse models (e.g., burn infection, persistent pneumonia).

[0423] The inventors proposed that multivalent vaccines will provide optimal protection against this pathogen, which led the inventors to examine the three most promising antigen candidates namely rFtsZ, rOpmH and rLptH, in trivalent and bivalent formulations. The trivalent vaccine was the most effective, as it significantly reduced lung bacterial loads by more than 2.33 iogio CFU. Several antigens conferred better protection against P. aeruginosa than the individual antigens likely because they contain a higher number of immunogenic epitopes than monovalent formulations. Of note is that the formulation of the multivalent vaccines included lower doses of each individual antigen relative to those used in the monovalent formulations.

[0424] Equivalents

[0425] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

Claims1. A composition comprising one or more isolated immunogens selected from P. aeruginosa FtsZ, OpmH, LptH, ArcA, LpdG, GuaB, BamB, TssK1, ALAD, HfIK, , AguA, GlnA, , HsIU, GlmU, FumC2, PchG, , MmsA, CycH, OprQ, GltA, HGDO, MetK, PrpD, llvC, PsIB, HemX, ApeB, FixH, YjiA and peptidase M16 (or a nucleotide or mRNA encoding same).

2. The composition of Claim 1 , comprising two or more of the immunogens.

3. The composition of Claim 1 or 2, comprising three or more of the immunogens.

4. The composition of any one of the preceding claims, comprising at least FtsZ immunogen (or a nucleotide or mRNA encoding same).

5. The composition of any one of the preceding claims, comprising at least OpmH immunogen (or a nucleotide or mRNA encoding same).

6. The composition of Claim 3, comprising at least FtsZ, OpmH and LptH immunogens (or a nucleotide or mRNA encoding same).

7. The composition of Claim 3, wherein the composition comprises only three immunogens and wherein the immunogens are FtsZ, OpmH and LptH.

8. The composition of any one of the preceding claims, wherein the immunogen is an immunogenic portion.

9. The composition of any one of the preceding claims, wherein the immunogen is a polypeptide or a peptide.

10. The composition of any one of the preceding claims, wherein the composition is a vaccine.

11. The composition of Claim 10, wherein the vaccine is a subunit vaccine.

12. The composition of Claim 10, which is a DNA vaccine and comprises one or more nucleic acid sequences.

13. The composition of Claim 10, which is an mRNA vaccine and comprises one or more mRNA sequences.

14. The composition of any one of the preceding claims, further comprising one or more adjuvants.

15. The composition of any one of the preceding claims, comprising P. aeruginosa ALAD, HfIK, , ArgA, FtsZ, AguA, GlnA, , OpmH, HsIU, GlmU, FumC2, PchG, LpdG, GuaB, MmsA, CycH, BamB, OprQ, LptH, TssK1 , GltA, HGDO, MetK, PrpD, llvC, PsIB, HemX, ApeB, FixH, YjiA and peptidase M16, isolated immunogens ( or a nucleotide or mRNA encoding same).

16. The composition of any one of the preceding claims, for use as a medicament.

17. The composition of any one of Claims 1 to 15, for use in a vaccine therapy to prevent or treat infection by P. aeruginosa in a subject.

18. The composition for use of Claim 17, wherein the subject is one with a comprised immune system, a damaged epithelial barrier, a pulmonary disorder, or is a critically ill subject in an intensive care unit.

19. The composition for use of Claim 18, wherein the subject has a pulmonary disorder selected from cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (nCFBE), or ventilator-associated pneumonia (VAP).

20. The composition for use of any one of Claims 17 to 19, wherein the P. aeruginosa infection is an infection in the lower respiratory tract of the subject.

21. The composition for use of any one of Claims 17 to 19, wherein the P. aeruginosa infection is a bloodstream infection (BSI).

22. A composition comprising an antibody directed to at least one of P. aeruginosa ALAD, HfIK, ArcA, , FtsZ, AguA, GlnA, OpmH, HsIU, GlmU, FumC2, PchG, LpdG, GuaB, MmsA, CycH, BamB, OprQ, LptH, TssK1 , GltA, HGDO, MetK, PrpD, llvC, PsIB, HemX, ApeB, FixH, YjiA and peptidase M16, immunogens.

23. The composition of Claim 22, for use as a medicament.

24. The composition of Claim 22, for use in a method of treating P. aeruginosa infection in a subject.

25. The composition of Claim 22, for use in passive immunisation of a subject against P. aeruginosa infection.

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