Field of invention

The invention relates to insecticidal compounds, particularly saccharides substituted by at least one O-alkyl phosphoramidate group. The invention also relates to an insect model for Campylobacter infection.

Background

Campylobacter jejuni is the leading bacterial cause of gastroenteritis in the world causing 2.4 million cases alone in the US per annum and affecting 1% of the population (Alios (2001) Clin. Infect. Dis. 32 1201-6). In developing countries Campylobacter is hyper-endemic, a leading bacterial cause of diarrhoeal disease and a major cause of infant mortality (Jannsen et al. (2008) Clin. Microbiol. Rev. 21 505- 18). Irritable bowel syndrome (IBS) is a post-infectious complication of campylobacteriosis compounding the economic burden of C. jejuni diarrhoeal disease (Jannsen et al. (2008). Furthermore, in some cases C. jejuni infection may result in complications such as meningitis, endocarditis, Reiter's syndrome and Guillain Barre Syndrome (GBS), including its variant Miller Fisher Syndrome (Jannsen et al. (2008)).

Given the medical and socioeconomic importance of Campylobacter infection, it is remarkable that C. jejuni is one of the least understood enteropathogens. The identity of its core virulence determinants and mechanisms of molecular pathogenesis have proved elusive. The absence of obvious enterotoxins or enterically active cytotoxins suggest that the organism causes diarrhoea in a manner quite distinct from that employed by well-established enteric pathogens such as Vibrio cholerae and the toxigenic pathovars of Escherichia coli. Association of C. jejuni with insects has previously been reported, with suggestions that insect vectors may play roles in the transmission between poultry or other animal reservoirs (Hald et al. (2008) Poult. Sci. 87 1428-34; Rosef & Kapperud (1983) Appl. Environ. Microbiol. 45 381-3). In support of this suggestion, C. jejuni has been isolated from up to 16% of fly species trapped at farm sites or in broiler houses (Hald et al. (2008)) and from over 43% of flies trapped at a piggery (Rosef & Kapperud (1983)). There are no reports of toxicity or killing of insects infected with C. jejuni, whether by natural or experimental infection.

Our poor understanding of C. jejuni is a consequence of two factors. Firstly, an historic lack of molecular tools for C. jejuni genetic manipulation which has now largely been overcome with the development of species-specific promoters and associated tools. Secondly, there is a lack of a suitable infection model. C. jejuni infection models previously used include a ferret diarrhoeal model, a colostrum deprived piglet model and a chick colonisation model. These have been used to identify potential virulence determinants. However, limitations such as reproducibility, cost, ease of use, breeding and specialised training associated with all of these models of infection have precluded their widespread use (Newell (2001) Symp. Ser. Soc. Appl. Environ. Microbiol. 57S-67S). In addition, because the avian gut is the normal site of carriage of the bacterium it is questionable whether avian colonisation models are able to identify virulence mechanisms involved in human infection (Jannsen et al. (2008)).

More recently, several transgenic mouse models have been reported which have applications for understanding C. jejuni mechanisms of infection, for example, limited flora, IL-10, MyD88 and Nrampl-/- mice (Champion et al. (2008) Microbes. Infect. 10 922-7; Mansfield et al. (2007) Infect. Immun. 75 1099-115; Watson et al. (2007) Infect. Immun. 75 1994-2003). However, these models are expensive and not widely accessible to the research community. In addition, due to genetic manipulations in mice that affect their immune status, the results must be carefully interpreted (Mansfield et al. (2007)).

An alternative to mammalian models of infection is the use of invertebrate hosts such as nematodes or insects. Caenorhabditis elegans has attracted attention as an infection model for a diverse range of bacterial pathogens including Burkholderia sp., Pseudomonas aeruginosa, Salmonella enterica, Yersinia pestis, Staphylococcus aureus, Acinetobacter baumannii and the fungal pathogen Cryptococcus neoformans. However C. elegans cannot survive at 37°C and lacks functional homologs of some components of the mammalian immune system, such as specialised phagocytic cells (Mylonakis et al. (2007) Plos. Pathogens 3 859-65). Furthermore, C. elegans dies rapidly under microaerophilic atmosphere, the conditions under which pathogens such as Helicobacter pylori and C. jejuni grow and survive.

Models of infection based on insects offer the advantage that they can be used at 37°C. In addition, insects possess specialised phagocytic cells also known as hemocytes or granulocytes (Mylonakis et al. (2007)). These cells are able to phagocytose pathogens and kill them using antimicrobial peptides and reactive oxygen species, which are generated during a respiratory burst. Therefore, insect hemocytes have many properties in common with mammalian phagocytes.

Insects are also relevant in agriculture, requiring control in view of the fact that many insects are responsible for significant damage to agricultural products such as crops. Insects can cause damage when in larval and/or adult form, for example, by reducing crop volume by eating leaves or burrowing in stems. Some insects can cause damage to other products or agricultural systems; for example, the greater wax moth (Galleria mellonella) lives in beehives when in larval form, feeding on wax and young bees and filling hive tunnels with silk threads. There is a need to develop alternative insecticides to those already in use, since many of these have unwanted effects on other flora and fauna and on the quality of soil, water and other resources. Similarly, insect control is often required in buildings, such as residential buildings, work buildings and storage facilities, as well as other human environments such as parks and gardens. There is a need for environmentally and human/animal friendly insecticide products for use in such environments.

Summary of invention

According to a first aspect of the invention, there is provided an insecticide compound comprising a saccharide substituted by at least one O-alkyl phosphoramidate group. The compound has the general structure represented by Formula I:

R ¹

O

I

-O— P— NH— R

O Formula I where R is hydrogen or Ci_ ₆ alkyl;

R ¹ is a saccharide; and

R ² is an alkyl group such Ci_ ₆ alkyl, for example, methyl.

In one embodiment, R is hydrogen and R ² is methyl. The saccharide may be a monosaccharide having 1-6 carbon atoms, such as an aldose (e.g., an aldotriose such as D-Glyceraldehyde, an aldotetrose such as D-Erythrose or D- Threose, an aldopentose such as D-Ribose, D-Arabinose, D-Xylose, D-Lyxose, and aldohexose such as D-Allose, D-Altrose, D-Glucose, D-Mannose, D-Gulose, D-Idose, D-Galactose, D-Talose) or a ketose (e.g., a ketotriose such as Dihydroxyacetone, a ketotetrose such as D-Erythrulose, a ketopentose such as D-Ribulose or D-Xylulose or a ketohexose such as D-Psicose, D-Fructose, D-Sorbose, D-Tagatose), a disaccharide (such as Sucrose, Lactulose, Lactose, Maltose, Trehalose, Cellobiose, Kojibiose, Nigerose, Isomaltose, β,β-Trehalose, Sophorose, Laminaribiose, Gentiobiose, Turanose, Maltulose, Palatinose, Gentiobiulose, Mannobiose, Melibiose, Melibiulose, Rutinose, Rutinulose or Xylobiose) or a polysaccharide (such as starch, glycogen, cellulose, chitin). All of the saccharides mentioned above are non-limiting examples only; the skilled person is readily able to determine whether a molecule is a saccharide.

In a preferred embodiment, the saccharide is a bacterial capsular polysaccharide. Such a polysaccharide may comprise repeating subunits of one to six monosaccharides and may have a molecular weight of 100-lOOOkDa and/or be water soluble and/or acidic. Alternatively or additionally, such a polysaccharide may lack the lipid A anchor of lipopolysaccharides.

Surprisingly, the inventors have found that the presence in C jejuni of saccharides modified with an O-alkyl phosphoramidate group is essential in order for exposure of an insect to C. jejuni to be fatal to the insect. Killing of insects by exposure to a Campylobacter bacterium has not previously been observed and the present inventors have also identified a saccharide modification which must be produced by the bacterium for this killing to occur. Therefore, the compound according to the invention may be obtainable from a Campylobacter bacterium, particularly a C. jejuni bacterium. The compound is preferably in an isolated and/or pure form, i.e., the compound is in the absence of a bacterium.

According to a second aspect of the invention, there is provided an insecticidal composition comprising a compound according to the first aspect of the invention, in combination with an environmentally acceptable carrier such as an agriculturally acceptable carrier or a carrier acceptable for use in household products. Therefore, the composition may be intended for use in agriculture, such as an insecticide. Alternatively, the composition may be intended for use in buildings or other non- agricultural environments such as gardens or parks, for example to purge a work, storage or home building or space from an infestation by insects, or to provide a general use composition for occasional insect repellent, deterrent or control. Insects which might be controlled in this way include bed bugs (e.g., Cimex lectularius), cockroaches (order Blattaria) and/or fleas (order Siphonaptera).

Such a composition may comprise solid and/or liquid dispersible carrier vehicles if desired, or be in the form of particular dosage preparations for a specific application made therefrom, such as solutions, emulsions, suspensions, powders, pastes and granules that are thus ready for use. The composition can be formulated or mixed with, if desired, conventional inert diluents or extenders of the type usable in conventional agricultural formulations or compositions, e.g., conventional dispersible carrier vehicles such as gases, solutions, emulsions, suspensions, emulsifiable concentrates, spray powders, ready-to-use (RTU) micro-emulsions, oil-in-water emulsions, pastes, soluble powders, dusting agents, granules, foams, pastes, tablets, aerosols, natural and synthetic materials impregnated with active compounds, microcapsules, coating compositions, and formulations used with burning equipment, such as fumigating cartridges, fumigating cans and fumigating coils, as well as ULV cold mist and warm mist formulations.

The formulations are preferred to be water soluble or miscible since they can be diluted in water before use to achieve an appropriate concentration.

Liquid treatments can be applied by spraying. Formulations include water-soluble powders (SP), soluble (liquid) concentrates, wettable powders (WP) or water- dispersable granules (WG). Solid formulations such as granules or briquettes, where the active ingredient is mixed with bulking agents such as sawdust, sand or plaster, can easily be used by introduction of the formulation into water containers such as tanks or latrines.

The composition may be formulated as an emulsifiable concentrate (EC). Generally, a 25-50% solution of the insecticide compound in a solvent is used and at least 10% solubility is typically needed to make the formulation economic to transport. The insecticide compound may be soluble in organic solvents but not in water. In addition to appropriate solvents, emulsifiers can be added to ensure that a fine oil drop (1-2 nm) in water emulsion is produced when the formulation is diluted with water. The resultant emulsion appears opaque and ideally does not settle for 24 hours. ECs are a convenient way of formulating water- insoluble ingredients and they do not cause nozzle abrasion.

Typical solvents for conventional emulsifiable concentrates are non-polar water- immiscible solvents or polar aprotic water miscible organic solvents. These solvents have very low solubilities in water and are capable of dissolving a wide range of active ingredients.

The non-polar solvents can be selected from the group consisting of aliphatic or aromatic hydrocarbons and esters of plant oils or mixtures thereof.

Aliphatic and aromatic hydrocarbons such as hexane, cyclohexane, benzene, toluene, xylene, mineral oil or kerosene or substituted naphthalenes, mixtures of mono- and polyalkylated aromatics are, for example, commercially available under the registered trademarks Solvesso, Shellsol, Petrol Spezial and Exxsol.

Esters of plant oils, which are used as nonpolar, water-immiscible solvents, can be alkyl esters obtainable from medium chained fatty acids by esterification with alkanols or by transesterification of the corresponding plant oils, preferably in the presence of a lipase. Preferred fatty acids of these plant oils have 5 to 20, in particular 6 to 15 carbon atoms. In one embodiment, the methyl ester of the plant oil used is the methyl ester of caprylic/capric ester or of capric ester having a distribution of fatty acid chain lengths around 10 units. Particular methyl esters of plant oils are Witconol 1095 and Witconol 2309 which are commercially available from the Witco Corporation, Houston, USA. Water-miscible polar aprotic organic solvents can be compounds which exhibit a dielectric constant of 2.5 or more at 25°C, in particular from 2.7 to 4.0 at 25°C. Particularly envisaged are cyclic amides and lactones, for example N-methylpyrrolidone, N-cyclohexylpyrrolidone and γ-butyro lactone and N-methylpyrrolidone or mixtures thereof.

Also envisaged are water-miscible polar aprotic solvents selected from the group consisting of alkyl lactates, in particular, isopropyl lactate such as plurasolv IPL which is obtainable from Plurac, alky carbonates, polyethylene glycols, polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, and most preferably particular isopropyl lactate, or mixtures thereof.

The emulsifiers may comprise at least one emulsifier which can be a non-ionic surfactant, ionic surfactant or a blend of both types of surfactants.

Examples of the nonionic surfactants which can be used include alkoxylate block polymers, alkoxylated alcohols, alkoxylated alkylphenols; alkoxylated amines, alkoxylated amides; alkoxylated fatty esters, alkoxylated oils, fatty esters, alkoxylated fatty acids and sorbitan derivatives. In a preferred embodiment the nonionic surfactants can include alkoxylated alcohols, ethoxylated glycerides and ethoxylated tristyryl. The nonionic emulsifier can be present in the emulsifiable concentrate in an amount of from about 1 to about 15% w/v. Examples of the ionic surfactants which can be used include: alkylaryl sulfonates; alkylaryl sulfonic acids; carboxylated alcoholethoxylates and alkylphenol ethoxylates; carboxylic acids/fatty acids; diphenyl sulfonate derivatives; olefin sulfonates; phosphate esters; phosphorous organic derivatives; quaternary surfactants; sulfates and sulfonates of oils and fatty acids; sulfates and sulfonates ethoxylated alkylphenols; sulfates of ethoxylated alcohols; sulfates of fatty esters; sulfonates of dodecyl and tridecylbenzenes; sulfonates of naphthalene and alkyl naphthalene; sulfonates of petroleum; sulfosuccinamates, alkanolamides and alkoxylated amine. In a preferred embodiment the ionic surfactant can be salts of dodecylbenzene sulfonic acid. The ionic emulsifier can be present in the emulsifiable concentrate in an amount of from about 0.5 to about 10% w/v.

An emulsifiable concentrate can also include an anti- freeze agent. Examples of suitable anti-freeze agents include relatively low molecular weight aliphatic alcohols such as ethylene glycol, propylene glycol, diethylene glycol, glycerine, urea, hexane diol, and sorbitol. Preferred anti-freeze agents include dipropylene glycol, diethylene glycol, glycerine, urea, hexylene glycol and propylene glycol. The anti-freeze agent can be present in the emulsifiable concentrate in an amount of from about 1 to about 10% w/v.

The compositions can also be used as ready-to-use (RTU) micro -emulsions. The RTU micro-emulsions can comprise at least one emulsifier, the examples of which are the same as used in emulsifiable concentrates as outlined above. The nonionic emulsifier can be present in the micro-emulsion in an amount of from about 0.002 to about 0.1% w/w. The ionic emulsifier can be present in the micro-emulsion in an amount of from about 0.002 to about 0.1 % w/v.

The RTU-micro-emulsions can also include an anti-freeze agent, the examples of which can be the same as used in emulsifiable concentrates as outlined above. The anti-freeze agent can be present in the micro-emulsion in an amount of from about 1 to about 10%) w/v.

The composition of the present invention may also be suited for aerosol-based applications, including aerosolized foam applications. Pressurised cans are the typical vehicle for the formation of aerosols. An aerosol propellant that is compatible with the composition is used. Preferably, a liquefied-gas type propellant is used. Suitable propellants include compressed air, carbon dioxide, butane and nitrogen. The concentration of the propellant in the insecticide composition can be from about 5% to about 40%) by weight of the composition, preferably from about 15% to about 30%> by weight of the composition. The formulation can also include one or more foaming agents. Foaming agents that can be used include sodium laureth sulphate, cocamide DEA and cocamidopropyl betaine. Preferably, the sodium laureth sulphate, cocamide DEA and cocamidopropyl betaine are used in combination. The concentration of the foaming agent(s) in the composition can be from about 10% to about 25% by weight, more preferably about 15%) to about 20%> by weight of the composition.

When the formulation is used in an aerosol application not containing foaming agent(s), the composition of the present invention can be used without the need for mixing directly prior to use. However, aerosol formulations containing the foaming agents do require mixing (e.g., shaking) immediately prior to use. In addition, if the formulations containing foaming agents are used for an extended time, they may require additional mixing at periodic intervals during use.

Alternatively or additionally to the inclusion of the compound according to the first aspect of the invention, the composition according to the second aspect of the invention may comprise at least one bacterium that produces the compound according to the first aspect of the invention. Such a bacterium may be a Campylobacter species such as C. jejuni (for example, strain NCTC11168, strain Gl, strain NCTC12517 or strain 81-176), or may be a bacterium of another species which produces the compound of the first aspect of the invention and/or may be a bacterium which comprises the cj 1416 gene (SEQ ID NO: l) or an equivalent gene from a C. jejuni strain which is not NCTC11168. The bacterium may be a recombinant bacterium or may be naturally occurring. The bacterium may be a Campylobacter species, for example, C. jejuni. The term "produces the compound" indicates that the compound is synthesised by the bacterium and may be retained within the bacterium, form part of the bacterial cell wall and/or cell membrane, and/or be secreted by the bacterium. In the latter case, the compound may, therefore, be detectable in a liquid medium in which the bacterium is cultured, i.e., living, growing and/or reproducing.

The present invention also encompasses the use of the compound or composition as an insecticide, or such a use of a bacterium which produces the compound. Therefore, according to a third aspect of the invention, there is provided a method of killing an insect comprising applying to the insect or an environment thereof, or administering to the insect, a compound according to the first aspect of the invention and/or a composition according to the second aspect of the invention and/or a bacterium that produces the compound according to the first aspect of the invention. The bacterium may comprise the cj 1416 gene or an equivalent gene from a C. jejuni strain which is not NCTC11168. The bacterium may be a Campylobacter species, for example, C. jejuni.

The step of "applying to the insect or an environment thereof may comprise any standard method of delivery of an environmental aid product (for example a household or agricultural product), such as by spreading, spraying, coating or other application of solid or liquid compounds or compositions.

The step of "administering to the insect" may comprise injection of the compound or composition into the insect, which may be at any stage of its lifecycle but may preferably be in larval form. The insect may be an insect of the family Pyralidae, for example, an insect of the genus Galleria, such as the species Galleria mellonella. In addition to Galleria mellonella, the family Pyralidae also includes the following species: sunflower moth (Homoeosoma nebulella), Indianmeal moth (Plodia interpunctella), meal moth (Pyralis farinalis) and lesser wax moth (Achroia grisella). The insect may be from another family in the order Lepidoptera, for example, the family Cossidae such as Chilecomadia moorei (or Chilean Moth). Alternatively, the insect may be, for example, of the family Stratiomyidae (for example, Hermetia illucens or Black Soldier fly) or the family Tenebrionidae (for example, Tenebrio molitor or Mealworm beetle) or the family Scarabaeidae (for example, Pachnoda marginata). In a further alternative, the insect may be a bed bug (for example, family Cimicidae such as Cimex lectularius), cockroaches (order Blattaria) and/or fleas (order Siphonaptera).

According to a fourth aspect of the invention, there is provided a method of assessing or determining the virulence of a Campylobacter bacterium comprising administering a sample containing the bacterium to an insect and observing whether the insect dies or has impaired activity within a given period.

The sample of the bacterium may be administered by injection of the bacterium into the insect. The insect may be at any stage of its lifecycle but may preferably be in larval form. The insect may be an insect of the family Pyralidae or other families in the order Lepidoptera, for example, an insect of the genus Galleria, such as Galleria mellonella or an insect of the family Cossidae such as Chilecomadia moorei (or Chilean Moth). Alternatively, the insect may be, for example, of the family Stratiomyidae (for example, Hermetia illucens or Black Soldier fly) or the family Tenebrionidae (for example, Tenebrio molitor or Mealworm beetle) or the family Scarabaeidae (for example, Pachnoda marginata). In a further alternative, the insect may be a bed bug (for example, family Cimicidae such as Cimex lectularius), cockroaches (order Blattaria) and/or fleas (order Siphonaptera). There are several advantages of G. mellonella larvae as an infection model. The larvae, which are the caterpillar stage of the wax moth, are commercially available and bred as live food for captive reptiles and amphibians. The larvae, which are typically 1-2 cm in length, are easy to handle and survive for up to 3 weeks before pupating. During this time they do not require feeding and require minimal maintenance. Unlike many invertebrate models of infection, it is possible to give precise doses of bacteria by injection. Therefore differences in virulence at 37°C can be revealed and this model is well suited to the screening of large panels of bacterial isolates or mutants. The Campylobacter bacterium may be, by way of non- limiting example, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hominis, C. hyointestinalis, C. insulaenigrae, C. jejuni, C. lanienae, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, C. upsaliensis. Preferably, the Campylobacter bacterium is a C. jejuni bacterium. The sample of bacterium may be prepared by growing bacteria on a surface such as an agar plate and transferring into liquid culture, or the sample may be prepared by growing bacteria in liquid culture. Samples prepared by growing bacteria in liquid culture are preferred. The sample may comprise 10-10 ⁸ CFU (Colony Forming Units, determinable by the skilled person) of bacteria, preferably 10 ² -10 ⁶ CFU, more preferably about 10 ² , about 10 ³ , about 10 ⁴ , about 10 ⁵ or about 10 ⁶ CFU. The "given period" may be from 20 minutes to 30 hours, preferably from 4-24 hours, for example, about 20 minutes or about 1, about 4, about 12 or about 24 hours. During the period, the insects may be maintained in an environment having a temperature of 20-4 FC, preferably 25-40°C, more preferably about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C or about 38°C.

The term "assessing or determining the virulence" indicates that the user of the method is assessing or determining whether the administration of the bacterium to the insect results in the death of the insect, or in altered behaviour or activity, such as altered speed or types of movement which can be achieved by the insect. "Impaired activity" may be measured as a reduction in the speed or the types of movement which can be achieved by the insect, or by a reduced or eliminated ability to reproduce. The infection of insects is accompanied by the generation of melanin. This response is especially marked in G. mellonella where the larvae change from a cream colour to a pale or dark brown colour. The precise role of melanisation in host defence is not known but during this process melanin becomes deposited around pathogens. Therefore, the "altered behaviour" may include the melanisation of the insect, especially when the insect is in the larval stage of its lifecycle, observable by a darkening in the external colour of the insect.

According to a fifth aspect of the invention, there is provided a method of selecting a test compound which prevents or reduces infection by a Campylobacter bacterium, comprising administering a sample containing the bacterium to an insect in the presence of the test compound. The compound is selected as a compound which prevents or reduces infection if the occurrence of death or impaired activity of the insect is reduced as compared to the occurrence in control insects to which a sample containing the bacterium has been administered in the absence of the test compound. According to a sixth aspect of the invention, there is provided a recombinant bacterium which comprises the NCTC1 1 168 cj 1416 gene (SEQ ID NO: l) and/or the NCTC1 1 167 cj l 132-1 152 gene cluster (SEQ ID NO:2) and/or the NCTC1 1 168 cj051 1 gene (SEQ ID NO:3). The bacterium may, alternatively or additionally, comprise a gene from a strain of C. jejuni which is not strain NCTC1 1 168 which is homologous to a gene having a nucleotide sequence of any of SEQ ID NOs: 1-3. Such a gene may, for example, have 95% or greater sequence identity to any of SEQ ID NOs: 1-3, determined, for example, using the BLASTP sequence comparison program (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 September 2010). The skilled person is readily able to determine the equivalent genes in other C. jejuni strains (see, for example, Karlyshev et al. (2005) Mol. Microbiol. 55 90-103). The bacterium may be a gram-negative bacterium. The bacterium may be a bacterium which is not C. jejuni or may be a non-Campylobacter bacterium and/or may be a bacterium known to have insecticidal activity in its naturally occurring form, for example, a bacterium such as Bacillus thuringiensis . The invention also provides the use of such a bacterium as an insecticidal agent.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

Brief description of Figures

Embodiments of the invention will now be described, by way of example only, with reference to Figures 1-4 in which:

Figure 1 demonstrates the ability of 10 ⁶ CFU of plate-grown human C. jejuni isolates (strain 11168H, Gl or 81-176) to kill G. mellonella at 37°C, with results shown after 24 hours;

Figure 2 demonstrates the ability of 10 ⁶ CFU of broth-grown C. jejuni mutants to kill G. mellonella at 37°C with results shown at 24 hours;

Figure 3 demonstrates killing of G. mellonella larvae by broth-grown C. jejuni 11168H, mutant cjl416 which lacks the O-methyl phosphoramidate capsule modification and its complemented derivative, results shown at 24 hours; and Figure 4 shows enumeration of C. jejuni 11168H, mutant cjl416 or its complemented derivative cjl416+ in G. mellonella larvae at times up to 24 h post challenge.

Examples

Methods and Materials

Strains and culture conditions

All bacterial strains and mutants used in this study are shown in Table 1. C. jejuni strain 11168H is a hypermotile variant of the sequenced strain NCTC11168 that readily colonises chickens. C. jejuni strains were cultured on Columbia agar plates (CBA) supplemented with either 5-9% (v/v) horse blood or with Skirrow selective supplement (Oxoid, UK) and 5-9% (v/v) horse blood in a variable atmosphere incubator (VAIN) (Don Whitley Scientific, UK) under microaerobic conditions (5% 0 ₂ , 85%) N ₂ , 10%) C0 ₂ ) at 37°C for 24 or 48 h. For some experiments bacteria were cultured in sealed jars in an atmosphere of 6%> O ₂ /10%> C0 ₂ conditions (CampyPak, Oxoid) for 48 h. Where necessary, CBA plates were supplemented with the antibiotics kanamycin (50 μg/ml) and/or chloramphenicol (15 or 30 μg/ml). H. pylori strains were grown in Brain Heart Infusion (BHI; Oxoid) broth supplemented with 10% v/v foetal calf serum or on Helicobacter selective agar (DENT), consisting of Blood Agar Base No. 2 (Oxoid) supplemented with 7% v/v lysed defribinated horse blood (TCS Microbiology, Botolph Claydon, UK) and DENT or Skirrow selective supplement (Oxoid), in a microaerobic atmosphere at 37°C.

For infections, bacteria were subcultured into 25ml of Mueller-Hinton broth (Oxoid) and grown under microaerobic conditions as before for 24 to 48 h. The bacteria were then adjusted to OD590 1.0 for infections. In some experiments bacteria were harvested from the plates into 1 ml of PBS and adjusted to OD ₅₉₀ 1.0. Infectious doses were confirmed by serially diluting the inoculum and plating out onto Columbia agar supplemented with horse blood. Infections at lower doses were adjusted accordingly. Bacteria collected from the hemolymph of infected larvae were serially diluted in PBS and plated onto Columbia agar supplemented with 5% (v/v) horse blood and Campylobacter selective supplement (Oxoid). G. mellonella killing assays

G. mellonella larvae were maintained on wood chips at 15°C. Larvae were infected with C. jejuni strains in 10 μΐ inocula by micro -injection (Hamilton) in the right foremost leg. The larvae were incubated at 25°C, 37°C or 42°C and survival and appearance recorded at 24 h intervals. PBS injection and no injection controls were used. Survival 24 h post-infection was recorded. To determine the numbers of bacteria and site of localization in the hemocoel, larvae were chilled on ice for 5 minutes. The rear 2 mm of larvae were aseptically removed and hemocoel was drained into a sterile 1.5 ml microcentrifuge tube. Bacteria were enumerated as described above. PBS injection and no injection controls were used. Bacteria were heat killed at 95°C for 5 min and sterility checked by streaking out. For each experiment 10 G. mellonella larvae were infected and experiments were repeated three times unless otherwise stated.

C. jejuni mutant construction

Mutants were constructed via insertion of the kan cassette into unique restriction sites present in the gene fragments from a pUC18 library. Gene cjl416 was inactivated via insertion of the kan cassette into Psil site of a fragment of this gene present in plasmid cam85cl 1. In order to mutate gene cjl324 the kan cassette was inserted into an EcoRV site of plasmid caml90. Derivatives with the correct orientation of the kan cassette (co-linear with the gene target) were selected by restriction analysis and used for transformation into C. jejuni strain 11168H. Allelic replacement, resulting in gene inactivation in Kan ^r clones selected after transformation, was confirmed by PCR analysis using gene-specific and kan specific primers. Insertional inactivation resulted in truncation of the cjl416, cjl324, cdtA and flaA gene products by 52%, 66%, 47%) and 97% from the C-terminus respectively.

C. jejuni complementation

Complementation of mutant 11168H/cjl416 was achieved via introduction of an intact copy of gene cjl416 into an intergenic region within the rrna gene cluster as. Briefly, the gene was PCR amplified using the primers: ak296: 5 ^* -GAATAATATTTAAATATGAATGCAATTATCTTAGCAGCAGG-3 (SEQ ID NO:4) ak297: 5 ^* -TAAGGATCTAGATTATTTTTCAATTTGCACCTCTAAATCACTC-3 (SEQ ID NO:5)

The product was digested by Swal and Xbal restriction enzymes and cloned into Swal/Xbal digested vector pRRC for expression under the control of a constitutive earn promoter. The recombinant plasmid containing cam ^r -cjl416 cassette was transformed into mutant 11168H/cjl416 strain followed by selection of Cm ¹ and Kn ^r colonies. Integration of the gene cassette into the chromosome was confirmed by PCR as described (Karlyshev & Wren (2001)).

Susceptibility of other insect larvae to 11168H

The susceptibility of other insect larvae (Tenebrio molitor (mealworms), Chilecomadia moorei (Chilean moth), Hermetia illucens (Phoenix worms or Black Soldier Fly) and Pachnoda marginata) to killing by Campylobacter jejuni strain 11168-H was tested. All larvae were maintained at 15°C.

Groups of 5 larvae were inoculated with C. jejuni 11168-H by ΙΟμΙ micro-injection with a Hamilton syringe. C. moorei were inoculated via the right foremost pro-leg; H. illucens and T. molitor were inoculated more randomly at gaps in their cuticle. Groups of larvae were also injected with ΙΟμΙ of PBS or left uninoculated. The groups were then incubated at 37°C, and their survival and appearance were noted at 24 hours post-infection. Effect of C. jejuni 11168A1416 on other insect larvae

The inventors next investigated whether C. jejuni 11168Δ1416 was able to kill these other insect larvae. After inoculation, the groups were then incubated at 37°C and their survival and appearance were noted at 24 hours post-infection.

Results

Genetically diverse C. jejuni strains kill G. mellonella

To establish the relevance of G. mellonella as an infection model to screen for variations of virulence in wild type strains of C. jejuni, the lethality of a panel of well- characterised C. jejuni strains was determined; 11168H (human diarrhoeal isolate), Gl (human GBS isolate) and 81-176 (human diarrhoeal isolate) in G. mellonella (Fig. 2). Figure 1 shows that infection with approximately 10 ⁶ CFU of C. jejuni strain Gl which had been cultured on an agar plate at 37°C resulted in a 25% average survival rate at 24 h post challenge. Similar doses (suspensions with an OD ₆ oo of 1.0) of C. jejuni strain 81-176 or strain 11168H resulted in 43% or 36%> respectively average survival rates at 24 h. In later studies it was found that the conditions under which C. jejuni strain 11168H was cultured affected virulence in the mortality virulence in G. mellonella larvae. Whereas some larvae survived a 10 ⁶ CFU challenge if the bacteria were grown on agar at 37°C, all of the larvae were killed if C. jejuni was cultured at 37°C in broth. All subsequent experiments reported below were carried out with broth-grown C. jejuni.

Cjlll68H actively kills G. mellonella in a dose responsive manner

The survival of G. mellonella larvae following injection of 1 x 10 ² CFU, 1 x 10 ⁴ CFU or 1 x 10 ⁶ CFU of C. jejuni 11168H and incubation at 37°C was monitored 4 h and 24 h post challenge. At the highest dose (1 x 10 ⁶ CFU) melanisation of all of the larvae was observed at 4 h, although all the larvae were scored as alive. Following challenge with 1 x 10 ² CFU, lxl 0 ⁴ CFU or lxl 0 ⁶ CFU the number of surviving larvae at 24 hours post challenge was 30%>, 10%> or 0%> respectively. A distinctive black melanisation was observed in all dead larva. When lxlO ⁶ CFU of C. jejuni 11168H were heat killed and then injected into G. mellonella larvae, no melanisation of larvae or deaths were observed at up to 24 h post challenge, indicating that C. jejuni has an active G. mellonella killing mechanism. In contrast, injection of lxlO ⁶ CFU of the related pathogen H. pylori (strains SSI and 26696) did not result in the death of larvae under the same conditions (data not shown).

Survival of G. mellonella following infection with C. jejuni can be monitored at 37°C but not 42°C

All of the G. mellonella larvae (N=10) challenged with approximately 10 ⁶ CFU of C. jejuni 11168H and incubated at 25°C or 37°C were dead at 24 h. Although all of the challenged larvae (N=10) incubated at 42°C were dead at 24 h, the uninfected controls also died at this temperature. Therefore, this model may not be suitable to investigate genes that are expressed at 42°C (avian gut temperature). G. mellonella model reveals variable levels of attenuation in C. jejuni 11168H mutants

To establish the relevance of G. mellonella as a C. jejuni virulence screen, larval survival was recorded following challenge with 10 ⁶ CFU of a range of C. jejuni strains with gene insertional inactivations (Table 1).

All of the uninfected control larvae or larvae challenged with PBS were alive at 24 h, whereas none of the larvae challenged with C. jejuni 11168H survived. The mutants showed differing abilities to kill G. mellonella larvae, as shown in Figure 2. A flagella glycosylation mutant (insertion in gene cjl321) and a mutant which lacks legioniminic acid modification to the O-linked glycan of the flagella (insertion in gene cjl324) were not significantly attenuated compared to wild type C. jejuni. The other mutants tested showed different degrees of attenuation. A mutant which lacked the LOS (lipooligosaccharide) biosynthesis cluster (insertion in gene cluster cjl l32c to cj 1152c) was the most highly attenuated mutant in G. mellonella. In this study, a panel of C. jejuni mutants was initially screened for virulence in G. mellonella. The results indicate that it is possible to discriminate attenuated mutants in G. mellonella and, moreover, it is possible to assess degrees of attenuation. The inventors have found that mutants cjl321 and cjl324, which are defective in glycosylation of flagella, are not attenuated in G. mellonella. Previously it has been reported that the flagellar glycosylation system plays a role in colonisation of chickens (Karlyshev et al. (2004)) but the role of this system in disease of humans is not known.

O-methyl phosphoramidate capsule modification is essential for virulence in G. mellonella model

It is known that most C. jejuni strains have capsular polysaccharides with an unusual modification of O-methyl phosphoramidate. To investigate whether the O-methyl phosphoramidate modification played a role in virulence of C. jejuni, G. mellonella larvae were challenged with C. jejuni 11168H mutant ls.cjl416, which specifically lacks the O-methyl phosphoramidate modification to the capsule, as well as with its complemented derivative cjl416/cjl416+. After 24 h all of the larvae challenged with the wild type or cjl416 complemented strains had died (see Figure 3). However, all of the larvae challenged with Acjl416 survived. Table 1 C. jejuni strains & mutants tested for virulence in this study

[Source publications: (a) Karlyshev et al. (2002) Microbiol. 148 473-80; (b) Korlath et al (1985) J. Infect. Dis. 152 592-6; (c) Karlyshev et al. (2000) Mol. Microbiol. 35 529-41; (d) Karlyshev et al, unpublished; (e) Oldfield et al. (2002) J. Bacteriol. 184 2100-7; (f) Howard et al (2009) Infect. Immun. 77 2544-2556; (g) Jones et al (2004) Infect. Immun. 72 3769-76; (h) McNally et al. (2007) J. Biol. Chem. 282 28566-76.] In a subsequent experiment, bacterial load was measured 20 min, 1 h, 4 h or 24 h post challenge. As can be seen in Figure 4, four hours post challenge a reduction in bacterial load was observed in larvae infected with ls.cjl416 compared with wild type and complemented cjl416. Due to larval death, the bacterial load of larvae infected with wild type and complemented cjl416 could not be measured 24 h post infection. However, no bacteria were recovered from larvae infected with ls.cjl416.

The rapid death of G. mellonella larvae infected with C. jejuni suggested the role of a secreted protein in killing. Some of the genetically defined mutants in this study have previously been shown to be attenuated in other infection models. For example, the cyto lethal distending toxin (CDT; c 0079) has been shown to play a role in persistent infection and promoting mucosal inflammation in susceptible mouse strains (Lara- Tejero & Galan (2000) Science 290 354-7) and the present inventors have shown that a CDT mutant was attenuated in G. mellonella. The attenuation of an aflagellate mutant of C. jejuni (cjl339) in G. mellonella may reflect the suggestion that CDT is secreted through the flagella (Zheng et al. (2008)).

The inventors' finding that a lipooligosaccharide (LOS) biosynthesis mutant was completely attenuated in G. mellonella is consistent with the observation that phase variation of LOS affects the invasion of cultured cells (Guerry et al. (2002) Infect. Immun. 70 787-93). However, these results are the first direct evidence that LOS is essential for virulence of C. jejuni. The role of other genes, revealed by the testing of mutants in this study, provides new insight into their possible roles in virulence. For example, cj0511 is defective in the production of a protease and the finding that it was markedly attenuated in G. mellonella indicates that it merits further attention as a virulence determinant. The inventors have shown that a mutant of C. jejuni defective in the production of capsular polysaccharide was markedly attenuated in G. mellonella. A mutant lacking a specific O-methyl phosphoramidate modification of the C. jejuni capsule was attenuated and virulence was fully restored on complementation. This modification is found in many, but not all strains of C. jejuni, and is especially associated with isolates from enteritis, Guillain Barre Syndrome and Miller Fisher Syndrome patients. All three wild type strains tested in that study possess the O-methyl phosphoramidate modification to the capsule. It seems possible that the killing of G. mellonella larvae is a consequence of the toxicity provided by the O-methyl phosphoramidate modification.

Although the currently available animal models of C. jejuni infection including ferret diarrhoeal model, colostrum deprived piglet model and chick colonisation model have been used to identify virulence determinants, the relevance of these models to human disease is not clear. An additional limitation of these models is that they are unsuitable for screening of large numbers of mutants. The results disclosed in the present application demonstrate that Galleria mellonella larvae can be a useful screen to identify potential virulence determinants. The effect of C. jejuni 11168H and mutant Acjl416 on other insects

After inoculation of insects with C. jejuni 11168-H and incubation at 37°C, their survival and appearance were noted at 24 hours post-infection. The number of survivors in recorded in Table 2. For some of the insect larvae mortality was seen even when injected with PBS. However, increased mortality was seen when larvae were challenged with C. jejuni 11168-H

Table 2 Survivors/total of uninfected insect larvae of larvae challenged with

PBS, C. jejuni 11168-H

The results after inoculation with C. jejuni 11168Δ1416 are shown in Table 3. The results indicate that C. jejuni 11168 Δ1416 was attenuated. Table 3 Survivors/total of uninfected insect larvae of larvae challenged with PBS, C. jejuni 11168-H or C. jejuni 11168-H Δ1416

Larvae Uninfected PBS C. jejuni C. jejuni 11168

11168-H Δ 1416

G. mellonella 5/5 5/5 0/5 Melanised 5/5

C. moorei 5/5 2/5 0/5 Darkened 3/5 Dead

desiccated

T. molitor 5/5 2/5 0/5 4 darkened 1/5 Dead

blackened

P. marginata 5/5 3/5 2/5 Dead 3/5 Dead

darkened darkened