WO2010064141A1 - Assay - Google Patents

Abstract

The present invention provides a method for determining modulatory activity of an agent on a test cell or organism, comprising providing a sample comprising a reporter cell and a test cell or organism; contacting the agent with the sample; and detecting a signal associated with the reporter cell, thereby determining the modulatory activity of the agent on the test cell or organism.

Description

ASSAY

FIELD

The present invention relates to the field of assays for studying the growth and/or survival of cells or organsims, including pathogens such as bacteria, protozoa, fungi, cancer cells or pests. The assays may be used to identify agents which act on the cells, for example to identify agents which may be used for treating a disease caused by a pathogen.

BACKGROUND

Infectious and parasitic diseases are the second most frequent death cause in the world (after cardiovascular diseases). Respiratory infections alone cause 6.95 % of all deaths [I]. Even in countries with well-developed health systems, the emergence of multiresistant bacterial strains such as Methicillin-resistant Staphylococcus Aureus (MRSA) cause major problems. Some reports suggest that in 2005 more people in the United States died due to an MRSA infection (18000 victims) than due to AIDS (16500 victims; [2]). Hence there is a huge demand for novel antibiotics and assay systems allowing the screening of large compound libraries for antimicrobial properties.

Assays for the screening of potential antibiotics can be divided into two main types:

Biochemical assays make use of isolated and purified proteins of the pathogen. For example, drug candidates can be assayed for the inhibition of essential metabolic pathways. However, this strategy requires well-characterized drug targets and might lead to the selection of compounds that do not show any significant activity on the whole pathogen (instead of the purified target). For example, the selection of compounds that do not penetrate or persist in cells constitutes a significant problem [3], especially since many bacterial strains express so-called drug efflux pumps whose properties on small molecules are hardly predictable.

These limitations can be circumvented using whole-cell assays. In those systems phenotypic changes of the pathogen (e.g. growth arrest) are monitored in response to a drug candidate. In the simplest case, bacterial growth directly results in an easily detectable readout signal. Corresponding systems based on absorption, fluorescence, bioluminescence (Bio-Siv) and the release of radioactively-labelled CO₂ (BACTEC) have been developed [4],[5],[6],[7]). More complex systems allowing drawing conclusions about the involved drug targets have also been established. For example, arrays of bacterial strains, each one hypersusceptible for the inhibition of one particular gene have been generated [8]. Furthermore, assays based on mass spectrometric analysis of cell lysates have been performed to screen compounds for their ability to specifically inhibit one particular drug target [9]. However, these kinds of assays required detailed knowledge about the pathogen and its potential drug targets as well as genetic modifications of the corresponding microorganisms.

One common limitation of biochemical and whole-cell assays is the potential selection of compounds that also harm human cells. Especially when using purified pathogen-specific drug targets, cytotoxic side effects on human cells cannot be ruled out completely. Therefore, a primary screen for hits is usually followed by a secondary screen to determine the cytotoxicity in human cells.

WO 2006/082385 discloses an assay for viral inhibitors, wherein viral entry is coupled to downregulation of a reporter gene. A positive signal therefore identifies candidate inhibitory compounds. However, there is no screening method for inhibitors of microbial pathogens such as bacteria which uses a positive reporter signal as indicative of inhibitory activity.

Accordingly there is a need for an improved assay for identifying compounds which act on microbial pathogens. Likewise, there is a need for an improved assay for identifying compounds which acts on other types of cells and organisms, including cancer cells, multicellular organisms and agricultural pests.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for determining modulatory activity of an agent on a test cell or organism, comprising providing a sample comprising a reporter cell and the test cell or organism; contacting the agent with the sample; and detecting a signal associated with the reporter cell, thereby determining the modulatory activity of the agent on the test cell or organism. In another aspect, the invention provides a method for identifying an agent which modulates growth and/or survival of a test cell or organism, comprising determining modulatory activity of a plurality of candidate agents by a method as described above, and selecting an agent showing elevated modulatory activity.

In another aspect, the invention provides a sample preparation comprising a reporter cell and a pathogenic cell or organism, wherein the reporter cell expresses an exogenous reporter gene, and wherein growth and/or survival of the pathogenic cell or organism is inversely related to expression of the exogenous reporter gene by the reporter cell.

In another aspect, the invention provides a kit for identifying an agent which modulates growth and/or survival of a pathogenic cell or organism, the kit comprising a reporter cell which expresses an exogenous reporter gene, a pathogenic cell or organism, and a growth medium and/or support surface suitable for supporting growth and/or survival of either the reporter cell or the pathogenic cell or organism, such that when the reporter cell and the pathogenic cell or organism are grown together in the presence of the growth medium and/or support surface, growth and/or survival of the reporter cell and the pathogenic cell or organism are inversely related.

Embodiments of the present invention provide a novel assay system based on the co- preparation (e.g. co-cultivation) of reporter (e.g. human) cells with a test cell or organism. The test cell or organism may, for example, be from a pathogen of interest. In this way, cytotoxic side effects of a candidate agent can be directly monitored during the primary screen. Furthermore the readout signal of the assay is generated by the reporter cells (instead of the test cell or organism), meaning that no genetic modifications of the test cell or organism (e.g. pathogen) have to be performed. Thus the method does not require any detailed knowledge about the employed pathogen and its potential drug targets.

In other words, the present assays are typically indirect inhibition assays. This means that the reporter signal for the assay readout is not generated by the test cell or organism (e.g. pathogen) which the agent is intended to inhibit, but instead by a further cell type (the reporter cell). This strategy allows the identification of agents which specifically inhibit the test cell without harming the further cell type. In one embodiment, the modulatory activity comprises modulation of the growth and/or survival of the test cell or organism, preferably inhibitory activity on the test cell or organism.

In one embodiment, growth and/or survival of the reporter cell and the test cell or organism are inversely related. For instance, the reporter cell and the test cell or organism may compete for nutrients, space or a support surface in the sample. Alternatively or in addition the growth of the test cell or organism may generate toxic products which inhibit the growth and/or survival of the reporter cell.

In one embodiment the test cell or organism comprises a pathogenic cell or organism. Preferably the test cell or organism comprises a microorganism, e.g. a bacterial, fungal or protozoal cell.

In one embodiment the bacterial cell is from a species which is pathogenic in a mammal, preferably a human. Preferably the bacterial cell is selected from Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphteriae, Enterococcus faecalis, Enterococcus faecum, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae and Yersinia pestis.

In one embodiment the fungal cell is from a genus selected from Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. In another embodiment the protozoal cell is from a genus selected from Plasmodium, Acanthamoeba, Giardia, Toxoplasma and Leishmania. In various embodiments the organism comprises a multicellular organism, an agricultural pest, a plant cell or a cancer cell.

In one embodiment the reporter cell comprises a mammalian cell, for example a human cell.

In one embodiment the reporter cell expresses an exogenous reporter gene, for example tissue plasminogen activator. The reporter gene may be expressed on the surface of the reporter cell.

In one embodiment the reporter cell expresses an affinity tag, for example a hemagglutinin peptide.

In one embodiment the signal is a fluorescent or luminescent signal. Preferably an increase in the signal in the presence of the agent, compared to a control level of the signal in the absence of the agent, is indicative of inhibitory activity of the agent on the growth and/or survival or the test cell or organism.

In one embodiment the reporter gene encodes an enzyme or active fragment thereof, and the signal is detected by:

a) contacting the sample with a substrate for the enzyme; and

b) detecting an enzymatic product of the enzyme.

Preferably the substrate is non-fluorescent and the enzymatic product emits a fluorescent signal.

In another embodiment the reporter gene encodes an enzyme or active fragment thereof, and the signal is detected by:

a) contacting the sample with a substrate for the enzyme; and

b) detecting light (e.g. luminescence) emitted due to conversion of the substrate by the enzyme.

Preferably the sample comprises a co-culture of the reporter cell and the test cell or organism. In one embodiment an agent is selected if a signal from the reporter cell is above a predetermined level. Preferably an agent which inhibits growth and/or survival of the test cell or organism is selected. It is additionally preferable that an agent which does not substantially inhibit growth and/or survival of the reporter cell is selected.

In one embodiment the sample preparation further comprises a growth medium comprising one or more nutrients required by both the reporter cell and the pathogenic cell or organism, such that the reporter cell and pathogenic cell or organism compete for nutrients in the sample preparation.

In another embodiment the sample preparation further comprises a support surface suitable for supporting growth and/or survival of either the reporter cell or the pathogenic cell or organism, such that the reporter cell and the pathogenic cell or organism compete for the support surface in the sample preparation.

In one embodiment the sample preparation or kit comprises one or more candidate agents for inhibiting growth and/or survival of the pathogenic cell or organism.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Assay for the selection of species-specific antibiotics. A) Human reporter cells are displaying a membrane-bound form of tissue plasminogen activator (tPA) on their surface. This enzyme converts plasminogen into plasmin which in turn converts a substrate into a fluorescent product. B) Competitive co-cultivation of a metabolically active pathogen with the human reporter cells. In absence of any potent antibiotic, the pathogen overgrows the indicator cells, which finally die due to the lack of nutrition and the accumulation of cytotoxic metabolites. Hence no fluorescence signal is generated. In contrast, the presence of a potent antibiotic results in cell death of the pathogen and proliferation of the indicator cells. Consequently, upon addition of the assay components a strong fluorescence signal is obtained.

Figure 2: Survival of human reporter cells exposed to sterile, bacterially- conditioned media. Top: Microscopic images of HEK293T-tPA cells grown in normal DMEM media or bacterially-conditioned media (pH8). For control purposes, the pH of the bacterially conditioned media was adjusted to 7.7 (by adding IM HCl) and/or glucose was added to a final concentration of 4g/l. Bottom: Survival rates of the HEK293T-tPA cells after 3 days of incubation as determined using a live/dead stain.

Figure 3: Fluorescence signals of the tPA-based readout system. The fluorescence was determined for reporter cells (C), S. aureus (S) and co-cultures of both (C+B). Streptomycin (S) or sodium azide (NaN₃) were added at the indicated concentrations.

Figure 4: Reproducibility of the co-cultivation assay. Two completely independent sets of experiments were performed on two different days (using the same sample compositions). The fluorescence signals for all samples of Runl were plotted against the corresponding fluorescence signals of Run2. R = determination coefficient.

DETAILED DESCRIPTION OF THE INVENTION

Modulatory activity

In one aspect, the present invention provides a method for determining modulatory activity of an agent on a test cell or organism. By "modulatory activity" it is meant, for example, that the method involves determining whether the agent shows an effect on at least one physiological property of the test cell or organism. For instance, the agent may influence growth and/or survival of the test cell or organism. Modulation may involve an increase or decrease in such a physiological property. Typically the method is employed to detect agents which inhibit growth and/or survival of the test cell or organism, especially where the test cell or organism is a pathogen.

In one embodiment the method involves determining inhibitory activity of an agent on the test cell or organism, e.g. determining antimicrobial, antibacterial, antibiotic, antifungal, pesticidal, cytostatic or cytotoxic activity. By "determining inhibitory activity" it is meant to encompass detecting any inhibitory effect of the agent on growth or viability of the test cell or organism, e.g. pathogenic cells. For instance, in specific embodiments the method may be used to determine bacteriocidal, or bacteriostatic or cytostatic activity of a candidate antibiotic or antiproliferative compound.

Agent The agent is typically a candidate inhibitor compound such as a chemical entity which it is desired to test. The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.

In specific embodiments, the agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may be a polynucleotide molecule. The agent may be an antibody. The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.

By way of example, the agent may be a natural substance, a biological macromolecule. or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised agent, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as using a peptide synthesiser or by recombinant techniques or combinations thereof).

Typically, the agent will be an organic compound. Typically, the organic compound will comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, alkyl groups, cyclic groups etc; substituent groups may be unbranched- or branched-chain. In addition to the possibility of the substituents being cyclic groups, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. For some applications, preferably the agent comprises at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. Preferably the candidate inhibitor is a polypeptide. When the candidate inhibitor is a polymer such as a polynucleotide or a polypeptide, preferably the candidate inhibitor is produced by the reporter cell. This may be by use of an expression library encoding candidate inhibitors such as a peptide library. For instance, a gene capable of directing expression of a candidate inhibitor may be introduced into a reporter cell. Transfection may be stable or transient, preferably stable.

Sample

The method involves providing a sample comprising a reporter cell and a test cell or organism, e.g. a pathogenic cell. By "sample" it is simply meant that the reporter cell and test cell or organism are comprised in the same preparation, e.g. are present in sufficient proximity such that the test cell or organism can influence the growth and/or survival of the reporter cell. Thus the sample may be any type of preparation, provided that the reporter cell and test cell or organism are both present.

Typically the sample is a preparation of cultured cells. For example, the reporter cell and a test (e.g. pathogenic) cell may be grown together in vitro in a culture medium, i.e. the reporter cell and test cell are co-cultured. Thus both the reporter cell and test cell are isolated cells. In particular embodiments, the sample may comprise an adherent cell culture or a suspension culture.

The sample preparation may further comprise a growth or culture medium, which is typically capable of supporting the growth and/or survival of either the reporter cell or the test (e.g. pathogenic) cell or organism. By this it is meant that preferably both the reporter cell and the test cell or organism can grow and/or survive in the medium, particularly when either is present in isolation. Thus the medium may comprise nutrients which are required by both the reporter cell and the test cell or organism. However, the growth or culture medium may be selected such that the supply of nutrients is limited, e.g. to the extent that consumption of nutrients during growth of the test cell or organism restricts availability of such nutrients to the reporter cell, thereby limiting its growth. A skilled person can easily select an appropriate medium comprising suitable concentrations of nutrients, taking into account the nature of both the reporter cell and the test cell or organism, in order to achieve this effect. In addition, particularly where the sample comprises an adherent cell culture, a support surface may be provided which is capable of supporting growth of either the reporter cell or the test (e.g. pathogenic) cell or organism. By this it is meant that both the reporter cell and the test cell or organism can grow on the support surface in isolation. However, the support surface area may be restricted such that the reporter cell and the test cell or organism compete for space on the support surface. The support surface may be provided, for example, by the wall of a flask, dish or well or a semi-solid medium on which the cells are grown.

Reporter cell

By "reporter cell" it is meant any type of cell which is capable of providing a detectable signal, e.g. as a read-out from the assay method. Typically the reporter cell is a eukaryotic cell, particularly a mammalian cell, more particularly a human cell. Preferred reporter cells are 293 EBNA T cells or HEK293T cells; preferably the reporter cells are derived from HEK293T cells. In one embodiment, the reporter cell may be an indicator cell as described in WO 2006/082385. In another embodiment, the reporter cell may be a plant cell.

The reporter cell may be, for example, a cell from a species which is susceptible to infection by the test (e.g. pathogenic) cell or organism. Typically growth and/or survival of the reporter cell is influenced by the test cell or organism in the sample. For instance, growth of the test cell or organism may have a negative effect on the reporter cell, such that growth/survival of the test cell or organism and reporter cell are inversely related.

This inverse relationship may arise through several alternative mechanisms or a combination thereof. The test cell or organism and the reporter cell may compete for one or more resources within the sample, e.g. nutrients which includes any substance necessary for the growth and/or survival of each cell type. Alternatively the test cell or organism and the reporter cell may compete for space or support within the sample. This may occur, for example, where both the test cell and reporter cell are adherent cells in competition for a limited surface area in the sample. The surface area in the sample may be constrained by the available surface area in a flask, dish or well within which the sample is contained. In another embodiment, an inverse growth relationship and/or competition between the test cell or organism and the reporter cell may arise as a result of production of one or more substances which inhibit cell growth or survival. For instance, the test cell or organism may produce toxic metabolites or other substances which inhibit the growth and/or survival of the reporter cell. In specific cases, infectious pathogens such as bacteria may produce toxins which selectively inhibit the growth of, or which kill the cells of a host species. Production of such toxins may be responsible at least partially for the pathological symptoms arising in vivo from such infections. Embodiments of the present invention may utilise such effects in order to provide an inverse relationship between growth of the test cell (e.g. a bacterium) and reporter cell (e.g. a recombinant human cell expressing a reporter gene). However, in various embodiments metabolites produced by the test cell or organism may negatively influence growth and/or survival of the reporter cell without being classed as recognised toxins of the type responsible for pathological effects.

The reporter cell may provide any type of signal. Preferably the signal is indicative of growth and/or survival of the reporter cell. The signal may be an endogenous signal produced naturally by the cells, or the cells may be genetically modified (e.g. using recombinant DNA techniques) to produce the signal. In one embodiment, the reporter cell expresses a reporter gene. The reporter cell may be produced by transfection, transformation or transduction of the reporter gene, i.e. the reporter cell expresses an exogenous or recombinant reporter gene. The transfection may be transient or stable, preferably stable. Most preferably the reporter cell expresses a reporter gene which has stably integrated into the genome of the cell. The reporter gene may be assayed by any suitable means.

As used herein, the term 'reporter genes' has its normal meaning in the art, i.e. of a gene whose product can be readily detected, for example so as to derive information about the expression state of said gene. Typical reporter genes include fluorescent proteins or enzymes. Preferred reporter genes include green fluorescent protein (GFP)₅ β-lactamase (beta-lactamase), β-galactosidase (beta-galactosidase) or tissue plasminogen activator (tPA) which are each well known in the art; preferably the reporter is tPA. Further reporter genes which may be used include luminescent proteins or luminescence-inducing enzymes, such as luciferase (e.g. from the firefly Photinus pyralis) which oxidizes luciferin to produce luminescence.

Preferably the reporter gene encodes an enzyme or active fragment thereof capable of converting a fluorogenic or chromogenic substrate to a fluorophore or chromophore whose presence can be detected thereby. The enzyme may be an intracellular enzyme or may be displayed on the cell surface. Alternatively, the enzyme may be released in soluble form (i.e. secreted) by the reporter cells.

Preferably the enzyme or fragment is displayed on the cell surface. This may be achieved by fusion to a cell surface protein such as CD4, or may be by incorporation (e.g. fusion) of a suitable signal sequence (such as that derived from Igκ). Preferably ^' a transmembrane domain e.g. PDGFR-TM is also included as a membrane anchor. Preferably that part of the reporter gene product which mediates detection is extracellular. This enables easy access to reagents/substrates used for detection without having to propel them across the cell membrane.

Li another embodiment, the reporter cell expresses a detectable marker such as an affinity tag (e.g. a peptide tag such as a hemagglutinin (HA) tag, myc tag, flag tag or any other suitable tag), typically on the cell surface, to facilitate its detection. A signal may be provided by detection of the marker.

In a preferred embodiment, the reporter cells express a membrane-bound and HA- tagged form of the human tissue plasminogen activator (tP A-HA).

Test cell or organism

The test cell or organism is the entity in the assay against which modulatory activity of the agent is desired to be determined. The nature of the test cell or organism is not particularly limited, other than that the test cell or organism differs from the reporter cell. Typically the test cell or organism is a pathogenic cell or pathogenic organism, e.g. a pathogenic microorganism. However, the method may also be used to detect agents which affect (e.g. inhibit or promote the growth/survival of) non-pathogenic organisms or cells, including non-pathogenic microorganisms (e.g. non-pathogenic bacteria, fungi or protozoa). By "cell" and "organism" it is intended to refer only to cellular organisms, including cellular microorganisms, i.e. viruses are not included. In one embodiment the test cell or organism is a microorganism. Herein throughout, the phrase 'microorganism' is used to describe a microscopic unicellular organism which may belong to any family of organisms such as, but not limited to prokaryotic organisms, eubacteriuni, proteobacterium, archaebacterium, eukaryotic organisms, yeast, fungi, algae, protists, protozoan, and other parasites, as exemplified hereinbelow.

Non-limiting examples of prokaryotic bacteria phyla include acidobacteria, actinobacteria, aquificae, bacteroidetes, chlamydiae, chlorobi, chloroflexi, chrysiogenetes, cyanobacteria, deferribacteres, deinococcus-thermus, dictyoglomi, fibrobacteres, firmicutes, fusobacteria, gemmatimonadetes, nitrospirae, planctomycetes, proteobacteria, spirochaetes, thermodesulfobacteria, thermomicrobia, thermotogae and verrucomicrobia.

Non-limiting examples of archaebacterium phyla include crenarchaeota, euryarchaeota, korarchaeota and nanoarchaeota. Non-limiting examples of proteobacteria include Alpha proteobacteria such as Caulobacterales (Caulobacter), Parvularculales, Rhizobiales (rhizobia), Rhodobacterales, Rhodospirillales (Acetobacter), Rickettsiales Rickettsia) and Sphingomonadales (Sphingomonas); Beta proteobacteria such as Burkholderiales (Bordetella), Hydrogenophilales, Methylophilales, Neisseriales (Neisseria), Nitrosomonadales, Rhodocyclales and Procabacteriales; Gamma proteobacteria such as Acidithiobacillales, Aeromonadales (Aeromonas), Alteromonadales (Pseudoalteromonas), Cardiobacteriales, Chromatiales purple sulfur bacterid, Enterobacteriales (Escherichia), Legionellales (Legionella), Methylococcales, Oceanospirillales, Pasteurellales (Haemophilus), Pseudomonadales (Pseudomonas), Vibrionales (Vibrio) and Xanthomonadales (Xanthomonas); Delta proteobacteria such as Bdellovibrionales (Bdellovibrio), Desulfobacterales, Desulfovibrionales, Desulfurellales, Desulfarcales, Desulfuromonadales (Geobacter), Myxococcales, Myxobacteria and Syntrophobacterales; and Epsilon proteobacteria such as Campylobacterales (Helicobacter) and Nautiliales.

In one embodiment the test cell is a pathogenic cell from any pathogenic microorganism. For instance, the pathogenic cell may be a bacterial, fungal or protozoal cell. By "pathogenic" it is meant that the organism (e.g. microorganism) is associated with a disease in another species, including diseases of animals and plants. In one embodiment the organism is causative of a mammalian (e.g. human) disease.

As used herein, the term "pathogenic cell" includes modified cells derived from a pathogenic species, including forms which have been modified to reduce or eliminate pathogenic activity for experimental or safety reasons. Preferably the pathogenic cell is a bacterium or a cancer cell, i.e. the method is for determining antibacterial or cytostatic activity of an agent.

Preferred pathogenic cell types include those from any of the following species, genera or other classification units: Escherichia, Helicobacter, Plasmodium falciparum and related malaria-causing protozoan parasites, Acanthamoeba and other free-living amoebae, Aeromonas hydrophila, Ascaris lumbricoides, Balantidium coli, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Coccidia (Cryptosporidium parvum) Cyclospora cayetanensis, Diphyllobothrium, Entamoeba histolytica, Eustrongylides, Giardia lainblia, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nanophyetus, Plesiomonas shigelloides, Pseudomonas, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis; Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.

In another embodiment the test organism is a multicellular organism. Suitable multicellular organisms include pathogenic organisms such as parasites, particularly parasites of humans and/or animals (e.g. livestock or domestic animals). Examples of parasites include nematodes (roundworms, hookworm), platyhelrnith.es (tapeworms, fiatworms), lice and mites, including Ascaris lumbricoides, Cestoda, Dracunculus medinensis, Enterobius vermicularis, Fasciola hepatica, Necator americanus, Pediculus humanus, Phthirus pubis, Sarcoptes scabiei, Strongyloides stercoralis, Toxocara cards, Toxocara cati, Trichinella spiralis, Trichuris trichiura and Trichuris vulpis. In these embodiments the test organism is preferably an endoparasite such as a helminth. The method may involve preparing a sample in which the multicellular organism (e.g. parasite) is added to the reporter cell, e.g. a cell which is derived from the host species of the parasite, including a tissue preparation which may play host to the parasite. Thus the reporter cell may be present as part of a host tissue which has been modified in order to produce a detectable signal, for instance by transfection of a reporter gene.

In another embodiment the test cell or organism may be a unicellular or multicellular pathogen of plants. Examples of plant pathogens include phytopathogenic fungi such as Ascomycetes (e.g. Fusarium, TMelaviopsis,Verticillium, Magnaporthe grisea) and Basidiomycetes (e.g. Rhizoctonia, Phakospora pachyrhizi, Puccinia); oomycetes (e.g. Pythium, Phytophthora); bacteria (e.g. Agrobacterium, Burkholderia, Xanthomonas, Pseudomonas, phytoplasmas, spiroplasmas); protozoa and nematodes. The reporter cell may be derived from e.g. an animal (e.g. human) or a plant cell. In this embodiment, the method may be used, for example, to detect agents which inhibit growth and/or survival of the plant pathogen, without producing toxic effects on the plant host and/or animals (e.g. humans) which consume the plant. For instance, the method may be used to detect pesticide compounds which inhibit an agricultural pest, by using the pest as the test cell or organism. In order to detect toxic effects on humans of the candidate pesticide compound, the reporter cells may be human cells. Toxic effects on plant cells may be detected by incubating a plant cell as the reporter cell together with the pest (test) cell or organism. The plant cell reporter may be used in a separate assay sample independently of the human cell reporter, or alternatively a plant cell reporter and human cell reporter may be used together with the pest cell or organism in the same assay sample, provided that the plant and human reporters produce distinguishable signals. Samples which produce signals corresponding to both the plant and human reporters may be indicative of the presence of an agent which inhibits the pest without toxicity on either the plant or humans.

In another embodiment, the test cell or organism may be a plant cell or tissue. In this embodiment, the reporter cell may be, for example, an animal (e.g. human) cell. For instance the method may be used to detect agents which have herbicidal activity without exerting toxic effects on human cells.

In another embodiment, the test (pathogenic) cell is a cancer cell. Examples of cancer cells include primary cells isolated from clinical tumour samples, e.g. cancer cells from breast, lung, prostate, stomach, liver, spleen, pancreas, blood, skin, bowel and cervical tissue. The cancer cell may be from a cancer cell line such as NUGC-4, NCI- N87, AGS₅ HCTl 16, HT-29, LoVo₅ HepLrZ, MOLT-4, Hep3B₅ SK-HEP-I₅ BxPC-3, MiaPaca-2₅ Panc-1₅ H460, A549, NCI-H69, MDA-MB-435, MCF-7₅MDA-MB-231₅ BT-474, PC-3, LnCap, DU145₅22RV1, SK-MEL-5, LOX₅ OVCAR-3, SKOV3, KU- 7₅UC14₅ FaDu₅ Hep2₅ SCC-25, U87, DAOY₅ SN12C, A498, HT1080, K562, OST₅ A431, HeLa or any other immortalized cancer cell line. Furthermore, the test cell may be derived from a non-cancer cell in which single or multiple tumour promoter genes have been inserted. Thus the pathogenic cell may be from a tumor tissue sample, or from a tumor cell line or the pathogenic cell might be a genetically-modified cell of non-pathogenic origin.

Contacting the agent with the sample

By "contacting the agent with the sample" it is meant that the agent is added to, mixed with or otherwise brought into contact with the reporter cell and test (e.g. pathogenic) cell or organism. In embodiments where the agent is produced by the reporter cell, e.g. where the agent is a recombinant polypeptide, this may mean that expression of the agent in the reporter cell is induced, or that the agent continues to be expressed by the reporter cell.

Typically the agent is incubated with the sample for a defined period of time, before detecting the signal. The incubation step is to allow time for the agent to affect growth or survival of the test cell or organism (e.g. pathogen), which subsequently affects the reporter cell and read-out of the signal. The time required for this will vary depending on the nature of the reporter cell and test cell or organism (e.g. pathogen). The precise time of incubation for a given system may be determined experimentally based on e.g. the time taken the signal to decrease in the absence of the agent and/or the timing of an increase in the signal in the presence of a known antimicrobial compound.

Detecting a signal

In embodiments of the present invention, the assay read-out is provided by detecting a signal associated with the reporter cell, rather than the test cell or organism (e.g. pathogenic cell) as such. Thus the reporter cell provides an indication of the growth and/or survival of the test (e.g. pathogenic) cell or organism. Thus the signal level typically shows a negative correlation with growth of the pathogen. Agents which show relatively high antimicrobial activity will produce a corresponding high signal in the sample with which they are contacted.

Any suitable detection method, depending on the nature of the signal, may be used. For instance, where the reporter cell comprises a reporter gene encoding an enzyme, or an active fragment thereof, detection of the signal may comprise contacting the indicator cell with a substrate for the enzyme, incubating to allow the enzyme to act on the substrate, and detecting the presence of enzymatic product, presence of the product indicating reporter gene activity.

The signal may comprise a fluorescent, luminescent or visible light signal. For instance, the reporter cell may express a reporter gene encoding a fluorophore or a chromophore or other entity capable of direct detection. In particular embodiments, detection may be by fluorescent resonance energy transfer (FRET), by change in fluorescence and/or absorbance, by abolition of fluorescence and/or absorbance or by generation/initiation of fluorescence and/or absorbance at the appropriate wavelengths. Preferably detection is by generation/initiation of fluorescence (or absorbance) wherein the substrate is non-fluorescent (or non-absorbent) but the cleaved product is fluorescent (or absorbent). In other words (or alternatively) detection may be by discernibly different fluorescence (or absorbance) spectra of substrate and product, hi another embodiment detection is by the generation of luminescence, wherein conversion of a substrate by the reporter gene product results in luminescence (e.g. oxidation of luciferin by luciferase).

In another embodiment wherein the reporter cell expresses a detectable marker (e.g. a peptide tag), detecting may involve contacting the reporter cell with an antibody capable of reacting with the marker. A signal associated with the reporter cell may be determined by detecting the presence of bound antibody on the reporter cell.

In one preferred embodiment the reporter cells are genetically engineered host cells that express (preferably constitutively) a membrane-bound affinity tag and/or reporter enzyme. Consequently, a signal can be detected in these cells by staining with antibodies and/or assaying for conversion of a non-fluorogenic substrate into a fluorogenic product. Thus, growth of the microorganism results in a decreased reporter signal, whereas inhibition of microbial growth (by activity of the agent) leads to the maximum signal intensity.

In a preferred embodiment, the current system is based on reporter cells expressing a membrane-bound and HA-tagged form of the human tissue plasminogen activator (tP A-HA). This enzyme converts plasminogen into plasmin which then converts a non-fluorogenic substrate into a fluorogenic product. Clearly in some embodiments the detection of tPA may be by its direct action on a chromogenic or fluorogenic substrate, rather than its action on plasmin and the subsequent action of plasmin on a chromogenic or fluorogenic substrate.

Applications

The invention finds application in many areas including high-throughput screens and directed evolution techniques. The assays of the invention allow screening of drug candidates for e.g. antibacterial or cytostatic activity.

In particular, the invention finds application in the screening of small molecules within microtitre plates or microfluidic devices (emulsions), and screening genetically-encoded libraries of peptides, shRNAs or antibodies using FACS. This application advantageously allows new drugs and also new drug targets to be identified.

Furthermore the invention may be used to detect a pathogen in a sample. In this embodiment, reporter cells according to the present invention are contacted with a test sample thought to comprise the pathogen of interest. The signal from the reporter cells will remain 'on' (i.e. giving continuous readout) in the absence of the pathogen, but would be shut off in the presence of the pathogen. Thus, if, following contact with the test sample, the signal is lost then it would indicate that the test sample is likely to have comprised the pathogen. Since the loss of signal can in theory be mediated by a variety of pathogens, in one embodiment a control sample with a specific inhibitor of the species of interest (the species to be detected) may be included. If this sample shows the signal while in absence of the specific inhibitor the signal is lost, the test sample is likely to have comprised the pathogen.

Assays of the invention In one embodiment, the present invention is based on genetically modified reporter cells, which may comprise a stable cell line. These cells may be genetically modified in the sense that they express a reporter gene, such as an affinity tag, a fluorogenic protein or an enzyme able to convert substrates into fluorogenic., chromogenic or luminometric products. The signal from the reporter cells is decreased by survival and/or growth of the test (e.g. pathogenic) cell or organism also present in the sample.

Assay formats

Microfluidic handling techniques, emulsion based droplet compartmentalisation and microtitre plate wells (such as 12, 24 or 96-well format) are all useful formats for the assays of the present invention. These techniques are well known in the art. In particular, reference is made to WO99/02671 and WO00/40712 which both describe optical sorting methods of application to the methods described herein. The way in which the readout is collected and the optimal assay formats depend upon operator preferences. Factors to be taken into account may include the number of samples to be processed. For example, if sample numbers are small, it may be convenient to process them manually in a microtitre plate with manual pipetting. However, where sample numbers are large, it may be more convenient to use an automated or semi- automated processing apparatus to conduct the screening and selection. These choices are well within the ordinary skill of the person working the invention.

Reporters/Substrates/Readout

The signal from the reporter cells may be detected directly (e.g. by antibody based detection) or indirectly (e.g. by assay of reporter activity). Direct detection of reporter gene activity may be based on the gene activity such as detection of transcription, translation or direct detection of the gene product. Indirect detection principally refers to assaying for activity of the gene product such as an enzymatic activity, e.g. by supplying a substrate and monitoring conversion of same or by some similar technique.

In choosing a reporter enzyme, preferably it should mediate a rapid turnover of substrate (ie. have high Kcat/Km). Preferably it should be an enzyme for which fluorogenic and/or chromogenic substrate(s) are available. Preferably the reporter enzyme or fragment thereof is displayed on the cell surface. Preferably the reporter gene comprises a surface targeting element such as a transmembrane domain or a signal peptide to achieve cell surface localization of the reporter enzyme or fragment thereof. Preferred cell surface targeting element is a single-spanning membrane protein, or a single spanning domain from a multiple membrane-spanning protein. For example, the reporter gene could be fused to the SU domain of retroviral env protein(s), preferably N-terminally fused thereto. Especially preferred cell surface targeting agents are fusion to CD4 receptor, or fusion to the transmembrane domain of PDGFR (Platelet Derived Growth Factor Receptor transmembrane domain, PDGFR-TM). Expression of the reporter gene should preferably be driven by a strong promoter. An example of a preferred signal peptide is the Ig-κ signal peptide found in immunoglobulin precursors or any other signal peptide mediating the transport to the endoplasmic reticulum and finally the cell surface.

Preferably the reporter gene encodes an enzymatic activity, which activity is retained at the cell surface. It will be noted that when the reporter enzyme activity is located at the cell surface, that the substrate for conversion to a chromogenic or fluorogenic product will also need to be available at the cell surface. Typically this is achieved by presenting the substrate extracellularly so that it will be able to be acted upon by the cell surface localized reporter enzyme activity. In these embodiments, it will be apparent that droplet co-compartmentalisation is advantageous in that it allows a pool of cleaved substrate to be detected in the extracellular part of the droplet and thereby associates that with the cell in the droplet. Thus, droplet format is advantageously used when selecting cells on the basis of extracellular readout. Alternatively the reporter gene product itself can be tagged, for example by reaction with an anti- reporter antibody. This advantageously allows individual cells to be selected without having to perform droplet co-compartmentalisation. The skilled worker may easily choose the format which best suits their application of the invention.

As is described herein, it will be noted that some reporter genes may give readout via intermediate steps. For example, when the reporter is tPA, then the readout is preferably via the action of tPA on plasminogen; this creates plasmin; the plasmin acts on the substrate such as HDLVK-Amc and this creates a fluorogenic product. Thus, when using multi-step readouts such as this, then each of the necessary elements must be provided to the indicator cells. In the case of tPA readout, this may involve supplying both plasminogen as well as HDLVK-Amc to the reporter cells to allow the readout to be produced.

Plasminogen may be obtained from Roche, Switzerland. The plasmin substrate HDLVK-Amc is preferably used and may be obtained from Bachem, Switzerland (see examples). Alternatively, other plasmin substrates such as Rhodamine 110-bisCBZ-L- Phe-L-Arg from Molecular Probes, USA may be used.

When the reporter gene is β-lactamase, preferably Fluorocillin™ Green 495/525 β- lactamase substrate (Molecular Probes) is the substrate.

When the reporter gene is β-galactosidase, preferably Fluorescein di-β-D- galactopyranoside (FDG) is the substrate.

In another embodiment, the invention may be used to determine optimal concentrations of a given antimicrobial agent. When reporter cells and test (e.g. pathogenic) cell or organisms are present together in a sample, the signal level correlates with the survival or growth of the reporter cells. A major advantage of the invention over prior art assays is the fact that within the present assays adverse side effects of the antimicrobial agent on the reporter cells will cause a decreased fluorescence signal. This is due to the fact that only viable reporter cells will express the reporter gene and thus generate a positive readout signal.

In a broad aspect the invention relates to techniques to select antibodies, peptides and small molecules which inhibit microorganisms such as bacteria, fungi and protozoa. Preferably said techniques are compartmentalization-based.

Further Aspects

The reporter cells, test (e.g. pathogenic) cell or organisms and agent may be incubated within microtiter plates.

In one embodiment the reporter cells, test (e.g. pathogenic) cell or organisms and agent are incubated within a microfluidics device. In another embodiment the reporter cells, test (e.g. pathogenic) cell or organisms and agent are incubated within aqueous droplets of an emulsion. The droplets may subsequently be sorted using fluorescence activated cell sorting (FACS) or a microfluidics sorting device. Alternatively the emulsion may subsequently be broken to enable affinity-based sorting of reporter cells.

Whether using direct or indirect detection of the reporter cells, signal amplification can be easily introduced. For example, using direct detection antibody sandwich techniques can be used to amplify the signal, and when using these or indirect techniques involving enzymatic activity, each enzyme molecule can repeatedly turn over substrate molecules to provide more signal.

Kits

The invention also provides kits for performing the methods described above. Typically such kits may comprise a reporter cell and a test (e.g. pathogenic) cell or organism as described above, together with a medium within which the method may be performed. Thus the kit may comprise a support surface or growth medium as described above. In some embodiments, the kit may further comprise additional reagents useful in the method, such as reagents for detecting reporter gene expression (e.g. a substrate for a reporter enzyme). The kit may further comprise suitable packaging and/or instructions for performing the method.

The invention is now described by way of examples, which are not intended to limit the scope of the invention but rather are intended to illustrate specific ways in which the invention can be worked.

Example 1

Infections with pathogens are a continuing threat to health throughout the world. Especially the generation of multidrug resistant bacterial strains such as Methicillin- resistant Staphylococcus Aureus (MRSA) causes a high number of fatalities in the hospital. To identify effective antibiotics against those species, reliable screens compatible with high throughput formats are required. Using 5^*. aureus as a model organism, we have developed a generic assay coupling a strong fluorescence signal with the inhibition of pathogens. As a special feature, the assay allows to directly identify compounds that do not harm human cells. The system is based on the competitive cocultivation of a metabolically-active pathogen with human reporter cells continuously generating a fluorescence signal. While in absence of any antibiotic the pathogen overgrows the human reporter cells and thus abolishes the fluorescence signal, the presence of antimicrobial compounds results in viable reporter cells and a strong fluorescence signal. The assay allows z-factors of >0.9, takes cytotoxic side effects into account and can be applied to any metabolically-active pathogen.

Materials and Methods

Cells. Reporter cells expressing a membrane-bound and HA-tagged form of the tissue plasminogen activator (HEK293T-tPA) were obtained by retroviral transduction of HEK293T cells with MLV(VSV-G) pseudotype particles comprising a suitable encoding vector (e.g. as described in WO2006/082385). Subsequently high-expressers were selected by FACS sorting (Dako) using goat polyclonal antibodies to tPA (Abeam).

HEK293T-TPA cells were grown in DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL) and 1% penicillin/streptomycin (GIBCO-BRL). Cells were incubated at 37°C in a 5% CO₂ atmosphere saturated with water.

Staphylococcus aureus was grown in DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL). Except when growing co-cultures, bacteria were incubated at 37°C at 230 rpm in a shaking incubator.

In vitro co-cultivation assay. To set up the co-cultures, S. aureus was grown in DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO- BRL) to an OD_δoo of 0.4 (corresponding to 3 x 10⁷ CFU/mL). Subsequently, the samples were diluted 10⁵ fold and lOOμl (30 bacterial cells) were added to each well of a 96-well plate. In parallel, HEK293T-tPA cells were washed twice with DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL) and seeded into the same wells at a density of 2 x 10⁴ cells/well (determined with a Neubauer counting chamber). Subsequently, antibiotics were added at the indicated concentrations. All samples were prepared as triplicates and incubated for 3 days at 37°C under a 5% CO₂ atmosphere saturated with water. For the fluorescence readout HDVLK-Amc (Bachem) and Plasminogen (Roche) were added to a final concentration of 1 mM and 1.67μM, respectively. All measurements were performed at an excitation/emission wavelength of 370nm/450nm with a Spectramax M5 spectrophotometer (Molecular Devices).

Antibiotics. Penicillin (Sigma) and Streptomycin (Sigma) were diluted with sterile 1 x PBS (Euromedex) to concentrations of lmg/mL, 100 μg/mL and lOμg/mL. For control purposes, sodium azide (Sigma) was also diluted with sterile 1 x PBS (Euromedex) to concentrations of lmg/mL, 100 μg/mL and lOμg/mL.

Determination of Z-Factors. Z-Factors were calculated using the following equation:

Z - factor

σ = standard deviation

μ = mean signal

X₍p₎ = parameter of the positive control

X_(π) = parameter of the negative control

Live/Dead staining. One day before starting the assay HEK293TPA cells were seeded at a density of 2 x 10⁴ cells/well into 6-well plates (VWR). In parallel, S. aureus was grown in DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL) to complete confluency (48h at 37°C) at 230 rpm in a shaking incubator. Then the bacterially conditioned DMEM was recovered by filtration trough a 0.22μM sterile filter (Millipore) and ImI was added to each well hosting HEK293T-tPA cells (after removing the primary media from those wells). For control purposes, the pH of the bacterially conditioned media was adjusted to 7.7 (by adding IM HCl) and/or glucose was added to a final concentration of 4g/l. Subsequently, the samples were incubated for 3 days at 37⁰C under a 5% CO₂ atmosphere and microscopic pictures were taken daily (using a LEICA DMIRB microscope and a Guppy camera; Allied Vision Technologies). On the third day of incubation the media was removed from the cells and 1 mL of 0.25% (w/v) trypsin (GIBCO-BRL) was added. After detachment the cells were centrifuged at 285g for 5min and resuspended in a live/dead staining solution (LIVE/DEAD Viability/Cytotoxicity Kit for animal cells, Invitrogen Kit L-3224). After 1 hour staining the cells were counted manually by using a microscope (Leica DMIRB) with a UV-lightsource (LEJ ebq 100). For each sample 1000 cells were counted to determine the fraction of living (green stain) and dead (red stain) cells.

Results

The first requirement for establishing the novel assay system was the use of human reporter cells continuously generating a fluorescence signal. For this purpose, we have chosen HEK293T-derived cells continuously expressing a membrane-bound and HA- tagged form of the tissue plasminogen activator (HEK293T-tPA). These cells were originally developed for viral inhibition assays (e.g. as described in WO2006/082385) and generate a strong fluorescence signal upon the addition of plasminogen and the fluorogenic substrate HDVLK-aminocoumarin (HD VLK- Amc, Bachem). During this reaction, the membrane-bound tPA converts plasminogen into plasmin, which subsequently releases the aminocoumarin group of HDVLK-Amc resulting in a fluorescent molecule (Fig. IA).

The next step was to find a way of competitively cocultivating these cells with a metabolically active pathogen. In particular, we wanted to set up a system in which in absence of any antibiotic these reporter cells get overgrown by the pathogen and die, while in presence of antibiotics the reporter cells survive and generate a strong fluorescent signal (Fig. IB).

Using S. aureus as a model organism, we determined if reporter cells seeded into 96- well plates indeed die upon growth of the pathogen. We inoculated the samples with different amounts of S. aureus in absence and presence of streptomycin (at concentrations of 10^'6 - 10¹ mg/ml) and performed microscopical analyses the following days. The biggest difference between the two samples (± antibiotics) and the most reproducible results were obtained when inoculating each well with approximately 30 bacterial cells: Within 3 days, the absence of any antibiotic resulted in the detachment and fragmentation of the reporter cells and the generation of big bacterial aggregates. In contrast, the addition of the antibiotics inhibited the bacterial growth and resulted in viable, proliferating reporter cells.

To elucidate the mechanism that ultimately killed the reporter cells, we performed experiments using sterile filtrated supernatants of dense S. aureus cultures grown in DMEM medium. We incubated human reporter cells with this bacterially-conditioned media and determined the survival rate by microscopical analysis and a commercially available live/dead stain (Fig. 2).

It turned out that the killing of the reporter cells was not dependent on the presence of viable bacteria. More likely, the reporter cells died due to the presence of toxic metabolites or the lack of nutrition. To prove this, we repeated the experiments with additional samples. In particular, we also included bacterially-conditioned media whose pH was adjusted to 7.7 (instead of pH 8 without any treatment), bacterially- conditioned media supplemented with 4g/l glucose and bacterially conditioned-media with glucose and pH 7.7. Adjustment of the pH as well as addition of glucose significantly increased the survival rates of the reporter cells (with the addition of glucose having the stronger effect). Performing both modifications at the same time allowed obtaining survival rates almost identical to the control samples with fresh DMEM medium (>97% viable cells). This clearly shows that within the co-cultures the reporter cells ultimately die due to the lack of nutrition and changes in the pH.

Next, we focussed on the fluorescence readout of the assay and characterized its suitability for high throughput screening of drug candidates. Within 96-well plates we seeded reporter cells, cultures of S. aureus and co-cultures of both species in presence and absence of streptomycin (an antibiotic which specifically kills bacteria) and sodium azide (a compound which non-specifically kills both species). After three days of incubation, we added all components of the fluorescence readout (plasminogen and HDVLK-Amc, (see Material and Methods for details) and determined the fluorescence signal (Fig. 3).

While the samples with the reporter cells alone generated a strong fluorescence signal, the 5! aureus cultures and the co-cultures of both species showed solely a very low fluorescence background. This changed when adding different concentrations of streptomycin (10^~6 - 10¹ mg/ml): For all concentrations equal or higher than 10^"3 mg/ml a significant increase in fluorescence was observed. A concentration of exactly 10^"3 mg/ml resulted in the strongest signal, with an intensity corresponding to that of the positive control (reporter cells without S. aureus). Higher concentrations of streptomycin mediated continuously decreasing fluorescence intensities, most likely due to increasing cytotoxicity. This is in good agreement with the samples containing the toxic compound sodium azide (mediating cell death of both species as determined microscopically) which showed only very low fluorescence. This clearly demonstrates that the assay can be used to select compounds that specifically kill the pathogen without doing harm to the human reporter cells.

To prove the quality of the assay we also determined the z-factors [10] using the fluorescence values of co-cultures in presence (positive control) and absence (negative control) of streptomycin (Table 1). For all concentrations equal to or above lO^'3 mg/ml, the z-factor was exceeding 0.79 with the best value being 0.95 when using 10^"2 mg/ml streptomycin. This clearly fulfils the requirements for an excellent assay [10]. The signal to background values (fluorescence of the positive control divided by the fluorescence of the negative control) for the assayed samples were in the range of 0.73 -25.24.

Table 1: Z-factors and signal to background ratios for different concentrations of streptomycin.

Concentration of Z-factor Signal to streptomycin background

10mg/mL 0.68 10.25

lmg/mL 0.84 18.76

lOOμg/mL 0.84 10.10

lOμg/mL 0.95 14.56

lμg/mL 0.79 25.24

100ng/mL -3.57 0.93 IOng/mL -2.00 0.80

lng/mL -2.11 0.73

As the next step, we determined the reproducibility of the assay by performing two completely independent sets of experiments (Runl and Run2) on two different days. Each experiment contained triplicates of all samples for which the fluorescence was determined (using streptomycin concentrations of 10^'6 - 10¹ mg/ml; 108 samples for each run). Subsequently the obtained values for Runl were plotted versus the corresponding values for Run2 (Fig. 4).

The resulting data points in the dot plot show a linear relation demonstrating a high reproducibility of the assay. The fitted trend line has a coefficient of determination (R²) of 0.881. In parallel, we determined the (mean) relative standard deviation (RSD) between the replicates in one run (intra comparison) and between the two runs (inter comparison). The obtained values of 9.41% and 9.91% (Table 2) clearly prove the reliability and robustness of the assay.

Table 2: Reproducibility of the assay

Mean RSD intra Mean RSD inter R¹

Discussion

We have developed a generic fluorescence assay for the inhibition of metabolically- active pathogens. The system is based on the competitive co-cultivation of human reporter cells with the pathogen of interest. Using & aureus as a model organism, we obtained z-factors of up to 0.95 and a relative standard deviation of 9.41% for all samples within one run and 9.91% when comparing equal samples between two runs.

The main advantage of the current system is the fact that it allows selection of specific antibiotics against metabolically-active pathogens, even if the pathogen is not well characterized. It neither requires any detailed knowledge about potential drug targets, nor any genetic modification of the pathogen (e.g. to establish a reporter system). Using live/dead stains we could show that without adding effective antibiotics to the co-cultures, the human reporter cells finally die due to the lack of nutrition. This generic mechanism is adaptable for any kind of metabolically-active pathogen.

Even if the pathogen of interest has been well-characterized and potential drug targets are known, it seems advantageous to use a reporter system based on human cells. This way, adverse side effects of the screened compounds are directly taken into account. We have shown here that sodium azide, a toxic compound doing harm to the pathogen and the human reporter cells, does not mediate a positive fluorescence signal. Hence the described assay system does not require a secondary screen for cytotoxicity, thus saving time and money.

Even though the described screening system does not elucidate the mode of action of the screened molecules, it is useful for the identification of novel drug targets. Since the assay is well compatible with poorly characterized pathogens, it can be used for the selection of compounds acting on so far uncharacterized targets which could ultimately be identified using labelled or immobilized molecules [11]).

Example 2

The assay as described in Example 1 can be used to select inhibitors of any metabolically-active pathogen. In this example, the method as described in Example 1 is adapted for the selection of cancer cell-specific inhibitors. For this purpose, human reporter cells (derived from non-cancer cells) are co-cultivated with cancer cells (instead of S. aureus) in absence or presence of drug candidates.

In the absence of a cancer cell-specific inhibitor, the cancer cells overgrow the human reporter cells quickly resulting in a lack of nutrition and/or the accumulation of potentially cytotoxic metabolites. A co-cultivation of adherent cancer and non-cancer cells additionally results in a competition for solid support, due to a limited surface area of the tissue culture flask or well. If the cancer cells grow to confluency, there is no (or at least less) space for the reporter cells. Hence at the time of readout, fewer reporter cells have grown in the absence of any cancer-cell specific inhibitor, resulting in a decreased signal (compared to the sample with a cancer cell-specific inhibitor in which all available surface area can be occupied by the reporter cells).

Consequently, in the absence of a cancer cell-specific inhibitor, the reporter cells die, resulting in an abolishment of the reporter signal. Cell death of the reporter cells also occurs if a non-specific cytotoxic compound is added, m contrast, the presence of a cancer cell-specific inhibitor (e.g. a specific cytostatic drug) mediates the growth of the human reporter cells, thus resulting in a strong reporter signal.

This assay strategy offers several advantages over conventional methods for the screening of potentially cytostatic drugs. First of all, it is very difficult to identify cancer cell-specific inhibitors since cancer cells are genetically almost identical to normal human cells (only a few mutations can render a human cell into a cancer cell). Hence, co-cultivation of the two cell types and monitoring the survival of the non- cancer cells seems to be ideal for the identification of specific drugs. Second, many different kinds of mutations can render a normal cell into a cancer cell. Consequently, promising drug targets are not always well known. Therefore, an assay that is not dependent on knowledge about the involved drug targets is highly desirable and part of the current invention. In fact, it can even be used to identify novel drug targets by first selecting specific inhibitors which (modified with fluorophores or binding tags) then serve as probes for the discovery of the corresponding binding partners.

Example 3

The assay described in Example 1 may also be adapted to select pesticides that do not harm human cells. For this purpose, human reporter cells are co-cultivated either with single cells of the pest or with multicellular organisms. In absence of a specific pesticide, the pest overgrows the human reporter cells quickly resulting in a lack of nutrition or support area available to the reporter cells, and/or the accumulation of potentially cytotoxic metabolites. Consequently, the reporter cells die, resulting in an abolishment of the reporter signal. Cell death of the reporter cells also occurs if a nonspecific cytotoxic compound is added. In contrast, the presence of a specific pesticide mediates the growth of the human reporter cells, thus resulting in a strong reporter signal.

References [1] ^■ T.W.H.O. (WHO), "The world health report 2004 - changing history," 2004.

[2] E.A. Bancroft, "Antimicrobial resistance - it's not just for hospitals," JAMA, vol. 298, no. 15, pp. 1803-1804, 2007.

[3] E.D. Brown and G.D. Wright, "New targets and screening approaches in antimicrobial drug discovery.," Chem Rev, vol. 105, pp. 159-11 A, Feb 2005.

[4] R.D.L. Fuente, N.D. Sonawane, D. Arumainayagam, and A.S. Verkman, "Small molecules with antimicrobial activity against E. CoIi and P. Aeruginosa identified by high-throughput screening.," Br J Pharmacol, vol. 149, pp. 551-559, Nov 2006.

[5] R. Srivastava, D.K. Deb, K.K. Srivastava, C. Locht, and B. S. Srivastava, "Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.," Biochem Biophys Res Commun, vol. 253, pp. 431-436, Dec 1998.

[6] T.M. Arain, A.E. Resconi, MJ. Hickey, and CK. Stover, "Bioluminescence screening in vitro (bio-siv) assays for high-volume antimycobacterial drug discovery.," Antimicrob Agents Chemother, vol. 40, pp. 1536—1541, Jun 1996.

[7] JJ. Tarrand and D.H. Groschel, "Evaluation of the bactec radiometric method for detection of 1% resistant populations of mycobacterium tuberculosis.," J CHn Microbiol, vol. 21, pp. 941-946, Jun 1985.

[8] J. A. DeVito, J. A. Mills, V. G. Liu, A. Agarwal, CF. Sizemore, Z. Yao, D.M. Stoughton, M.G. Cappiello, M.D. F.S. Barbosa, L.A. Foster, and D.L. Pompliano, "An array of target-specific screening strains for antibacterial discovery.," Nat Biotechnol, vol. 20, pp. 478-483, May 2002.

[9] K.D. Greis, S. Zhou, R. Siehnel, C Klanke, A. Curnow, J. Howard, and G. Layh-Schmitt, "Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization- time of flight mass spectrometry.," Antimicrob Agents Chemother, vol. 49, pp. 3428- 3434, Aug 2005. [10] Zhang, Chung, and Oldenburg, "A simple statistical parameter for use in evaluation and validation of high throughput screening assays.," JBiomol Screen, vol. 4, no. 2, pp. 67-73, 1999.

[11] Y. Oda, T. Owa, T. Sato, B. Boucher, S. Daniels, H. Yamanaka, Y. Shinohara, A. Yokoi, J. Kuromitsu, and T. Nagasu, "Quantitative chemical proteomics for identifying candidate drug targets.," Anal Chem, vol. 75, pp. 2159-2165, May 2003.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments and that many modifications and additions thereto may be made within the scope of the invention. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims. Furthermore, various combinations of the features of the following dependent claims can be made with the features of the independent claims without departing from the scope of the present invention.

Claims

1. A method for determining modulatory activity of an agent on a test cell or organism, comprising:

a) providing a sample comprising a reporter cell and the test cell or organism;

b) contacting the agent with the sample; and

c) detecting a signal associated with the reporter cell, thereby determining the modulatory activity of the agent on the test cell or organism.

2. A method according to claim I₅ wherein the modulatory activity comprises modulation of the growth and/or survival of the test cell or organism.

3. A method according to claim 1 or claim 2, wherein the modulatory activity comprises inhibitory activity on the test cell or organism.

4. A method according to any preceding claim, wherein growth and/or survival of the reporter cell and the test cell or organism are inversely related.

5. A method according to any preceding claim, wherein the reporter cell and the test cell or organism compete for nutrients, space or a support surface in the sample.

6. A method according to any preceding claim, wherein the growth of the test cell or organism generates toxic products which inhibit the growth and/or survival of the reporter cell.

7. A method according to any preceding claim, wherein the test cell or organism comprises a pathogenic cell or organism.

8. A method according to any preceding claim, wherein the test cell or organism comprises a microorganism.

9. A method according to any preceding claim, wherein the test cell or organism comprises a bacterial, fungal or protozoal cell.

0. A method according to claim 7, wherein the bacterial cell is from a species selected from Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphteriae, Enterococcus faecalis, Enter ococcus faecum, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae and Yersinia pestis.

11. A method according to claim 10, wherein the bacterial cell is Staphylococcus aureus.

12. A method according to claim 9, wherein the fungal cell is from a genus selected from Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.

13. A method according to claim 9, wherein the protozoal cell is from a genus selected from Plasmodium, Acanthamoeba, Giardia, Toxoplasma and Leishmania.

14. A method according to any of claims 1 to 7, wherein the organism comprises a multicellular organism.

15. A method according to any of claims 1 to 9 or 14, wherein the test cell or organism comprises an agricultural pest.

16. A method according to any of claims 1 to 7, wherein the test cell comprises a cancer cell.

17. A method according to any of claims 1 to 9 or 14, wherein the test cell or organism comprises a plant cell.

18. A method according to any preceding claim, wherein the reporter cell comprises a mammalian cell.

19. A method according to claim 18, wherein the reporter cell is a human cell.

20. A method according to any preceding claim, wherein the reporter cell expresses an exogenous reporter gene.

21. A method according to claim 20, wherein the reporter gene is tissue plasminogen activator.

22. A method according to claim 20 or claim 21, wherein the reporter gene encodes a product which is expressed on the surface of the reporter cell or which is secreted.

23. A method according to any preceding claim, wherein the reporter cell expresses an affinity tag.

24. A method according to claim 23, wherein the affinity tag comprises a hemagglutinin peptide.

25. A method according to any preceding claim, wherein the signal is a fluorescent or luminescent signal.

26. A method according to any preceding claim, wherein an increase in the signal in the presence of the agent, compared to a control level of the signal in the absence of the agent, is indicative of inhibitory activity of the agent on the growth and/or survival or the test cell or organism.

27. A method according to any preceding claim, wherein the reporter gene encodes an enzyme or active fragment thereof, and the signal is detected by:

a) contacting the sample with a substrate for the enzyme; and

b) detecting an enzymatic product of the enzyme.

28. A method according to claim 27, wherein the substrate is non-fluorescent and the enzymatic product emits a fluorescent signal.

29. A method according to any of claims 1 to 26, wherein the reporter gene encodes an enzyme or active fragment thereof, and the signal is detected by:

a) contacting the sample with a substrate for the enzyme; and

b) detecting light emitted due to conversion of the substrate by the enzyme.

30. A method according to any preceding claim, wherein the sample comprises a co-culture of the reporter cell and the test cell or organism.

31. A method for identifying an agent which modulates growth and/or survival of a test cell or organism, comprising determining modulatory activity of a plurality of candidate agents by a method as described in any preceding claim, and selecting an agent showing elevated modulatory activity.

32. A method according to claim 31, wherein an agent is selected if a signal from the reporter cell is above a predetermined level.

33. A method according to claim 31 or claim 32, wherein an agent which inhibits growth and/or survival of the test cell or organism is selected.

34. A method according to any of claims 31 to 33, wherein an agent which does not substantially inhibit growth and/or survival of the reporter cell is selected.

35. A sample preparation comprising a reporter cell and a pathogenic cell or organism, wherein the reporter cell expresses an exogenous reporter gene, and wherein growth and/or survival of the pathogenic cell or organism is inversely related to expression of the exogenous reporter gene by the reporter cell.

36. A sample preparation according to claim 35, further comprising a growth medium comprising one or more nutrients required by both the reporter cell and the pathogenic cell or organism, such that the reporter cell and pathogenic cell or organism compete for nutrients in the sample preparation.

37. A sample preparation according to claim 35 or claim 36, further comprising a support surface suitable for supporting growth and/or survival of either the reporter cell or the pathogenic cell or organism, such that the reporter cell and the pathogenic cell or organism compete for the support surface in the sample preparation.

38. A kit for identifying an agent which modulates growth and/or survival of a pathogenic cell or organism, the kit comprising:

a) a reporter cell which expresses an exogenous reporter gene;

b) a pathogenic cell or organism;

c) a growth medium and/or support surface suitable for supporting growth and/or survival of either the reporter cell or the pathogenic cell or organism, such that when the reporter cell and the pathogenic cell or organism are grown together in the presence of the growth medium and/or support surface, growth and/or survival of the reporter cell and the pathogenic cell or organism are inversely related.

39. A sample preparation or kit according to any of claims 35 to 38, further comprising one or more candidate agents for inhibiting growth and/or survival of the pathogenic cell or organism.

40. A method according to any of claims 1 to 34, wherein the reporter cell comprises a plant cell.