Method for identifying and isolating keystone bacteria in an ecosystem

Abstract

The present invention relates to a method for isolating and identifying a keystone microorganism in an ecosystem of interest. This keystone microorganism carries at least one metabolic function involved in an altered metabolic pathway in a representative sample of the ecosystem. This altered metabolic pathway is then responsible for dysbiosis.

Description

Title of the invention: Method for identifying and isolating keystone bacteria in an ecosystem. Technical field

[0001] The invention relates to the field of the microbiome and concerns a method for isolating and / or identifying a keystone microorganism in an ecosystem of interest. This keystone microorganism carries at least one metabolic function involved in an altered metabolic pathway in a representative sample of an ecosystem of interest. This altered metabolic pathway may lead to dysbiosis. Prior art

[0002] Microbiomes are present in all non-sterile environments (air; water, soil, plants, animals, surfaces, mucous membranes, skin...). Their proper functioning contributes to the balance of the ecosystem composed of the microbiota and its environment, which may be the intestine, the oceanic or marine environment, the soil or the subsoil.

[0003] For example, the proper functioning of the intestinal ecosystem ensures the maintenance of the good health of its host, the proper functioning of the rhizobiome ecosystem guarantees the good health of the roots and therefore of the plant, while the soil microbiome protects it in particular against erosion and retains water.

[0004] However, it is known that microbiomes tend to become impoverished overall in all ecosystems, notably due to a loss of diversity. The main causes of this impoverishment are exposure to chemical molecules or the overexploitation of environments. Chemical molecules, in particular, modify the microbiota habitat, for example, through the accumulation of heavy metals in soils (i.e., copper in viticulture), or through the presence of antibiotic molecules used in human and animal health that are released into the environment, thus prolonging their antibiotic effects. The exploitation or modification of environments, such as intensive agriculture, deforestation, soil exploitation, chemical pollution, etc., also tends to impoverish these environments.

[0005] Such microbiological impoverishment of the ecosystem inevitably leads to its dysfunction. In particular, the host-microbiota interaction is altered, leading to a breakdown of the symbiosis between the host and its microbiota; this is known as dysbiosis. This state of dysbiosis is characterized by a loss of essential functions provided by the microbiota to its host. In this situation, it is critical to restore the balance between the host and its microbiota by restoring the altered functions.

[0006] The stakes for human, animal and environmental health are important since dysbiosis between a living being and its microbiomes is increasingly associated with chronic pathological conditions, such as the onset of metabolic disorders, chronic inflammation in humans and animals; limited plant growth, reduced yields, soil erosion, etc.

[0007] In humans and animals, it has been shown that commensal microbiomes act in symbiosis with the organism, fulfilling specific metabolic or biochemical functions such as the synthesis of vitamins, the digestion of nutrients like complex carbohydrates, the stimulation of the immune system, or protection against pathogens.

[0008] In the environmental field, soil microbiomes form the basis of the ecosystem, supporting soil health. Indeed, the proper functioning of soil microbiomes enables the production of substances that act as a "glue" between sand grains, thus combating soil erosion (accelerated soil erosion affects 70% of arable land worldwide and is one of the main factors in crop yield loss). Furthermore, these substances that constitute this glue also help retain water in the soil (most of the world's accessible drinking water is contained in soils). Finally, the proper functioning of this community of microorganisms allows for the fixation of carbon and nitrogen in the soil, making them available to plants, thereby reducing greenhouse gas emissions and regulating the climate.

[0009] Consequently, the stakes for the proper functioning of microbiomes are numerous and affect all living elements on Earth because they form the link between the environment, animals, and humans. They are therefore at the heart of the global planetary ecosystem, which constitutes a single "health."

[0010] This results in a strong need to restore the proper functioning of ecosystems, in particular bacterial ecosystems, and thus to optimize and restore the interaction between individuals (i.e. microorganisms) in order to produce substances of interest through the joint action of several microorganisms.

[0011] However, there is no satisfactory industrial solution adapted to microbial ecosystems that allows for the restoration of their functioning.

[0012] In soil health, the restoration of a microbiota is primarily achieved through the modulation of agricultural practices, particularly by implementing crop rotation. Failing such a solution, it is known to restore complex ecosystems by replacing or supplementing a portion of the altered ecosystem with a healthy exogenous ecosystem (i.e., soil transplantation in agriculture, organic amendment).

[0013] When dysbiosis occurs, it is known to directly administer probiotic or prebiotic bacteria. Probiotics, in particular, will allow The goal is to increase the number of beneficial microorganisms and thus decrease the population of those that are potentially harmful or responsible for dysbiosis. In human and animal health, these bacteria can, for example, stimulate the host's immune system (e.g., fecal microbiota transplantation in humans and administration of bacterial communities in the form of bacterial consortia). In plant health, there are symbiotic root bacteria that enable nitrogen fixation. Some microorganisms are also used as biocontrol agents to limit the proliferation of certain pathogens. However, these solutions do not allow for precise and controlled action on the entire microbial ecosystem.

[0014] Prebiotics, unlike probiotics, are fibers or complex carbohydrates that facilitate the growth of certain beneficial organisms. They also promote microbial diversity in the ecosystem. However, these solutions are not very specific and cannot restore functions performed by microorganisms that have disappeared from the ecosystem.

[0015] Finally, synbiotics are combinations of probiotics and prebiotics. Thus, they can complement each other to provide a common solution whose action is targeted on the host, but they present the same drawbacks.

[0016] For example, in the field of agriculture, it is known to carry out organic amendments rich in carbon and microorganisms (manure, slurry, guano etc); there are also bacterial biostimulants which are bacteria targeting the rhizobiome of the plant (i.e. root system) to facilitate the absorption of nutrients (i.e. nitrogen) and therefore its growth.

[0017] However, all of these solutions do not allow for the specific targeting of microorganisms carrying an essential metabolic or biochemical function within a metabolic or biochemical pathway, these microorganisms being called keystone microorganisms, for example, those enabling the synthesis of a substance of interest to combat soil erosion.

[0018] To date, to identify a microorganism likely to be of interest, it is possible to perform a differential analysis of the presence / absence of microorganisms in healthy and dysbiotic ecosystems. This approach can be carried out based on the quantification of these microorganisms or using neural networks that reconstruct a map of microorganism groups based on their co-abundance. However, these methods do not allow for the identification of one keystone microorganism from another.

[0019] Indeed, these methods do not take into account the biological complementarity of microorganisms. However, ecosystems collaborate in order to function together in a metabolic or biochemical pathway. This collaboration relies on the complementarity of their biochemical or metabolic capacities (i.e., their biological functions) allowing, for example, the community of microorganisms to degrade complex substances that could not be degraded by a single microorganism.

[0020] Such an approach is particularly suitable when the microorganism is known and listed in a database. However, it is estimated that only 0.001% of microbial biodiversity is known. For example, the vast majority of soil bacteria (-99%) have never been cultured and are therefore unknown. Furthermore, when the microorganism is unknown, it is not possible to isolate it in a targeted manner using the usual techniques known to those skilled in the art. It is therefore not possible to identify a keystone microorganism using this type of approach.

[0021] There is therefore a need for a method for identifying and / or isolating a microorganism carrying a metabolic function involved in an essential metabolic or biochemical pathway, said pathway being altered, in an ecosystem of interest. This method would allow, firstly, the identification in a biological sample of an altered metabolic or biochemical pathway, then the metabolic or biochemical function(s) responsible for this alteration, and finally the identification of at least one microorganism carrying said metabolic or biochemical function. Given our limited knowledge of all the microorganisms present in a given ecosystem, it is necessary to develop a method for identifying and isolating a microorganism of interest unknown to those skilled in the art and to databases listing them. This is the object of the present invention. Summary of the invention

[0022] Thus, the present invention relates to a method for isolating and / or identifying a microorganism of interest in a biological sample, said microorganism carrying at least one metabolic or biochemical function (F) involved in an altered metabolic or biochemical pathway (V) within the microbial ecosystem localized in that biological sample. Said altered pathway (V) may be either a disrupted or missing metabolic or biochemical pathway, because an essential metabolic or biochemical function (F) involved in pathway (V) is not carried by any microorganism within that ecosystem, or altered because the essential metabolic or biochemical function (F) involved in pathway (V) is carried by a limited number of microorganisms.

[0023] The present method aims to identify an altered metabolic or biochemical pathway (V), and then the essential metabolic or biochemical function(s) (F) involved in said pathway (V). Once identified, the present method aims to identify at least one microorganism carrying said metabolic or biochemical function(s) (F) determined, which is / are involved in the altered metabolic or biochemical pathway (V). Said method according to the invention is not carried out by means of a quantitative approach, for example by the use of a neural network type approach which reconstructs a map of groups of microorganisms on the basis of their co-abundance.

[0024] Indeed, given the limited number of known and cataloged microorganisms, it is not possible to definitively identify the species of microorganism(s) carrying at least one function (F) involved in the altered metabolic or biochemical pathway (V). However, such information is essential for restoring an impoverished ecosystem.

[0025] Also, the present method includes the identification of a microorganism of interest, more preferably when it is unknown in the databases.

[0026] To achieve this, the present invention therefore relates to a method for isolating and / or identifying a microorganism of interest in a sample from a biological ecosystem, said microorganism carrying at least one metabolic or biochemical function (F) involved in an altered metabolic or biochemical pathway (V), said method comprising the following steps: a. Sequencing the DNA contained in a biological sample from the ecosystem, b. annotate the sequenced genes and / or operons and determine the set of functions (F) associated with said genes and / or operons, c. Reconstruct a path (V) from the functions (F) determined in the previous step, d. determine an altered pathway (V), by comparison to a reference sample e. determine at least one missing or deficient function (F) in the pathway (V) determined in step d), by comparison to a reference sample, preferably by differential comparison to a reference sample, f. reconstitute at least a part of the genome of at least one microorganism carrying at least one function (F) determined in step e) and involved in the pathway (V) determined in step d), g. identify at least one immunogenic surface protein (P) expressed by at least one microorganism carrying the function (F) determined in the previous step, h. generate at least one antibody targeting said protein (P) and label said antibody with at least one fluorophore, i. to label the microorganism of interest using said antibody, in the biological sample, and j. Isolate the microorganism of interest by cell sorting.

[0027] Thus, the method according to the invention aims to identify a keystone microorganism, that is, one whose abundance has a disproportionate impact on its environment, namely on the other members of the ecosystem and on the factors regulating that environment. Indeed, the defining characteristic of keystone microorganisms is their ability to carry rare, infrequent, or even unique metabolic or biochemical functions (F), which are nevertheless essential in certain metabolic or biochemical pathways (V), making them specific regulators of their ecosystem. Consequently, these microorganisms are very often scarce, and current identification and isolation methods, namely co-occurrence or co-abundance networks coupled with culturomics, are not suitable.These methods therefore do not allow for their effective detection and isolation, in order to reintroduce them into ecosystems where these microorganisms are absent or poorly represented.

[0028] Thus, according to a preferred object of the invention, the function (F) determined in step e), is absent or weakly expressed in the biological sample compared to a reference sample.

[0029] The method therefore comprises a first step of sequencing all the DNA contained in a biological sample of interest representative of an ecosystem of interest. Advantageously, the sequencing of a reference sample, i.e., considered healthy, is carried out under the same conditions as that of the sample of interest. This reference sample will subsequently allow for a comparative differential analysis.

[0030] Sequencing can be of all types, this one being suitable for sequencing thousands or millions of DNA or RNA molecules simultaneously. In this respect, in the context of the invention, a next-generation sequencing method is preferred, such as NGS (next-generation sequencing).

[0031] Also, the sequencing step of the method according to the invention is preferably sequencing performed by NGS. According to an even more preferred object, the sequencing is performed by short-read NGS and / or long-read NGS.

[0032] According to one variant, the method includes a sequencing step performed by combining NGS short read and NGS long read sequencing.

[0033] Once the sequencing has been completed, a large amount of raw sequencing data is obtained. This data then requires at least one additional step to allow for its processing.

[0034] Thus, the method according to the invention includes a step of annotating the previously sequenced genes and / or operons. The annotation of the genes and / or operons is Performed by bioinformatics analysis using standard human-informatics knowledge, examples include the Prokka, DFAST, and PGAP algorithms for annotating prokaryotic genomes (Tonkin-Hill et al. Microbial Genomics 2023;9:00&021 (doi 10.1099 / mgen.0.001021)). This bioinformatics analysis allows for the assembly of sequenced genes and / or operons from a biological sample representative of a given ecosystem, followed by annotation to determine their complete metabolic or biochemical functions (F).

[0035] The method therefore includes a step to annotate the sequenced genes and / or operons followed by a step to determine and reconstruct the set of functions (F) associated with said genes and / or operons.

[0036] This set of metabolic or biochemical functions (F) determined from sequencing data then makes it possible to reconstruct the different metabolic or biochemical pathways (V) present and involved in the ecosystem.

[0037] Thus, the present method also includes a step to reconstruct at least one metabolic or biochemical pathway (V) from the functions (F) determined during the gene and / or operon annotation step.

[0038] Most preferably, the reconstitution of pathway (V) is carried out using a GSM method, also sometimes called GEM (Genome-scale metabolic modeling; (Tarzi et al. Trends in Endocrinology & Metabolism 2024 (DOI: https: / / doi.org / 10.1016 / j.tem.2024.02.018)).

[0039] Therefore, it is possible to determine, to identify at least one altered pathway (V) in the ecosystem, by comparison to a reference sample, preferably by differential comparison.

[0040] Once at least one altered pathway (V) has been identified or determined, the present method includes an additional step to determine at least one missing or deficient function (F) in the pathway (V) determined in the previous step, namely step d), by comparison to a reference sample, preferably by differential comparison to a reference sample.

[0041] For the sake of clarity, the person skilled in the art will easily understand that the preceding steps are implemented both on the biological sample of interest and a reference sample, thus allowing comparison, by differential analysis, of the metabolic or biochemical pathways (V) present in each of the samples and thus, to determine at least one altered metabolic or biochemical pathway (V), as well as at least one missing or deficient function (F) in said altered pathway (V).

[0042] Thus, from the determined altered path (V), the person skilled in the art can identify and determine at least one missing or deficient function (F) in said altered path (V).

[0043] Finally, the present method includes a step of reconstituting at least a part of the genome of at least one microorganism carrying at least one function (F) determined in step e) and involved in the pathway (V) determined in step d).

[0044] Therefore, from the sequencing data, in particular from the reconstructed genome of the microorganism carrying at least one deficient or missing function (F), the method includes an additional step of identifying an immunogenic protein carried by the microorganism carrying said missing or deficient function (F).

[0045] Preferably, the immunogenic or immunogenic surface protein is a membrane protein comprising an extracellular domain, which allows the subsequent generation of antibodies, in particular polyclonal antibodies.

[0046] The method according to the invention then comprises an additional step of generating antibodies, preferably polyclonal antibodies, and labeling them, advantageously with a fluorescence label, thus enabling the identification of the microorganism by a microfluidic cell sorting technique. Preferably, the cell sorting is performed by a microfluidic method or flow cytometry.

[0047] Finally, according to another particularly preferred object of the invention, the biological sample is a soil sample, which includes anaerobic and aerobic microorganisms.

[0048] In the context of the invention, the microorganism of interest, possessing at least one metabolic or biochemical function (F) involved in an altered metabolic or biochemical pathway (V), can be either an anaerobic or an aerobic microorganism. In a particularly preferred embodiment, the microorganism is an anaerobic microorganism, which improves the performance and efficiency of the present method for identifying a so-called keystone microorganism. Indeed, the majority of bacterial diversity lives in anaerobic conditions (absence of oxygen). However, this intolerance to oxygen poses a number of difficulties, as it requires equipment capable of maintaining anaerobic conditions throughout the isolation method.The present invention aims, very preferably, at identifying such anaerobic microorganisms, unlike prior art methods, further improving the efficiency and isolation of microorganisms of interest capable of enriching an ecosystem of interest and thus treating dysbiosis.

[0049] According to another object of the invention, the microorganism is preferably chosen from among a bacterium, an archaeon, a microalga, a protist and a fungus.

[0050] Other features and advantages will become apparent from the detailed description of the invention which will follow. Detailed description of the invention

[0051] Definition

[0052] The term “metabolic or biochemical function” or “function (F)” in the context of the invention refers to a biological or biochemical activity carried out by an enzyme or group of enzymes in an organism. This activity contributes to the metabolism of a cell or organism, playing an essential role in its growth, development, survival, and environmental interactions with its ecosystem. This function is very often implemented in a metabolic pathway leading to the biosynthesis or degradation of substances of interest.

[0053] By "metabolic or biochemical pathway" or "pathway (V)" in the sense of the invention, we mean a series of interconnected biological or biochemical reactions that occur within a cell, an organism, or a community of organisms in an ecosystem. For example, the production of exopolysaccharides depends on the availability of glucose precursors in the environment, which are themselves released through the action of specific enzymes that may be carried by microorganisms distinct from the community. Thus, in a metabolic pathway, an initial substance (e.g., glucose) is modified by a sequence of reactions, resulting in the production of another final substance (e.g., the transformation of glucose into ADP-glucose, which is a component of a chain of a complex polysaccharide forming an exopolysaccharide).Each reaction is carried out using at least one specific enzyme, which allows the reaction to proceed efficiently and under controlled conditions.

[0054] For the purposes of this invention, a "keystone microorganism" is defined as a microorganism within an ecosystem that has a fundamental impact on its overall functioning and stability. These microorganisms carry out rare, or even unique, metabolic functions within the ecosystem in which they live. These microorganisms play an essential role in certain metabolic pathways, such as the cycling of certain nutrients, or in maintaining the ecosystem's resilience to disturbances. For example, a keystone microorganism may express a specific enzyme that breaks down certain bonds in a complex polysaccharide to release rare sugars. Finally, the metabolic functions carried out by these keystone microorganisms are not easily replaced if the keystone microorganism disappears or is eliminated.

[0055] Method according to the present invention

[0056] The present invention therefore relates to a method for isolating and / or identifying a microorganism of interest in a sample from a biological ecosystem, said microorganism carrying at least one metabolic or biochemical function (F) involved in an altered metabolic or biochemical pathway (V), said method comprising the following steps: a. Sequencing the DNA contained in a biological sample from the ecosystem, b. annotate the sequenced genes and / or operons and determine the set of functions (F) associated with said genes and / or operons, c. Reconstruct a path (V) from the functions (F) determined in the previous step, d. determine an altered pathway (V), by comparison to a reference sample e. determine at least one missing or deficient function (F) in the pathway (V) determined in step d), by comparison to a reference sample, preferably by differential comparison to a reference sample, f. reconstitute at least a part of the genome of at least one microorganism carrying at least one function (F) determined in step e) and involved in the pathway (V) determined in step d), g. identify at least one immunogenic surface protein (P) expressed by at least one microorganism carrying the function (F) determined in the previous step, h. generate at least one antibody targeting said protein (P) and label said antibody with at least one fluorophore, i. to label the microorganism of interest using said antibody in the biological sample, j. Isolate the microorganism of interest by tri-cell analysis.

[0057] The initial step of the present method therefore comprises the extraction and sequencing of all the DNA contained in the biological sample, which is taken from and representative of an ecosystem of interest. In the context of the invention, the present method comprises the collection of a biological sample of interest in which a metabolic or biochemical function (F) carried by a microorganism is to be identified, said function (F) being involved in a metabolic pathway (V) altered in the ecosystem of interest. A second reference sample is also required, serving as a control. This reference sample will be representative of the ecosystem of interest and considered healthy. For example, in human health, the biological sample could be feces. Human feces from a subject suffering from a metabolic disorder, compared to human feces not exhibiting a metabolic disorder and therefore considered healthy. In soil health, the biological sample can be a soil sample from a crop and the control sample, a soil sample taken near the crop that has received little or no phytosanitary treatment, organic amendments, etc. (for example, a forest or grove in the immediate vicinity of said crop).

[0058] It is well known to those skilled in the art that sequencing methods exist for managing the complexity and diversity of the microbiome in a sample. According to a particularly preferred object of the invention, next-generation sequencing (NGS) technologies are used. Indeed, these technologies make it possible to simultaneously sequence thousands or millions of DNA molecules, thus enabling a thorough and comprehensive analysis of all the DNA contained in a microbiome of interest.

[0059] According to a preferred object of the invention, sequencing can be carried out using short read NGS platforms, which are capable of generating a large number of sequences with high accuracy but having limited read lengths.

[0060] Alternatively, according to another object of the invention, sequencing can be carried out using long read NGS or NGS long read platforms, which provide longer sequences that can cover complex genomic regions and facilitate genome assembly.

[0061] According to one embodiment, particularly preferred, given the complexity of the biological samples of interest, the method according to the invention comprises a combination of short read and long read sequencing techniques, thus making it possible to take advantage of the benefits of each platform, thereby improving the accuracy and completeness of the microbial genomic data obtained, in particular of the genes and / or operons present in the biological sample of interest.

[0062] Two main technologies are preferentially used for long-read sequencing, namely the SMRT (Single Molecule, Real-Time) sequencing technology developed by Pacific Biosciences (PacBio) and that developed by Oxford Nanopore Technologies.

[0063] After the sequencing step, the collected genetic data undergo bioinformatic analysis to annotate the sequenced genes and / or operons. Annotation aims to assign biological information related to the sequenced genes and / or operons. Annotation thus consists of assigning functional information to gene sequences, such as gene functions, cellular locations, and biological functions.

[0064] This step then makes it possible to translate the raw sequencing data into an understanding of the functions carried by these genes and / or operons present in the sample of interest, in order to associate the sequencing data and the biological knowledge associated with these genes and / or operons.

[0065] The annotation step thus makes it possible to go beyond the simple identification of nucleotide sequences and to understand what these sequences do, how they interact and what their implications are for the organism or ecosystem studied.

[0066] In this context, gene annotation is particularly useful and of interest for the analysis of metabolic or biochemical pathways in an ecosystem of interest. By identifying the genes involved in specific metabolic or biochemical functions, researchers can reconstruct metabolic or biochemical pathways and understand how they are altered (e.g., the onset of dysbiosis).

[0067] This annotation step consists of comparing the sequenced DNA to known sequences listed in comprehensive databases, such as those containing annotated genomes and metabolic functions. This makes it possible to determine the metabolic functions associated with genes and / or operons. The result of this step provides a catalog of the metabolic or biochemical functions (F) present in the biological sample, which serves as the basis for reconstructing metabolic or biochemical pathways (V) and thus identifying the microorganisms responsible for these functions.

[0068] The bioinformatics tools and algorithms used for annotation are well known to those skilled in the art. They will be selected, in particular, based on their ability to handle the complexity of microbial communities and the quality of sequencing data, for example, Prokka, DFAST, or PGAP. Advantageously, these tools may include sequence alignment programs and gene prediction algorithms associated with databases such as NCBI's GenBank, UniProt, KEGG, and MetaCyc.

[0069] This annotation step makes it possible to obtain a detailed map of all the metabolic or biochemical functions (F) of the microbiome of the biological sample of interest, as well as of the reference sample. This information is then used to reconstruct and identify the metabolic or biochemical pathways (V) associated with said functions (F).

[0070] Once the functions (F) have been determined, they are used to reconstruct and determine the metabolic or biochemical pathway(s) in which said functions (F) are involved. In other words, from the mapping of the known functions (F) and metabolic or biochemical pathways, it is possible to determine where said functions (F) are integrated into a pathway (V) and how said functions (F) interact with other components of the path (V). Also, a person skilled in the art can reconstruct the paths (V).

[0071] In the context of the present invention, the method also includes an additional step of comparing the data obtained, namely the pathways (V) present in the biological sample with the data from a reference sample. This differential analysis of metabolic or biochemical pathways is performed to identify at least one altered pathway (V) and, consequently, the deficient, missing, or modified functions (F) involved in the alteration of said altered pathway (V). This allows for an understanding of the changes in metabolic or biochemical pathways that can lead to dysbiosis.

[0072] According to one embodiment of the invention, such data can be integrated with other "omics" type data, such as transcriptomics or proteomics, in order to provide a more precise analysis of metabolic functions and their regulation within the ecosystem.

[0073] Thus, by comparison according to a differential analysis between the data of the biological sample and the data of the reference sample, it is possible to determine the altered or deficient or missing metabolic pathways which can then contribute to the state of dysbiosis in the ecosystem of interest.

[0074] Once determined, the method includes a step to determine the missing or underrepresented functions involved in the altered (V) pathway.

[0075] The method includes a step to isolate at least one microorganism carrying said function (F) in order to subsequently restore it in the ecosystem and thus regenerate the latter. Thus, the method includes a step of reconstituting at least a part of the genome of the microorganism carrying at least one deficient or missing function (F) determined in step e) and involved in the altered pathway (V) determined in step d).

[0076] Finally, the method includes the isolation of the microorganism of interest. This is the subject of the remainder of the present method.

[0077] To isolate this, the method according to the invention implements the following steps, namely: a. identify at least one immunogenic surface protein (P) expressed by at least one microorganism carrying the function (F) determined in the previous step, b. generate at least one antibody targeting said protein (P) and label said antibody with at least one fluorophore, c. to label the microorganism of interest using said antibody, in the biological sample, and d. Isolate the microorganism of interest by tri-cell analysis.

[0078] Once the missing or deficient function (F) has been identified, it is possible, using sequencing data, to identify at least one immunogenic surface polymer (P) expressed by at least one microorganism carrying said function (F).

[0079] The identification of at least one immunogenic surface polymer (P) then makes it possible to determine a target for the further development of antibodies, in particular polyclonal antibodies. These antibodies can be used to label and isolate the microorganism of interest carrying the metabolic function (F) involved in the modified metabolic pathway (V).

[0080] Immunogenic surface polymers are complex polymers located on the outer membrane of microorganisms and capable of triggering an immune response. These polymers are therefore accessible to antibodies and can serve as markers for specific microorganisms.

[0081] Preferably, the immunogenic surface polymer is a membrane protein comprising an extracellular domain, more preferably a glycoprotein or a lipoprotein.

[0082] By way of example, a person skilled in the art, through bioinformatic analysis and dedicated tools, can predict the localization and immunogenicity of such proteins, for example a tool such as SignalP which makes it possible to predict the presence of signaling peptides and transmembrane domains, which are indicative of surface localization.

[0083] According to another preferred object of the invention, a bioinformatics tool, such as epitope prediction software, is used to identify regions of the protein that are likely to be recognized by polyclonal antibodies.

[0084] Once the potential immunogenic surface proteins are identified, and their expression on the surface of the microorganism validated, said proteins are selected for antibody production, according to the knowledge of those skilled in the art. These proteins will serve as antigens for the production of polyclonal antibodies.

[0085] The method then includes a step to generate at least one antibody, preferably a polyclonal antibody targeting said protein (P) and to label said antibodies, preferably polyclonal antibodies, with at least one fluorophore.

[0086] These selected immunogenic surface proteins are then used to immunize animals, generally rabbits or mice, in order to produce polyclonal antibodies. The host animal's immune system then produces these antibodies, which specifically recognize the immunogenic surface proteins.

[0087] Advantageously, a person skilled in the art will be able to synthesize the immunogenic protein or its epitope in the form of a recombinant protein in a suitable microorganism, such as Escherishia coli.

[0088] From the serum of immunized animals, the generated polyclonal antibodies are purified. They are then labeled with fluorophores to allow visualization and isolation of microorganisms of interest.

[0089] Advantageously, the labeling of microorganisms is carried out under a controlled atmosphere, the oxygen concentration of which is between 0 and 21%.

[0090] These labeled antibodies are then used to bind to the immunogenic surface proteins of the microorganisms of interest in the biological sample. This allows for the specific labeling of microorganisms carrying the metabolic or biochemical function (F) of interest, thus enabling their isolation by cell sorting. Preferably, cell sorting is performed by flow cytometry or microfluidics.

[0091] According to another object of the invention, the biological sample can be of any type, however, the biological sample is very preferably a soil sample.

[0092] According to another aspect of the invention, the microorganism is an anaerobic or aerobic microorganism. Advantageously, the microorganism is an anaerobic microorganism, which optimizes the performance of the present method. Anaerobic microorganisms, in particular, possess a large number of rare functional groups and are therefore more likely to be keystone microorganisms.

[0093] More preferably, the microorganism is a bacterium, an archaeon, a protist or a fungus, even more preferably a bacterium.

Claims

Demands

1. Method for isolating a microorganism of interest from a sample taken from a biological ecosystem, said microorganism carrying at least one metabolic or biochemical function (F) involved in an altered metabolic or biochemical pathway (V) of said ecosystem, said method comprising the following steps: a. Sequencing the DNA contained in a biological sample is derived from the ecosystem, b. annotate the sequenced genes and / or operons and determine the set of functions (F) associated with said genes and / or operons, c. Reconstruct a path (V) from the functions (F) determined in the previous step, d. determine an altered pathway (V), by comparison to a reference sample e. determine at least one missing or deficient function (F) in the path (V) determined in step d), by comparison to a reference sample, preferably by differential comparison to a reference sample, f. reconstitute at least a part of the genome of at least one microorganism carrying at least one function (F) determined in step e) and involved in the pathway (V) determined in step d), g. identify at least one immunogenic surface protein (P) expressed by at least one microorganism carrying the function (F) determined in the previous step, h. generate at least one antibody targeting said protein (P) and label said antibody with at least one fluorophore, i. label the microorganism of interest using said antibody, in the biological sample, j. Isolate the microorganism of interest by tri-cell analysis.

2. Method according to the preceding claim, characterized in that the function (F) determined in step e), is absent or weakly expressed in the biological sample compared to a reference sample.

3. Method according to any one of the preceding claims, characterized in that the sequencing is carried out by NGS short read and / or NGS long read.

4. Method according to any one of the preceding claims, characterized in that the annotation of genes and / or operons is carried out by bioinformatic analysis.

5. Method according to any one of the preceding claims, characterized in that step c) of reconstitution of pathway (V) is carried out using a GSM (Genome-scale metabolic modeling) method.

6. Method according to any one of the preceding claims, characterized in that the biological sample is a soil sample.

7. Method according to any one of the preceding claims, characterized in that the microorganism is an anaerobic microorganism.

8. Method according to the preceding claim, characterized in that the microorganism is a bacterium, an archaeon, a protist or a fungus.

9. A method according to any one of the preceding claims, characterized in that the immunogenic surface protein is a membrane protein comprising an extracellular domain.

10. Method according to any one of the preceding claims, characterized in that the tri-cell is a flow cytometry.