Patent Application
20240408170
The gastrointestinal tract is a unique organ that is constantly exposed to innumerable molecules derived from food and microorganisms. The gastrointestinal tract also encounters host-derived molecules such as digestive enzymes, in addition to external contents. In the upper gastrointestinal tract, digestive enzymes play an important role in breaking down large amounts of ingested nutrients from food into smaller components and absorption into the body. On the other hand, the large intestine mainly absorbs water, and no digestive enzymes are required, but rather this disturbance of the activity is thought to be associated with the change in the composition of the microbiota, the disruption of the mucosal barrier, and the generation of inflammation.
The intestinal tissue has various regulatory mechanisms and protective mechanisms, such as the production of mucin and an enzyme-inactivating molecule, in order to maintain homeostasis and a barrier function. In addition, it is known that the intestinal microbiota greatly contribute to the maintenance of a stable environment by reducing or changing substances in the lumen (J. L. Round and S. K. Mazmanian, The gut microbiota shapes intestinal immune responses during health and disease, Nature reviews immunology, 9, 313-323, 2009, for example).
However, the details of the regulation of intraluminal proteins by microorganisms have not been fully elucidated. In particular, little has been elucidated about the properties of microorganisms relating to the regulation of digestive enzymes remaining in the large intestine. An object of the present invention is to provide a technique for regulating the activity of proteases remaining in the large intestine by elucidating the functions of microorganisms relating to the regulation of the proteases.
The present invention includes the following aspects.
1. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; or bacteria that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
2. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; and bacteria that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
3. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 1, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability; or bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 2, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
4. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 1, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability; and bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 2, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
5. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 4, in which the bacteria have a type IX secretion system (T9SS).
6. The composition for decomposition of trypsin or TMPRSS2 according to 5, in which the T9SS contains PorV protein, PorU protein, PorN protein, PorM protein, PorL protein, PorK protein, or PorP protein.
7. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 6, in which the bacteria are bacteria belonging to the genus Paraprevotella, the genus Prevotella, the genus Prevotellamasilia, or the genus Bacteroidetes.
8. The composition for decomposition of trypsin or TMPRSS2 according to [7], in which the bacteria belonging to the genus Paraprevotella are Paraprevotella clara, and the bacteria belonging to the genus Prevotella are at least one type of bacteria selected from the group consisting of Prevotella rara, Prevotella rodentium, and Prevotella muris.
9. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 8, in which the bacteria are bacteria that have a 16S rRNA gene consisting of a base sequence set forth in SEQ ID NO: 3 or a base sequence set forth in SEQ ID NO: 4, or bacteria that have a 16S rRNA gene consisting of a base sequence having 97% or higher sequence identity with the base sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 4.
10. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 6, in which the bacteria are at least one type of bacteria selected from the group consisting of Paraprevotella sp. MSP 0303, Paraprevotella sp. MSP 0335, Prevotellamassilia timonensis, Bacteroidetes sp. MSP 0288, Bacteroidetes sp. MSP 0410, Bacteroidetes sp. MSP 0435, and Porphyromonas gingivalis.
11. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 10, in which the bacteria are live bacteria.
12. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 10, in which the bacteria are dead bacteria.
13. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; or 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
14. A composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; and 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
15. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 14, in which the composition for decomposition of trypsin or TMPRSS2 is for a treatment of a disease caused by trypsin or TMPRSS2.
16. The composition for decomposition of trypsin or TMPRSS2 according to any one of 1 to 15, in which the disease caused by trypsin or TMPRSS2 is an inflammatory bowel disease (ulcerative colitis, Crohn's disease), irritable bowel syndrome, an infectious disease, acute pancreatitis, or chronic pancreatitis.
17. The composition for decomposition of trypsin or TMPRSS2 according to 16, in which the infectious disease is a virus infectious disease or a bacterial infectious disease.
18. The composition for decomposition of trypsin or TMPRSS2 according to 16 or 17, in which the inflammatory bowel disease, the irritable bowel syndrome, or the infectious disease is a disease associated with TMPRSS2 or IgA.
19. A diagnostic drug for diseases caused by trypsin or TMPRSS2, the diagnostic drug containing: a specific binding substance that detects 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or that detects 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
20. A diagnostic drug for diseases caused by trypsin or TMPRSS2, the diagnostic drug containing: a primer set or a probe that detects a gene consisting of a base sequence set forth in SEQ ID NO: 1, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability, or that detects a gene consisting of a base sequence set forth in SEQ ID NO: 2, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
21. A quasi-drug for diseases caused by trypsin or TMPRSS2, the quasi-drug containing, as an active ingredient: bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
22. A quasi-drug for diseases caused by trypsin or TMPRSS2, the quasi-drug containing, as an active ingredient: 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; or 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
According to the present invention, a technique for regulating the activity of proteases can be provided.
In one embodiment, the present invention provides a composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; or bacteria that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
As will be described later in Examples, the inventors of the present invention clarified that bacteria having 00502 protein or bacteria having 00509 protein adsorb trypsin or TMPRSS2 on the bacterial cell surface to decompose trypsin or TMPRSS2 by autolysis of trypsin or TMPRSS2.
Accordingly, the composition containing bacteria having 00502 protein or bacteria having 00509 protein as an active ingredient can be used for the usage of the decomposition of trypsin or TMPRSS2. Accordingly, it can be said that the composition of the present embodiment is a decomposition agent for trypsin or TMPRSS2.
The decomposition of trypsin or TMPRSS2 can be used for industrial applications or, can be used for pharmaceutical applications as will be described later.
The accession number of UniProtKB of the 00502 protein of a Paraprevotella clara (YIT 11840) strain (catalog number “JCM: 14859”) is G5SNC9, which will be described later. In addition, the 00502 protein of the Paraprevotella clara (YIT 11840) strain is encoded by an HMPREF9441_00858 gene (UniProtKB). The NCBI accession number for the HMPREF9441_00858 gene is NZ_JH376591 REGION: complement (87340 . . . 91131). The amino acid sequence of the 00502 protein of the Paraprevotella clara (YIT 11840) strain is set forth in SEQ ID NO: 5, and the base sequence of the cDNA of the HMPREF9441_00858 gene is set forth in SEQ ID NO: 1.
In addition, the amino acid sequence of the 00502 protein of a Paraprevotella clara (1C4) strain is set forth in SEQ ID NO: 6, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Paraprevotella clara (1C4) strain is set forth in SEQ ID NO: 7.
In addition, the amino acid sequence of the 00502 protein of a Paraprevotella xylaniphila (82A6) strain is set forth in SEQ ID NO: 8, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Paraprevotella xylaniphila (82A6) strain is set forth in SEQ ID NO: 9.
In addition, the amino acid sequence of the 00502 protein of a Paraprevotella xylaniphila (YIT 11841) strain (catalog number “JCM: 14860”) is set forth in SEQ ID NO: 10, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Paraprevotella xylaniphila (YIT 11841) strain is set forth in SEQ ID NO: 11.
In addition, the amino acid sequence of the 00502 protein of a Prevotella rara (109) strain (catalog number “DSM: 105141”) is set forth in SEQ ID NO: 12, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Prevotella rara (109) strain is set forth in SEQ ID NO: 13.
In addition, the amino acid sequence of the 00502 protein of a Prevotella rodentium (PJ1A) strain (catalog number “DSM: 105243”) is set forth in SEQ ID NO: 14, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Prevotella rodentium (PJ1A) strain is set forth in SEQ ID NO: 15.
In addition, the amino acid sequence of the 00502 protein of a Prevotella muris (PMUR) strain (catalog number “DSM: 103722”) is set forth in SEQ ID NO: 16, and the base sequence of the cDNA of the gene encoding the 00502 protein of the Prevotella muris (PMUR) strain is set forth in SEQ ID NO: 17.
The accession number of UniProtKB of the 00509 protein of a Paraprevotella clara (YIT 11840) strain (catalog number “JCM: 14859”) is G5SNC1, which will be described later. In addition, the 00509 protein of the Paraprevotella clara (YIT 11840) strain is encoded by an HMPREF9441_00850 gene (UniProtKB). The NCBI accession number for the HMPREF9441_00850 gene is NZ_JH376591 REGION: 73848 . . . 76931. The amino acid sequence of the 00509 protein of the Paraprevotella clara (YIT 11840) strain is set forth in SEQ ID NO: 18, and the base sequence of the cDNA of the HMPREF9441_00850 gene is set forth in SEQ ID NO: 2.
In addition, the amino acid sequence of the 00509 protein of a Paraprevotella clara (1C4) strain is set forth in SEQ ID NO: 19, and the base sequence of the cDNA of the gene encoding the 00509 protein of the Paraprevotella clara (1C4) strain is set forth in SEQ ID NO: 20.
In addition, the amino acid sequence of the 00509 protein of a Paraprevotella xylaniphila (82A6) strain is set forth in SEQ ID NO: 21, and the base sequence of the cDNA of the gene encoding the 00509 protein of the Paraprevotella xylaniphila (82A6) strain is set forth in SEQ ID NO: 22.
In addition, the amino acid sequence of the 00509 protein of a Paraprevotella xylaniphila (YIT 11841) strain (catalog number “JCM: 14860”) is set forth in SEQ ID NO: 23, and the base sequence of the cDNA of the gene encoding the 00509 protein of the Paraprevotella xylaniphila (YIT 11841) strain is set forth in SEQ ID NO: 24.
In addition, the amino acid sequence of the 00509 protein of the Prevotella rara (109) strain (catalog number “DSM: 105141”) is set forth in SEQ ID NO: 25, and the base sequence of the cDNA of the gene encoding the 00509 protein of the Prevotella rara (109) strain is set forth in SEQ ID NO: 26.
In addition, the amino acid sequence of the 00509 protein of the Prevotella rodentium (PJ1A) strain (catalog number “DSM: 105243”) is set forth in SEQ ID NO: 27, and the base sequence of the cDNA of the gene encoding the 00509 protein of the Prevotella rodentium (PJ1A) strain is set forth in SEQ ID NO: 28.
As will be described later in the Examples, the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment may contain, as an active ingredient, bacteria having the homologue of 00502 protein or bacteria having the homologue of 00509 protein.
Examples of proteins as the homologue of 00502 protein include proteins having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the amino acid sequence set forth in SEQ ID NO: 5, and having a trypsin-binding ability. It is preferable that the bacteria having the homologue of 00502 protein have a trypsin-binding ability or TMPRSS2-binding ability and further have an activity of decomposing trypsin or TMPRSS2.
Examples of the homologue of 00509 protein include proteins having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, and having a trypsin-binding ability. It is preferable that the bacteria having the homologue of 00509 protein have a trypsin-binding ability or TMPRSS2-binding ability and further have an activity of decomposing trypsin or TMPRSS2.
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, the phrase “containing as an active ingredient” means that bacteria having 00502 protein or the homologue of the 00502 protein or bacteria having 00509 protein or the homologue of the 00509 protein are contained in an amount sufficient to decompose trypsin or TMPRSS2. Alternatively, the phrase means that bacteria having 00502 protein or the homologue of the 00502 protein or bacteria having 00509 protein or the homologue of the 00509 protein are contained as a major active ingredient.
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, the bacteria having 00502 protein or the homologue of the 00502 protein mean bacteria expressing the 00502 protein or the homologue of the 00502 protein.
The bacteria expressing 00502 protein may be bacteria having the HMPREF9441_00858 gene (SEQ ID NO: 1) encoding the 00502 protein. In addition, the bacteria having the homologue of the 00502 protein may be bacteria having a gene having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding the cDNA encoding a protein having a trypsin-binding ability. The 00502 protein may be derived from a gene endogenous to bacteria or may be derived from an exogenous gene.
Similarly, the bacteria having 00509 protein or the homologue of the 00509 protein mean bacteria expressing the 00509 protein or the homologue of the 00509 protein.
The bacteria expressing 00509 protein may be bacteria having the HMPREF9441_00850 gene (SEQ ID NO: 2) encoding the 00509 protein. In addition, the bacteria having the homologue of the 00509 protein may be bacteria having a gene having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding the cDNA encoding a protein having a trypsin-binding ability. The 00509 protein may be derived from a gene endogenous to bacteria or may be derived from an exogenous gene.
As long as the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment has the activity of decomposing trypsin or TMPRSS2, the composition may contain only bacteria having 00502 protein or the homologue of the 00502 protein, may contain only bacteria having 00509 protein or the homologue of the 00509 protein, or may contain both the bacteria having 00502 protein or the homologue of the 00502 protein and the bacteria having 00509 protein or the homologue of the 00509 protein. Alternatively, the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment may contain bacteria having 00502 protein or the homologue of the 00502 protein and having 00509 protein or the homologue of the 00509 protein. Such bacteria can be produced by genetic modification or the like.
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, it is preferable that bacteria having 00502 protein or the homologue of the 00502 protein or bacteria having 00509 protein or the homologue of the 00509 protein have a type IX secretion system (T9SS).
Furthermore, the T9SS preferably contains PorV protein, PorU protein, PorN protein, PorM protein, PorL protein, PorK protein, or PorP protein.
As will be described later in the Examples, the inventors of the present invention clarified that 00502 protein or the homologue of the 00502 protein, or 00509 protein or the homologue of the 00509 protein is transported by T9SS to the bacterial surface across the outer membrane, thereby binding to the bacterial surface.
For example, in a Paraprevotella clara strain to be described later, the NCBI accession number for PorV protein is WP_008622445.1, the NCBI accession number for PorU protein is WP_008622443.1, the NCBI accession number for PorN protein is WP_008623210.1, the NCBI accession number for PorM protein is WP_008623211.1, the NCBI accession number for PorL protein is WP_008623213.1, the NCBI accession number for PorK protein is WP_008623215.1, and the NCBI accession number for PorP protein is WP_008623217.1. In addition, in bacteria other than the Paraprevotella clara strain, the homologues of these proteins in the bacteria constitute T9SS.
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, bacteria having 00502 protein or the homologue of the 00502 protein or having 00509 protein or the homologue of the 00509 protein may be bacteria belonging to the genus Paraprevotella, the genus Prevotella, the genus Prevotellamasilia, or the genus Bacteroidetes.
As will be described later in the Examples, the inventors of the present invention clarified that bacteria belonging to the genus Paraprevotella, the genus Prevotella, the genus Prevotellamasilia, or the genus Bacteroidetes decompose trypsin or TMPRSS2. Examples of bacteria belonging to the genus Paraprevotella include Paraprevotella clara, Paraprevotella xylaniphila, Paraprevotella sp. MSP 0303, and Paraprevotella sp. MSP 0335. Examples of bacteria belonging to the genus Prevotella include Prevotella rara, Prevotella rodentium, and Prevotella muris.
The bacteria belonging to the genus Paraprevotella are bacteria which have a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 4, or have a 16S rRNA gene consisting of a base sequence having 97% or higher sequence identity with the base sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 4. In addition, when the sequence identity of the 16S rRNA gene is 97% or higher, the bacteria are determined to belong to the same species.
The base sequence set forth in SEQ ID NO: 3 is the base sequence of the 16S rRNA gene of a Paraprevotella clara (YIT 11840) strain (catalog number “JCM: 14859”), which will be described later. In addition, the base sequence set forth in SEQ ID NO: 29 is the base sequence of the 16S rRNA gene of a Paraprevotella clara (1C4) strain. In addition, the base sequence set forth in SEQ ID NO: 4 is the base sequence of the 16S rRNA gene of a Paraprevotella xylaniphila (82A6) strain, which will be described later. In addition, the base sequence set forth in SEQ ID NO: 30 is the base sequence of the 16S rRNA gene of a Paraprevotella xylaniphila (YIT 11841) strain (catalog number “JCM: 14860”).
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, bacteria having 00502 protein or the homologue of the 00502 protein or having 00509 protein or the homologue of the 00509 protein may be at least one type of bacteria selected from the group consisting of Paraprevotella sp. MSP 0303, Paraprevotella sp. MSP 0335, Prevotellamassilia timonensis, Bacteroidetes sp. MSP 0288, Bacteroidetes sp. MSP 0410, Bacteroidetes sp. MSP 0435, and Porphyromonas gingivalis.
Examples of Prevotella rara include a Prevotella rara (MSP 0081) strain and a Prevotella rara (109) strain. Examples of Prevotella rodentium include a Prevotella rodentium (PJ1A) strain (catalog number “DSM: 105243”). Examples of Prevotella muris include Prevotella muris (PMUR) strain (catalog number “DSM: 103722”). Examples of Prevotellamassilia timonensis include a Prevotellamassilia timonensis (MSP 0224) strain. Examples of Porphyromonas gingivalis include a Porphyromonas gingivalis (ATCC 33277) strain.
As will be described below in the Examples, the inventors of the present invention clarified that these bacteria decompose trypsin or TMPRSS2.
The base sequence set forth in SEQ ID NO: 31 is the base sequence of the 16S rRNA gene of the Prevotella rara (109) strain (catalog number “DSM: 105141”). In addition, the base sequence set forth in SEQ ID NO: 32 is the base sequence of the 16S rRNA gene of the Prevotella rodentium (PJ1A) strain (catalog number “DSM: 105243”). In addition, the base sequence set forth in SEQ ID NO: 33 is the base sequence of the 16S rRNA gene of the Prevotella muris (PMUR) strain (catalog number “DSM: 103722”).
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, the above-mentioned bacteria may be live bacteria or dead bacteria as long as they have the activity of decomposing trypsin or TMPRSS2.
For example, the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment may contain the above-mentioned bacteria in a powder form, a freeze-dried form, or the like, which are embedded in an aqueous form such as a solution or a suspension or a semi-solid form. In one aspect, the composition or the bacteria are freeze-dried. In one aspect, a subset of the bacteria in the composition is freeze-dried. A method of freeze-drying the composition containing the bacteria is well known in the technical field. For example, U.S. Pat. Nos. 3,261,761, 4,205,132, and PCT International Publication No. WO2012/098358 can be referred to. These documents are incorporated in the present specification by reference.
The bacteria may be freeze-dried as a combination or may be separately freeze-dried and combined. The bacteria may be combined with a pharmaceutically acceptable carrier before being combined with the other bacteria. A plurality of the freeze-dried bacteria may be combined in a freeze-dried form. A mixture of the bacteria combined once may be subsequently combined with a pharmaceutically acceptable carrier. In one aspect, the bacteria are a solid substance that has been freeze-dried. In one aspect, the composition is a solid substance that has been freeze-dried.
In one embodiment, the present invention provides a composition for decomposition of trypsin or TMPRSS2 containing, as an active ingredient: 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; or 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
As will be described below in the Examples, the inventors of the present invention clarified that the 00502 protein or the 00509 protein decomposes trypsin or TMPRSS2. It is preferable that the 00502 protein or the 00509 protein be immobilized on a solid phase surface.
The solid phase is not particularly limited, and examples thereof include particles made of resin, glass, metal, or the like; surfaces of containers such as plates and tubes; and films made of resin. The particles may be magnetic particles.
The solid phase may be a pharmaceutically acceptable solid phase. Examples of the pharmaceutically acceptable solid phase include liposomes; high-molecular-weight nanoparticles such as protein nanoparticles and lipid nanoparticles; iron nanoparticles; nanoemulsions such as lipid microspheres; micelles; vaccine adjuvants; and nanocrystals. The pharmaceutically acceptable solid phase is preferably one approved by the Pharmaceuticals and Medical Devices Agency (PMDA, an independent administrative institution), the United States Food and Drug Administration (FDA), and the European Medicines Agency (EMA).
A method of immobilizing the 00502 protein or the 00509 protein on a solid phase is not particularly limited, and examples thereof include bonding with a chemical linker, bonding with avidin-biotin, and physical adsorption.
As will be described later in the Examples, the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment may contain, as an active ingredient, the homologue of the 00502 protein or the homologue of the 00509 protein.
Examples of the homologue of the 00502 protein are the same as those described above and include proteins having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the amino acid sequence set forth in SEQ ID NO: 5, and having a trypsin-binding ability. It is preferable that the homologue of the 00502 protein have a trypsin-binding ability or TMPRSS2-binding ability.
Examples of the homologue of the 00509 protein are the same as those described above and include proteins having 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, and having a trypsin-binding ability. It is preferable that the homologue of the 00509 protein have a trypsin-binding ability or TMPRSS2-binding ability.
In the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment, the phrase “containing as an active ingredient” means that the 00502 protein or the homologue of the 00502 protein or the 00509 protein or the homologue of the 00509 protein is contained in an amount sufficient to decompose trypsin or TMPRSS2. Alternatively, the phrase means that the 00502 protein or the homologue of the 00502 protein or the 00509 protein or the homologue of the 00509 protein is contained as a major active ingredient.
The 00502 protein or the homologue of the 00502 protein, or the 00509 protein or the homologue of the 00509 protein may be added with a peptide tag for protein purification, for protein detection, or for binding to a solid phase. The peptide tag is not particularly limited, and examples thereof include a histidine tag, a FLAG tag, and a MYC tag.
As long as the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment has the activity of decomposing trypsin or TMPRSS2, the composition may contain only the 00502 protein or the homologue of the 00502 protein, may contain only the 00509 protein or the homologue of the 00509 protein, or may contain both the 00502 protein or the homologue of the 00502 protein and the 00509 protein or the homologue of the 00509 protein.
When the composition for decomposition of trypsin or TMPRSS2 according to the present embodiment contains both the 00502 protein or its homologue and the 00509 protein or its homologue, the 00502 protein or its homologue and the 00509 protein or its homologue may be bound.
The 00502 protein or its homologue and the 00509 protein or its homologue may linearly bound or may be cyclically bound. In addition, the 00502 protein or its homologue and the 00509 protein or its homologue may be directly bound or may be bound by a linker. The linker is not particularly limited, and examples thereof include a peptide consisting of an amino acid sequence in which GGGGS (SEQ ID NO: 34) is repeated 1 to 4 times.
The 00502 protein or its homologue and/or the 00509 protein or its homologue may be in the form of a fusion protein with an antibody constant region. The antibody constant region may be a constant region derived from a human antibody or may be a constant region derived from a human IgG-type antibody.
As shown in
In one embodiment, the above-mentioned composition for decomposition of trypsin or TMPRSS2 may be used for a treatment of diseases caused by trypsin or TMPRSS2. In other words, in one embodiment, the present invention provides a pharmaceutical composition for a treatment of diseases caused by trypsin or TMPRSS2. The pharmaceutical composition of the present embodiment contains, as an active ingredient, bacteria having 00502 protein or the homologue of the 00502 protein, bacteria having 00509 protein or the homologue of the 00509 protein, the 00502 protein or the homologue of the 00502 protein, or the 00509 protein or the homologue of the 00509 protein.
In the pharmaceutical composition of the present embodiment, the 00502 protein, the homologue of the 00502 protein, the 00509 protein, the homologue of the 00509 protein, and the bacteria having these are the same as those described above.
In the pharmaceutical composition of the present embodiment, examples of the diseases caused by trypsin or TMPRSS2 include an inflammatory bowel disease (ulcerative colitis, Crohn's disease), irritable bowel syndrome, acute pancreatitis, and chronic pancreatitis.
Alternatively, in the pharmaceutical composition of the present embodiment, examples of the diseases caused by trypsin or TMPRSS2 include infectious diseases. Examples of the infectious diseases include a virus infectious disease and a bacterial infectious disease. In addition, examples of the inflammatory bowel disease, the irritable bowel syndrome, or the infectious diseases mentioned above include diseases associated with TMPRSS2 or IgA. Examples of infectious diseases in which infection is associated with TMPRSS2 include coronavirus infectious diseases. Examples of infectious diseases associated with IgA include Salmonella infectious disease.
The pharmaceutical composition of the present embodiment may be formulated together with a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, one that is used for the formulation of a common pharmaceutical composition can be used without particular limitation. More specific examples thereof include binding agents such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; and swelling agents such as alginic acid.
The pharmaceutical composition of the present embodiment may further contain additives. Examples of the additives include lubricants such as calcium stearate and magnesium stearate; sweetening agents such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and Gaultheria adenothrix oil; stabilizers such as benzyl alcohol and phenol; and buffering agents such as a phosphoric acid salt and sodium acetate.
The pharmaceutical composition of the present embodiment can be formulated by appropriately combining the above-mentioned carriers and additives, and admixing them in a unit dosage form that is required for commonly accepted pharmaceutical practice.
A method of administering the pharmaceutical composition of the present embodiment is not particularly limited and may be appropriately determined depending on the symptom, the body weight, the age, the gender, and the like of patients. Examples thereof include tablets, powders, capsules, liquid medications, enemas, and suppositories. Tablets, powders, capsules, and liquid medications are orally administered. Enemas and suppositories are intestinally administered. The pharmaceutical composition is preferably in the dosage form capable of delivering the active ingredient (bacteria having 00502 protein or the homologue of the 00502 protein, bacteria having 00509 protein or the homologue of the 00509 protein, the 00502 protein or the homologue of the 00502 protein, or the 00509 protein or the homologue of the 00509 protein) to the intestine.
The dose of the pharmaceutical composition varies depending on the symptom, the body weight, the age, the gender, and the like of patients and cannot be unconditionally determined, but when the active ingredient is live bacteria, it is considered that the active ingredient is administered one time to several times at a dose of 0.1 to 100 mg/kg body weight per dosage unit form, for example. In addition, when the active ingredient is dead bacteria or protein, it is considered that the active ingredient is administered one time to several times per day at a dose of 0.1 to 100 mg/kg body weight per dosage unit form, for example.
The pharmaceutical composition of the present embodiment may be a quasi-drug. The quasi-drug is a product with a specifically recognized efficacy or effect and has a mild action on the human body. Examples of the quasi-drug of the present embodiment include, but are not limited to, forms such as drink agents, stomach medicines, and intestinal drugs.
In one embodiment, the present invention provides a diagnostic drug for diseases caused by trypsin or TMPRSS2, the diagnostic drug containing: a specific binding substance that detects 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or that detects 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In other words, the diagnostic drug of the present embodiment contains a specific binding substance that detects the 00502 protein, the homologue of the 00502 protein, the 00509 protein, and the homologue of the 00509 protein at a protein level.
Examples of the specific binding substance include antibodies, antibody fragments, and aptamers. Examples of the antibody fragments include Fab, F(ab′)2, Fab′, and single chain antibodies (scFv). An antibody may be a monoclonal antibody or a polyclonal antibody. A commercially available antibody may also be used.
In the diagnostic drug of the present embodiment, the 00502 protein, the homologue of the 00502 protein, the 00509 protein, the homologue of the 00509 protein, and the bacteria having these are the same as those described above. Furthermore, the same applies to the diseases caused by trypsin or TMPRSS2 as described above.
By using the diagnostic drug of the present embodiment, the presence or absence of the 00502 protein, the homologue of the 00502 protein, the 00509 protein, the homologue of the 00509 protein, or the bacteria having these can be detected from a biological specimen derived from a subject.
The detection principle by the diagnostic drug of the present embodiment is not particularly limited, and examples thereof include an enzyme-linked immunosorbent assay (ELISA) method, a lateral flow immunoassay, a western blot, and a flow cytometry (FACS).
Examples of the biological specimen derived from the subject include faeces specimens. When the presence of the 00502 protein, the homologue of the 00502 protein, the 00509 protein, the homologue of the 00509 protein, or the bacteria having these is detected from a biological specimen derived from a subject by using the diagnostic drug of the present embodiment, it can be determined that the subject has not developed the diseases caused by trypsin or TMPRSS2.
When the presence of the 00502 protein, the homologue of the 00502 protein, the 00509 protein, the homologue of the 00509 protein, or the bacteria having these is not detected from a biological specimen derived from a subject, it can be determined that there is a possibility that the subject has developed the diseases caused by trypsin or TMPRSS2. In this case, the symptom of the diseases caused by trypsin or TMPRSS2 can be treated or alleviated by administering the above-mentioned pharmaceutical composition or quasi-drug to the subject.
In one embodiment, the diagnostic drug of the present embodiment may detect the 00502 protein, the homologue of the 00502 protein, the 00509 protein, and the homologue of the 00509 protein at a gene level.
In other words, in one embodiment, the present invention provides a diagnostic drug for diseases caused by trypsin or TMPRSS2, the diagnostic drug containing: a primer set or a probe that detects a HMPREF9441_00858 gene encoding 00502 protein, or a gene having 30% or higher sequence identity with the base sequence of the HMPREF9441_00858 gene and encoding a protein having a trypsin-binding ability, or that detects a HMPREF9441_00850 gene encoding 00509 protein, or a gene having 30% or higher sequence identity with the base sequence of the HMPREF9441_00850 gene and encoding a protein having a trypsin-binding ability. In other words, the diagnostic drug of the present embodiment contains: a primer set or a probe that detects a gene consisting of a base sequence set forth in SEQ ID NO: 1, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability, or that detects a gene consisting of a base sequence set forth in SEQ ID NO: 2, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
The HMPREF9441_00858 gene and the HMPREF9441_00850 gene are the same as those described above.
The gene having 30% or higher sequence identity with the base sequence of the HMPREF9441_00858 gene and encoding a protein having a trypsin-binding ability is a gene encoding the homologue of the 00502 protein described above.
Furthermore, the gene having 30% or higher sequence identity with the base sequence of the HMPREF9441_00850 gene and encoding a protein having a trypsin-binding ability is a gene encoding the homologue of the 00509 protein described above.
By using the diagnostic drug of the present embodiment, the presence or absence of bacteria having the 00502 protein, bacteria having the homologue of the 00502 protein, bacteria having the 00509 protein, and bacteria having the homologue of the 00509 protein can be detected from a biological specimen derived from a subject.
The detection principle by the diagnostic drug of the present embodiment is not particularly limited, and examples thereof include PCR, RNA sequencing (RNA-Seq), metagenomics analysis, and DNA microarray analysis.
The biological specimen derived from the subject is the same as that described above, and examples thereof include faeces specimens. When the presence of bacteria having the 00502 protein, bacteria having the homologue of the 00502 protein, bacteria having the 00509 protein, and bacteria having the homologue of the 00509 protein is detected from a biological specimen derived from a subject by using the diagnostic drug of the present embodiment, it can be determined that the subject has not developed the diseases caused by trypsin or TMPRSS2.
When the presence of bacteria having the 00502 protein, bacteria having the homologue of the 00502 protein, bacteria having the 00509 protein, and bacteria having the homologue of the 00509 protein is not detected from a biological specimen derived from a subject, it can be determined that there is a possibility that the subject has developed the diseases caused by trypsin or TMPRSS2.
In this case, the symptom of the diseases caused by trypsin or TMPRSS2 can be treated or alleviated by administering the above-mentioned pharmaceutical composition or quasi-drug to the subject.
In one embodiment, the present invention provides a method for treating diseases caused by trypsin or TMPRSS2, the method including: a step of administering, to a patient in need of treatment, an effective amount of bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or an effective amount of bacteria that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In one embodiment, the present invention provides a method for treating diseases caused by trypsin or TMPRSS2, the method including: a step of administering, to a patient in need of treatment, an effective amount of bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 1, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability, or an effective amount of bacteria that have a gene consisting of a base sequence set forth in SEQ ID NO: 2, or a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
In one embodiment, the present invention provides a method for treating diseases caused by trypsin or TMPRSS2, the method including: a step of administering, to a patient in need of treatment, an effective amount of 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or an effective amount of 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In one embodiment, the present invention provides a method for diagnosing and treating diseases caused by trypsin or TMPRSS2, the method including: a step of detecting the presence or absence of the 00502 protein, the homologue of the 00502 protein, the 00509 protein, and the homologue of the 00509 protein from a biological specimen derived from a subject and indicating that the subject has developed the disease caused by trypsin or TMPRSS2 when the presence of the protein or the homologue is not detected; and a step of administering, to the subject when the presence of the protein or the homologue is not detected, an effective amount of bacteria that have 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, an effective amount of bacteria that have 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability, an effective amount of the 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or an effective amount of the 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In one embodiment, the present invention provides a method for diagnosing and treating diseases caused by trypsin or TMPRSS2, the method including: a step of detecting the presence or absence of bacteria having 00502 protein, bacteria having the homologue of the 00502 protein, bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 1, bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability, bacteria having 00509 protein, bacteria having the homologue of the 00509 protein, bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 2, or bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability from a biological specimen derived from a subject, and indicating that the subject has developed the disease caused by trypsin or TMPRSS2 when the presence of the bacteria having the protein, the homologue or the gene is not detected; and a step of administering, to the subject when the presence of the protein, the homologue or the bacteria having the gene is not detected, an effective amount of bacteria that have the 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, an effective amount of bacteria having a gene consisting of the base sequence set forth in SEQ ID NO: 1, an effective amount of bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability, an effective amount of bacteria that have the 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability, an effective amount of bacteria having a gene consisting of the base sequence set forth in SEQ ID NO: 2, an effective amount of bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability, an effective amount of the 00502 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability, or an effective amount of the 00509 protein, or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In one embodiment, the present invention provides a composition for treating diseases caused by trypsin or TMPRSS2, the composition containing, as an active ingredient: bacteria having 00502 protein; bacteria having a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 1; bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability; bacteria having 00509 protein; bacteria having a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability; bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 2; or bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
In one embodiment, the present invention provides a pharmaceutical composition for treating diseases caused by trypsin or TMPRSS2, the pharmaceutical composition containing, as an active ingredient: 00502 protein; a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; 00509 protein; or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
In one embodiment, the present invention provides use of the following bacteria for producing a pharmaceutical composition for treating diseases caused by trypsin or TMPRSS2: bacteria having 00502 protein; bacteria having a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 1; bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 1 and encoding a protein having a trypsin-binding ability; bacteria having 00509 protein; bacteria having a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability; bacteria having a gene consisting of a base sequence set forth in SEQ ID NO: 2; or bacteria having a gene having 30% or higher sequence identity with the base sequence set forth in SEQ ID NO: 2 and encoding a protein having a trypsin-binding ability.
In one embodiment, the present invention provides use of the following proteins for producing a pharmaceutical composition for treating diseases caused by trypsin or TMPRSS2: 00502 protein; a protein having 30% or higher sequence identity with an amino acid sequence of the 00502 protein and having a trypsin-binding ability; 00509 protein; or a protein having 30% or higher sequence identity with an amino acid sequence of the 00509 protein and having a trypsin-binding ability.
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
C57BL/6 mice bred under a specific-pathogen-free (SPF) condition or a germ-free (GF) condition were purchased from Sankyo Laboratories Japan, Japan SLC, Inc., Charles River Laboratories Japan, Inc., or CLEA Japan, Inc. GF mice and gnotobiotic mice were bred and maintained in a gnotobiotic facility of the RIKEN Center for Integrative Medical Sciences. All animal experiments were approved by the Committee of Experimental Animals of the RIKEN Yokohama Branch.
Paraprevotella clara (JCM 14859), Paraprevotella xylaniphila (JCM 14860), Paraprevotella copri (JCM 13464), Paraprevotella denticola (JCM 13449), Paraprevotella stercorea (JCM 13469), and Paraprevotella oulorum (JCM 14966) were obtained from the Japan Collection of Microorganisms (JCM). Paraprevotella clara (P237E3b) and (P322B5) were provided by Vedanta Biosciences, Inc. Paraprevotella xylaniphila (82A6) was isolated by the inventors of the present invention.
The protein was extracted from cecal contents by pipetting and mixing with tris buffered saline with Tween-20 (TBST) containing a protease inhibitor. Subsequently, insoluble matter was removed by centrifugation at 15,000×g for 20 minutes at 4° C. Thereafter, the supernatant was transferred to a new tube, 25% trichloroacetic acid (final concentration: 12.5% v/v) was added thereto, and incubation was performed at 4° C. for 1 hour. Subsequently, the supernatant was removed by centrifugation at 15,000×g for 15 minutes at 4° C. Thereafter, the precipitate was washed with acetone twice, and the lid was opened for drying. Subsequently, the dried specimen was redissolved in 0.5% sodium dodecanoate and 100 mM Tris-HCl (pH 8.5) using a water bath type sonicator (Bioruptor UCD-200, Sonic Bio Corp.). The protein concentration of the redissolved sample was measured by a bicinchoninic acid (BCA) assay, and the protein concentration was adjusted to 1 μg/μL. A pretreatment for shotgun proteome analysis was performed according to the description previously reported (Kawashima Y., et al., Optimization of Data-Independent Acquisition Mass Spectrometry for Deep and Highly Sensitive Proteomic Analysis, Int J Mol Sci., 20 (23), 5932, 2019.).
Peptides were directly injected into a 75 μm×15 cm PicoFrit emitter (New Objective) filled in-house with 2.7 μm core-shell C18 particles (CAPCELL CORE MP 2.7 μm, 160 Å material, Osaka Soda Co., Ltd.), and separation was performed by applying a gradient for 180 minutes at a flow velocity of 300 nL/minute using Eksigent ekspert nanoLC 400 HPLC system (Sciex Corporation).
The peptides eluted from the column were subjected to shotgun MS and SWATH-MS analysis (SWATH: Sequential Window Acquisition of All Theoretical Mass spectra) using a TripleTOF 5600+ mass spectrometer (Sciex Corporation). In the experiment using the shotgun MS, an MS1 spectrum was acquired for 250 milliseconds at a range of 400 to 1000 m/z. The top 25 precursor ions exceeding 150 counts/second with a charge state of 2+ to 5+ were selected, and fragmentation was performed by a rolling collision energy to acquire an MS2 spectrum for 100 milliseconds at a range of 100 to 1500 m/z. The dynamic exclusion time was set to 24 seconds.
In the experiment using the SWATH-MS, a mass spectrometer was operated in a continuous data-independent acquisition mode, and a range was increased by 12 m/z with precursor isolation windows. Using an isolation width of 13 m/z (1 m/z for overlapping windows), a set of 50 windows covering a precursor mass range of 400 to 1000 m/z was constructed. A SWATH MS2 spectrum was performed for 60 milliseconds at a range of 100 to 1500 m/z for each MS2 experiment. In each MS2 experiment, the rolling collision energy was used for fragmentation of precursor ions.
All shotgun MS files were cross-checked against the mouse UniProt reference proteomes of mice (Uniprot id. UP000000589, reviewed, canonical) using ProteinPilot Software v. 4.5 and the Paragon Algorithm (Sciex Corporation) to perform protein identification.
The threshold value of the reliability of the protein was such that an unused score of ProteinPilot was 1.3, and at least one peptide had a reliability of 95%. In the present study, the global false discovery rates for both peptides and proteins were less than 1%. The identified proteins were quantitatively determined from SWATH-MS data using PeakView v. 2.2 (Sciex Corporation).
Proteome Analysis of Culture Supernatant of P. clara
25% trichloroacetic acid (final concentration: 12.5% v/v) was added to the culture supernatant of P. clara, and incubation was performed at 4° C. for 1 hour. Subsequently, the supernatant was removed by centrifugation at 15,000×g for 15 minutes at 4° C. Thereafter, the precipitate was washed with acetone twice, and the lid was opened for drying. Subsequently, the dried specimen was redissolved in 0.5% sodium dodecanoate and 100 mM Tris-HCl (pH 8.5) using a water bath type sonicator (Bioruptor UCD-200). The protein concentration of the redissolved sample was measured by a BCA assay, and the protein concentration was adjusted to 1 μg/μL. The pretreatment for the shotgun proteome analysis was performed as described above.
Peptides were directly injected at 50° C. into a 75 μm×20 cm PicoFrit emitter filled in-house with 2.7 μm core-shell C18 particles, and thereafter separation was performed by applying a gradient for 80 minutes at a flow velocity of 100 nL/minute using UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific K.K.). The peptides eluted from the column were analyzed by overlapping window DIA-MS using Q Exactive HF-X (Thermo Fisher Scientific K.K.). An MS1 spectrum was acquired in a range of 495 to 785 m/z with a resolution of 30,000. The target value of automatic gain control was set at 3×e6, and a maximum injection time was set to 55.
An MS2 spectrum was acquired at a range of 200 m/z or more with a resolution of 30,000, the target value of automatic gain control was set at 3×e6, a maximum injection time was set to “auto”, and stepped normalized collision energies were set to 22%, 26%, and 30%. An isolation width of the MS2 was set to 4 m/z, and an overlapping window pattern at 500 to 780 m/z was used. The window layout was optimized by using Skyline software.
The MS files were cross-checked against the spectral library of P. clara by using Scaffold DIA (Proteome Software). The spectral library was generated from a protein sequence database of P. clara (Uniprot id. UP000000589, reviewed, canonical) by using Prosit software.
The protein sequence database of P. clara was independently created by metagenomics analysis. The search parameters of the Scaffold DIA were as follows. Experimental data search enzyme: trypsin, maximum number of missed cleavage sites: 1, allowable range of precursor mass: 8 ppm, allowable range of fragment mass: 8 ppm, and static modification: carbamidomethylation of cysteine.
The threshold value for protein identification was set such that the false discovery rates of peptides and proteins were 1% or less. The quantitative determination of the peptides was calculated by the EncyclopeDIA algorithm in the Scaffold DIA. For each peptide, four highest quality fragment ions were selected and quantitatively determined. The quantitative values of the proteins were estimated from the sum of the quantitative values of the peptides.
An acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) was added to the cecal contents and dried with a centrifugal evaporator. Acetone was added to the dried sample, and a low-molecular-weight compound that was soluble in lipids was extracted with a water bath type sonicator. Thereafter, centrifugation was performed at 15,000×g for 15 minutes at 4° C.
After removing the supernatant, 70% ACN-HCl was added to the precipitate, and the peptides were redissolved in a water bath type sonicator. Thereafter, centrifugation was performed at 15,000×g for 15 minutes at 4° C. Subsequently, the supernatant was transferred to a new tube and dried with a centrifugal evaporator. Subsequently, the dried sample was redissolved in 100 mM Tris-HCl containing a protease inhibitor, and then treated with 10 mM dithiothreitol at 50° C. for 30 minutes. Thereafter, alkylation was carried out with 30 mM iodoacetamide for 30 minutes at room temperature in a dark place. Thereafter, acidification was carried out with 0.5% trifluoroacetic acid (final concentration). The acidified sample was desalted with MonoSpin C18 (GL Sciences Inc.).
Peptides were directly injected at 50° C. into a 75 μm×25 cm PicoFrit emitter (New Objective) filled in-house with core-shell C18 particles (CAPCELL CORE MP 2.7 μm, 160 Å, Osaka Soda Co., Ltd.), and thereafter separation was performed by applying a gradient for 90 minutes at a flow velocity of 100 nL/minute using UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific K.K.).
The peptides eluted from the column were analyzed by DDA-MS using Q Exactive HF-X (Thermo Fisher Scientific K.K.). An MS1 spectrum was acquired at a range of 350 to 1500 m/z with a resolution of 120,000 to achieve 3×106 as the target value of automatic gain control (AGC).
The strongest 30 ions of more than 4.4×103 with a charge state of 2+ to 5+ were fragmented by collision-induced dissociation with a normalized collision energy of 26% in a data-dependent mode. Tandem mass spectra were acquired with an Orbitrap mass spectrometer having a mass resolution of 30,000 at 200 m/z. The AGC target was set to 1×105.
The MS files were searched with the mouse UniProt reference proteome (Uniprot id. UP000000589, reviewed, canonical) PEAKS Studio.
The search conditions were as follows: mass allowable range of precursor: 8 ppm, mass allowable range of fragment ions: 0.01 Da, presence or absence of enzyme, carbamidomethylation of fixed modification, and oxidation (M) of variable modification. Identification was performed by filtering such that the false discovery rate of peptides was 1% or less.
Mouse faecal samples were suspended and diluted 50-fold with phosphate buffered saline (PBS) to which a protease inhibitor cocktail (Roche) was added. Subsequently, the suspended sample was centrifuged at 15,000×g for 10 minutes at 4° C., and the supernatant was subjected to western blot. Mouse pancreatic tissue was rapidly frozen in liquid nitrogen. Proteins were extracted by TRIzol Reagent (Thermo Fisher Scientific K.K.), and the final protein concentration was adjusted to 4 μg/μL. For SDS-PAGE and blotting, Novex (registered trademark) NuPAGE (registered trademark) SDS-PAGE Gel system (Thermo Fisher Scientific K.K.) and iBlot™ 2 Dry Blotting System (Thermo Fisher Scientific K.K.) were used according to the manufacturer's instructions.
In some of the experiments, SDS-PAGE and PVDF membrane transfer (0.2 μm Transfer Membranes Immobilon-PSQ (Merck Millipore)) were performed according to the manufacturer's instructions (XV PANTERA SYSTEM (DRC)).
iBind™ Western Systems (Thermo Fisher Scientific K.K.) was used for staining.
The antibodies used are as follows. A rabbit anti-mouse PRSS2 antibody (Cosmo Bio Co., Ltd.), a rabbit anti-mouse HSP90 antibody (#4877, Cell Signaling Technology, Inc.), a rabbit anti-human PRSS2 antibody (LS-B15726, LSBio), a rabbit anti-human PRSS1 antibody (LS-331381, LSBio), a rabbit anti-mouse TMPRSS2 antibody (LS-C373022, LSBio, against the sequence of the protease domain), a rabbit anti-6-His antibody (A190-214A, Bethyl Laboratories, Inc.), a goat anti-mouse IgA a chain antibody (HRP) (ab97235, Abcam plc.), a rat anti-mouse κ chain antibody (HRP) (ab99632, Abcam plc.), a rabbit anti-mouse CELA3b antibody (OACD03205, Aviva Systems Biology Corporation), an anti-rabbit IgG antibody (HRP) (#7074, Cell Signaling Technology, Inc.), and a rabbit anti-mouse Reg3 antibody (51153-R005, Sino Biological, Inc.).
For a chemiluminescence assay, Chemi-Lumi One (Nacalai Tesque, Inc.) was used. For imaging, Molecular Imager (registered trademark) ChemiDoc™ XRS+System (Bio-Rad Laboratories, Inc.) or iBright™ FL1500 was used.
RNA of mouse pancreas was extracted by TRIzol Reagent (Thermo Fisher Scientific K.K.). The extracted RNA was converted to cDNA using ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (TOYOBO CO., LTD.).
RT-qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO CO., LTD.) and LightCycler 480 (Roche) by a ΔΔCt method. GAPDH was used as an endogenous control. The base sequences of the primers used are as follows.
Mouse large intestine tissue (including faeces) was collected and fixed in a Carnoy's solution (60% methanol, 30% chloroform, 10% glacial acetic acid) overnight at 4° C. Tissue Processor (Leica Microsystems) was used for paraffin embedding. Subsequently, a paraffin block was cut into thin sections (5.0 μm) with a microtome. Thereafter, paraffin was removed to perform immunostaining.
The antibodies used in the immunofluorescence method are as follows. A rabbit anti-PRSS2 antibody (LSBio), an Alexa 488-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific K.K.), 4′-6-diamidino-2-phenylindole (DAPI) (DOJINDO LABORATORIES), and rhodamine-labeled UEA 1 (ULEX Europaeus Agglutinin 1, Vector Laboratories, Inc.). For immunofluorescence imaging, Leica TCS SP5 co-focused with Leica AF600 was used.
Mouse intestinal contents or a mice faecal sample was diluted 500-fold (w/v) with a 0.9% NaCl solution. Human faeces were diluted 200-fold (w/v) with a 0.9% NaCl solution. The diluted solution was vortexed in a mini shaker for 20 minutes at 2,000 rpm, homogenized by pipetting, and then centrifuged at 10,000×g for 15 minutes at 4° C. Subsequently, the supernatant was recovered to measure the trypsin activity by a Trypsin Activity Assay Kit (Colorimetric) for 100 tests (ab102531) according to the manufacturer's protocol. The absorbance at 405 nm was measured in a kinetic mode by using a PerkinElmer 2030 Multilabel Reader.
Human faecal samples were collected at RIKEN and Keio University according to a research protocol approved by the Research Ethics Review Committee. Informed consent was obtained from each subject. A human faecal sample (preserved in 20% (v/v) glycerol) was transferred to an anaerobic chamber, thawed, and then sieved with a 100 μm mesh, transferred to a GF isolator, and orally administered to GF mice by 200 μL/mouse.
For an antibiotic treatment, each solution of 0.5 g/L ampicillin (Nacalai Tesque, Inc.), 0.5 g/L metronidazole (Nacalai Tesque, Inc.), and 1.0 g/L tylosin (Sigma-Aldrich) was produced with autoclaved tap water. The antibiotic solution was given, for 12 days, to mice to which the faecal contents derived from a donor C were orally administered. The antibiotic solution was changed once a week.
Separation and Identification of Colony-Forming Species from Mouse Faecal Contents
Mouse intestinal contents were mixed with PBS containing glycerol (20%), put in an anaerobic chamber, and stocked at −80° C. The stock was mixed with an equal volume of TS broth (BD) in the anaerobic chamber and plated on different agar plates described below. EG, ES, M10, NBGT, VS, and TS (BD), BL (Eiken Chemical Co., Ltd.), BBE (KYOKUTO PHARMACEUTICAL INDUSTRIAL CO., LTD), Oxoid CM0619 (Thermo Fisher Scientific K.K.), CM0619-supplemented SR0107 (Thermo Fisher Scientific K.K.), CM0619-supplemented SR0108 (Thermo Fisher Scientific K.K.), mGAM (Nissui Pharmaceutical Co., Ltd.), and Schaedler (BD). After culturing for 2 days, colonies having a different external appearance were transferred to a new EG plate. Thereafter, the colonies were cultured overnight in an EGEF liquid medium, mixed with glycerol (final concentration: 20% ((v/v)), and stocked at −80° C.
The formulation of an Eggerth Gagnon (EG) agar medium was as follows. Protease Peptone No. 3 (10.0 g), yeast extract (5.0 g), Na2HPO4 (4.0 g), glucose (1.5 g), soluble starch (0.5 g), L-cysteine HCl (0.5 g), L-cysteine (0.2 g), Tween 80 (0.5 g), agar (4.8 g), a horse meat extraction liquid (500 mL), and water up to 1000 mL+defibrinated horse blood (50 mL). In the EGEF medium, agar was removed, and defibrinated horse blood (50 mL) was replaced with a Fildes solution (40 mL).
Subsequently, the DNA genome of bacteria was extracted from the isolated strain by the same protocol as for DNA isolation from faeces. 16S rRNA was amplified by PCR using a KOD Plus Neo kit (TOYOBO CO., LTD.) according to the manufacturer's protocol. The Sanger sequence was outsourced to Eurofins Scientific. The base sequence was cross-checked against the NCBI database. The primer sequences for the Sanger sequence was as follows.
Sequence of 16S rRNA
The frozen mouse faecal sample was thawed, and 100 μL of the suspension was mixed with 900 μL of a TE10 (10 mM Tris-HCl, 10 mM EDTA) buffer solution containing RNase A (final concentration: 100 μg/mL, Thermo Fisher Scientific K.K.) and lysozyme (final: 3.0 mg/mL, Sigma-Aldrich). The suspension was incubated at 37° C. for 1 hour by gentle stirring. Purified Achromopeptidase (FUJIFILM Wako Pure Chemical Corporation) was added such that a final concentration was 2,000 units/mL, and incubation was further performed at 37° C. for 30 minutes. Subsequently, sodium dodecyl sulfate (final: 1%) and Proteinase K (final: 1 mg/mL, Nacalai Tesque Inc.) were added to the suspension, and incubation was performed at 55° C. for 1 hour.
Subsequently, high-molecular-weight DNA was extracted with phenol:chloroform: isoamyl alcohol (25:24:1), precipitated with isopropanol, washed thereafter with 70% ethanol, and resuspended in 100 μL of TE.
PCR was performed using Ex Taq (Takara Bio Inc.). The primer sequences for amplifying the V1-V2 region of the 16S rRNA gene were as follows.
PCR products were purified by Agencourt AMPure XP (Beckman Coulter, Inc.) according to the manufacturer's protocol. A 16S rRNA library was produced according to the manufacturer's protocol using a KAPA Library Quantification Kit (Kapa Biosystems, Inc.). The 16S rRNA was sequenced according to the standard protocol of MiSeq Reagent Kit ver3.
The isolated strains except for Phasolactobacterium faecium (3G4) were cultured in an anaerobic chamber at a temperature of 37° C. for 1 to 2 days. Phasolactobacterium faecium was cultured for 2 to 3 days in an Oxoid CM0619 agar plate to which 80 mM sodium succinate was added. The colonies were recovered and resuspended in EGEF. The bacterial density was adjusted based on an OD600 value, and the mixture of the cultured strains was orally administered to GF mice (150 μL/mouse, total bacterial count: about 1 to 2×108 CFU).
For quantitative determination of the DNA of P. clara in faeces, mouse faecal DNA was purified, and quantitative real-time PCR was performed to amplify sequences specific to the 16s rRNA gene of the P. clara strain with LightCycler 480 System (Roche) using THUNDERBIRD SYBR qPCR Mix (TOYOBO CO., LTD.). A standard curve was created from serial diluent solution of genomic DNA of the P. clara strain (JCM 14859). The base sequences of the primers used are as follows.
Citrobacter rodentium (C. rodentium) Infection
Any of 200 μL of 2-mix (B. uniformis and P. merdae)+P. clara (WT), 2-mix+P. clara (Δ00502), and 2-mix alone was orally inoculated to GF mice. After 14 days, the mice were infected with C. rodentium (150 μL/mouse) cultured overnight by oral administration, and euthanized on day 7.
For histological analysis, the cervical part of the mice was fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin block was incised and stained with hematoxylin and eosin.
The degree of colitis was evaluated by a gastroenterology technician according to the following criteria: infiltration of inflammatory cells (score: 0 to 4), mucosal thickening (score: 0 to 4), depletion of goblet cells (score: 0 to 4), crypt abscess (score: 0 to 4), and disruption of tissue structure (score: 0 to 4). The final histological score was defined as the sum of the scores for these parameters.
For a colony-forming unit (CFU) assay, cecal patches or cecal luminal contents were collected, and after homogenization with PBS, serially diluted homogenates were plated on LB agar plates. CFU was counted after overnight culture at 37° C. under aerobic conditions.
For the ex vivo evaluation of C. rodentium-specific IgA, the cecal contents were resuspended in a 5× (w/v) LB medium and centrifuged. The supernatant was filtered through a sterilization filter unit with a PVDF film having a pore size of 0.22 μm, and then mixed with an equal amount of a C. rodentium culture solution cultured overnight in vitro. This mixture was gently shaken at room temperature and incubated for 1 hour to examine the aggregation effect with a confocal microscope (Leica TCS SP8). Alternatively, after the incubation, the mixture was centrifuged and washed once with PBS. Thereafter, the bacterial cell pellet was dissolved in a 1% SDS solution (diluted with a 50 mM Tris-HCl buffer solution to which 5 mM EDTA was added). The lysate was stained by western blot with a goat anti-mouse IgA a chain antibody (HRP) (ab97235) to evaluate the relative amount of C. rodentium-binding (C. rodentium-specific) IgA in the faecal contents.
For a vaccine administration experiment, 200 μL of 2-mix (B. uniformis and P. merdae)+P. clara (WT), or 2-mix+P. clara (Δ00502) was preliminarily administered to GF mice. After 4 days, C. rodentium (1010 cells/mouse) inactivated with peracetic acid was orally administered to the mice once a week for 3 weeks. After 3 weeks of immunization, the mice were infected with C. rodentium (150 μL/mouse) cultured overnight by oral administration, and euthanized on day 14.
The C. rodentium inactivated by peracetic acid was produced as follows. C. rodentium cultured overnight was recovered by centrifugation (16,000×g, 10 minutes) and resuspended in sterilized PBS at a density of 1010 cells per 1 mL. Peracetic acid (240990, Sigma-Aldrich) was added such that a final concentration was 0.4%, and incubation was performed at room temperature for 1 hour. After washing 3 times with sterilized PBS, the final pellet was resuspended in PBS at a final concentration of 1011 particles/mL and preserved at 4° C. Before using a vaccine, 200 mL of an LB medium was inoculated with 100 μL of an inactivated vaccine and cultured at 37° C. overnight to confirm complete inactivation.
MHV-2 was provided by Makoto UJIKE (Nippon Veterinary and Life Science University). Five-week-old GF C57BL/6N male mice were obtained from CLEA Japan, Inc. or Sankyo Labo Service Corporation, and were separately bred in stainless steel isolators. 200 μL of 2-mix (B. uniformis and P. merdae)+P. clara (WT) or 2-mix+P. clara (Δ00502) was orally administered to the GF mice. Two weeks after inoculation, the mice were orally infected with 4.5×106 PFU of MHV-2, and the survival rate was observed every day for 10 days.
The liver and brain were collected on 4 or 5 days after infection to measure virus titers, and homogenization was performed with DNA/RNA Shield (Zymo Research Corporation). Viral RNA was extracted using a Quick-RNA Viral Kit (Zymo Research Corporation) according to the manufacturer's instructions, and cDNA was synthesized using ReverTra Ace (TOYOBO CO., LTD.) and a random primer (TOYOBO CO., LTD.).
Quantitative real-time PCR was performed with LightCycler 480 System (Roche) using THUNDERBIRD SYBR qPCR Mix (TOYOBO CO., LTD.) to amplify an orf1a gene. The relative amount of MHV was obtained from a standard curve created from a serial diluent solution of MHV genomic cDNA. The base sequences of the primers used are as follows.
The bacteria cultured overnight were incubated with recombinant mouse trypsin (final concentration: 1 μg/mL) for 1 hour or with human trypsin (final concentration: 20 μg/mL) for 4 hours. Recombinant trypsin isoforms used were as follows: recombinant mouse PRSS2 (50383-M08H, Sino Biological, Inc.), recombinant human PRSS1 (LS-G135640), recombinant human PRSS2 (LS-G20167), and recombinant human PRSS3 (His-tag) (NBP2-52220).
In some experiments, recombinant mouse PRSS2 was first treated with one of the following trypsin inhibitors for 30 minutes before incubation with a P. clara culture solution. AEBSF (Sigma-Aldrich, final concentration: 2 mM), Leupeptin (Sigma-Aldrich, final concentration: 100 μM), and TLCK (Abcam plc., final concentration: 100 μM).
In some experiments, P. clara was cultured overnight in the presence of Tunicamycin (Sigma-Aldrich, final concentration: 10 μg/mL), 2-Fluoro-L-fucose (Cayman Chemical Company, final concentration: 250 μM), or the corresponding DMSO control, before incubation with recombinant mouse PRSS2.
In the experiment of evaluating the effect of Ca2+, P. clara was cultured in a low Ca2+ mGAM medium supplemented with or without 1 mM of Ca2+, before incubation with recombinant mouse PRSS2. In the experiment in which the supernatant of P. clara was used, P. clara cultured overnight was filtered through a sterilization filter unit formed using a PVDF film having a pore size of 0.22 μm.
Recombinant mouse PRSS2 was labeled with Alexa Fluor™ 488 using an Alexa Fluor™ 488 Antibody Labeling Kit (Δ20181, Thermo Fisher Scientific K.K.), and was pretreated with an AEBSF inhibitor (150 μg/mL rmPRSS2 with 20 mM AEBSF).
Mouse PRSS2 labeled with Alexa Fluor™ 488 was incubated in an anaerobic chamber for 20 minutes with bacteria cultured at a final concentration of 5 μg/mL. The mixture was centrifuged, washed once with PBS, and then resuspended in PBS. A Leica TCS SP8 confocal microscope was used for capturing a confocal image.
DSSO (Δ33545) was purchased from Thermo Fisher Scientific K.K. A P. clara (1C4) strain cultured overnight was incubated with AEBSF-treated mouse recombinant PRSS2 for 20 minutes, washed once with PBS, and then resuspended in 10 mM of DSSO. After incubation at room temperature for 10 minutes, a tris-hydrochloride buffer solution (final concentration: 20 mM) was added to stop the reaction.
After washing with PBS, the pellet was dissolved in a 1% SDS solution (diluted with a 50 mM Tris-HCl buffer solution to which 5 mM EDTA was added). Only the P. clara (1C4) strain (without incubation with PRSS2) was treated in the same manner to be served as a negative control. The supernatant was stained with a rabbit anti-6-His antibody (Δ190-214A, Bethyl Laboratories, Inc.) and an anti-rabbit IgG antibody (HRP) (#7074, Cell Signaling Technology, Inc.), and was analyzed by western blot.
The P. clara (1C4) strain was cultured overnight in the presence of Tunicamycin (Sigma-Aldrich, final: 10 μg/mL), 2-Fluro-L-fucose (Cayman Chemical Company, final: 250 μM), or the corresponding DMSO control. Thereafter, the cultured bacteria were pelletized, washed once with PBS, and dissolved in a 1% SDS solution (obtained by dilution with a 50 mM Tris-HCl buffer solution to which 5 mM EDTA was added). SDS-PAGE was performed using Novex (registered trademark) NUPAGE (registered trademark) SDS-PAGE Gel system (Thermo Fisher Scientific K.K.). Proteins including the sugar chain were stained using a Pro-Q™ Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific K.K.) according to the manufacturer's protocol. The proteins of a whole-cell lysate were stained with a Colloidal Blue Staining Kit (Thermo Fisher Scientific K.K.). The protein in the supernatant was concentrated with an Amicon Ultra Centrifugal Filter (10 kD NMWL) and then stained with a Colloidal Blue Staining Kit (Thermo Fisher Scientific K.K.).
Deletion mutants (Δ03049 to 03053, Δ000502, and Δ000509) of the P. clara (JCM 14859) strain were produced as follows. First, a sequence of about 1 kb including a coding region was amplified by PCR and incorporated into a suicide vector (pLGB30) using a HiFi DNA Assembly (NEB) according to the manufacturer's protocol. Subsequently, 1 μL of each reaction liquid was transformed into electrocompetent Escherichia coli S17-1 λpir.
Subsequently, the transformant was bound to the P. clara (JCM 14859) strain as follows. A donor strain was cultured in an LB medium and a recipient strain was cultured in an EGEF medium such that an OD600 was 0.5, and mixing was performed at a ratio of 1:1. This mixture was added dropwise to an EGEF agar plate and was aerobically cultured at 37° C. for 16 hours. Subsequently, the exconjugants were selected on the EGEF agar medium plate containing tetracycline (10 μg/mL). The exconjugants were partially sensitive to the expression of ss-bfe1 toxin induced by rhamnose, and the growth was inhibited in the presence of 10 mM of rhamnose (overnight OD600 of up to 0.3). Thereafter, to select the disappearance from the plasmid genome by second crossover, the exconjugants were cultured in EGEF broth, to which 10 mM of rhamnose was added, for at least three generations until the exconjugants lost to the restored cells (OD600 reached up to 1.0 overnight). Thereafter, bacterial culture was performed, and a single colony was collected to confirm normal deletion by PCR.
A homologous sequence of about 0.5 to 1 kb of the coding region was incorporated into the suicide vector (pLGB30) to be transformed into an electrocompetent Escherichia coli S17-1 strain, and an insertion mutant was produced by the same procedure.
The transformant was conjugated with the P. clara (JCM 14859) strain by the same protocol. Exconjugants were selected on an EGEF agar plate containing tetracycline (10 μg/mL), confirmed by PCR, and then maintained with EGEF broth to which tetracycline (10 μg/mL) was added. The base sequences of all of the primers used to create the mutants are shown in Tables 1 to 5 below.
A wild-type (WT) strain or a Δ00502 P. clara (JCM 14589) strain was incubated for 20 minutes with mouse recombinant PRSS2 (50383-M08H, Sino Biological, Inc., final concentration: 5 μg/mL), washed with PBS, and then fixed at room temperature for 2 hours using a 4% paraformaldehyde-1% glutaraldehyde solution. After washing with 0.05 M PBS, the pellets were gradually dehydrated with ethanol (50%, 70%, 80%, 90%, 95%, 100%). The dehydrated pellets were infiltrated with an LRW resin (with 100% ethanol and LRW at 1:1 for 1 hour, thereafter with 100% ethanol and LRW at 1:2 overnight, and further with 100% LRW for 5 hours). After the infiltration, the sample was cured with a gelatin capsule (53° C., 24 hours). The polymerized LFW block was incised with a Leica Ultracut UCT to obtain an 80 nm section.
For immunogold staining, the section was first blocked with 0.05 M of PBS, to which 1% BSA was added, and then stained with a rabbit anti-6-His antibody (A190-214A, Bethyl Laboratories, Inc.) for 60 minutes. After washing with 0.05 M of PBS, the section was stained with a 12 nm colloidal gold-labeled goat anti-rabbit IgG antibody for 60 minutes. After washing again with 0.05 M of PBS, the section was fixed with 1% glutaraldehyde dissolved in 0.05 M of PBS, washed with water, and then stained with uranyl acetate for 5 minutes. All images were captured with a JEOL JEM-1400 transmission electron microscope.
In order to produce recombinant 00502 protein and recombinant 00509 protein, the coding regions (excluding the N-terminal sequence encoding a signal peptide) of the genes of both proteins were cloned into an expression vector pET-28b (+) (#69865, Novagen), and a C-terminal His-tag was introduced according to the supplier's protocol.
Subsequently, the expression vector was transformed into Rosetta-gami B(DE3) Competent Cells (#71136, Novagen). Subsequently, the transformant was grown exponentially, and 0.4 mM IPTG (16758, Sigma-Aldrich) was added to induce the expression of the protein. After culturing overnight at 25° C., the cells were lysed using B-PER™ Bacterial Protein Extraction Reagent (#78243, Thermo Fisher Scientific K.K.), and the recombinant 00502 protein and the recombinant 00509 protein were prepared using Pierce™ Ni-NTA Magnetic Agarose Beads (#78605, Thermo Fisher Scientific K.K.), and Pierce™ Polyacrylamide Spin Desalting Columns (#89849, Thermo Fisher Scientific K.K.).
Subsequently, the purified recombinant 00502 protein, the purified recombinant 00509 protein, or bovine serum albumin (#23209, Thermo Fisher Scientific K.K.) were bound to micromagnetic beads (Dynabeads™) using Dynabeads™ Antibody Coupling kit (14311D, Thermo Fisher Scientific K.K.) according to the manufacturer's protocol. 15 μg of the protein was added per 1 mg of the beads.
Subsequently, 1 mg of the protein-bound Dynabeads' was resuspended in 200 μL of an EGEF medium and mixed with recombinant mouse PRSS2 (final concentration: 3 μg/mL), Alexa Fluor™ 488-labeled mouse PRSS2 (final concentration: 5 μg/mL) pretreated with AEBSF, or 50 μL of GF cecal contents (50-fold diluted with PBS). The base sequences of all of the primers used to produce the recombinants are shown in Table 6 below.
The protease activity of the P. clara culture solution, the P. clara culture solution supernatant, the recombinant 00502 protein, and the recombinant 005009 protein were measured using Pierce™ Fluorescent Protease Assay Kit (#23266, Thermo Fisher Scientific K.K.) according to the manufacturer's protocol.
Using PerkinElmer 2030 Multilabel Reader equipped with an excitation filter and a fluorescence filter of fluorescein (485/538 nm), an increase in total fluorescence due to digestion of the FITC-casein substrate to smaller fluorescein-labeled fragments was detected. The protease activity was measured as a change in relative fluorescence units (RFUs).
Metagenomics Analysis of Published Human Faecal Samples from Cohorts
The metagenomes obtained from human faecal samples of each cohort of PRISM, HMP2, FHS, 500FG, CVON, and Jie were assembled into a non-redundant gene catalog de novo and aggregated into metagenomic species using MSPminer to quantitatively determine relative abundances.
USEARCH ublast (at protein level) was adopted to search the gene catalog for the homologues of the genes of the P. clara strain and the genes of a P. xylanphila strain at a trypsin-associated locus including the gene encoding the 00502 protein and the gene encoding the 00509 protein, and for other six nearby genes. Hits with a minimum e-value of 0.1 were maintained. As a result, it was confirmed in the gene catalog that all eight genes of each type were present.
In order to identify additional putative homologues and species encoding this locus, first, the similarity between the homologues corresponding to the P. clara strain and the P. xylanphila strain was evaluated, and the minimum identity (Id) and the coverage (Cov) threshold value of the ublast hits corresponding to each gene at the locus were set as follows. 00502: Id=25%, Cov=90% 00503: Id=70%, Cov=90% 00504: Id=60%, Cov=90% 00505: Id=60%, Cov=90% 00506: Id=50%, Cov=90% 00507: Id=25%, Cov=90% 00508: Id=45%, Cov=80% 00509: Id=20%, Cov=30%
Subsequently, whether or not other metagenome species (MPS) encode the homologues of the 00502 to 00509 genes of the P. clara strain and the P. xylanphila strain was evaluated, and MSP 0355 having a homologue of 8 and MSP 0305 having a homologue and 7 were identified. MSP 0355 and MSP 0305 had previously been annotated only to Bacteroidetes, but at this time, these proteomes were compared to the Unified Human Gastrointestinal Genome (UHGG) collection using ublast. As a result, MSP 0355 was annotated as GUT_GENOME 140082 and MSP 0305 was annotated as GUT_GENOME 016875, and the majority of genes (>90%) mapped to a single species representative of the UHGG with high reliability (median value of amino acid identity>99% and e value<1×e-184). In the UHGG, both were phylogenetically classified as a Paraprevotella species.
All statistical analyzes were performed using GraphPad Prism software (GraphPad Software, Inc.). One-way ANOVA which was used in combination with Tukey's test was used for the multiple comparison. For comparison between the two groups, a Mann-Whitney test (non-parametric) or a paired t test (parametric) with Welch's correction was used. In order to examine the correlation between two variables, Spearman's rank correlation was used. A Log-rank (Mantel-Cox) test was used for the survival analysis.
In order to examine the effect of the intestinal microbiota on the protein distribution in the large intestine, the cecal contents of germ-free (GF) mice and specific-pathogen-free (SPF) mice were collected to perform proteomics analysis mass spectrometry. It was found that 324 types out of 713 types of host-derived proteins detected were larger in the SPF mice than in the GF mice (>2-fold, p<0.05). They also contained immune-related molecules such as α-defensin 21 (Defa21) and peptidoglycan recognition protein 1 (Pglyrp1). On the other hand, in the GF mice, 45 types of proteins were abundantly present (>2-fold, p<0.05) than in the SPF mice.
As a result, it was found that the results of the trypsin activity test on the faeces of the SPF mice and the GF mice, the western blot analysis of the faeces of the SPF mice and the GF mice, and the immunostaining of the large intestine sections were also the same as the results of the proteomics analysis, and found that, in the GF mice, the anionic trypsin protease was abundantly present in the cecal contents and the faeces as compared to the SPF mice. Hereinafter, the anionic trypsin protease may be simply referred to as trypsin.
Trypsin is produced in the pancreas as an inactive precursor (trypsinogen), and then secreted into the duodenum to be activated by enteropeptidase. Therefore, the expression of trypsinogen in the pancreas of the GF mice and the SPF mice was examined. As a result, it was found that GF and SPF had equivalent levels of proteins and mRNA of PRSS2 in the pancreas. From this result, a possibility that the difference in trypsin levels observed in the large intestine was due to the difference in production amounts in the pancreas was ruled out.
Subsequently, intraluminal trypsin activity was examined at different sites in the small intestine and the large intestine of the GF mice and the SPF mice.
As a result, it was found that the amounts of trypsin of the SPF mice and the GF mice were similar up to the distal tip of the small intestine. On the other hand, in the large intestine, the trypsin activity of the SPF mice was significantly decreased as compared to that of the GF mice, clarifying that microbiota play an important role in the regulation of trypsin in the large intestine.
It has been previously reported that the microbiota of the large intestine inactivate pancreatic proteases, but bacteria involved in this process have not been identified. Therefore, an attempt was made to isolate and identify bacteria that reduce trypsin from human microbiota.
First, faecal samples collected from 6 healthy Japanese donors (donors A to F) were administered to the GF mice. Subsequently, the trypsin activity in the faeces of the GF mice was measured.
As a result, it was clarified that that the human faecal samples examined differed in their abilities to reduce faecal trypsin activity in the mice. Specifically, the faecal trypsin activity was not decreased in the mice to which the microbiota of the donor B were administered, whereas the faecal trypsin activity was significantly decreased in the mice to which the microbiota of the donors C, D, E, and F were administered.
Subsequently, the mice (mice C#5) to which the microbiota of the donor C was administered were selected, and the cecal contents thereof were collected. Furthermore, these cecal contents were orally administered to new GF mice (GF+C#5 mice). In order to narrow down the microbiota, the GF+C#5 mice were divided into 4 groups. Ampicillin (Amp), metronidazole (MNZ), tylosin (Tyl), or control (no antibiotic, No Abx) was administered to each of the groups via drinking, and the trypsin activity in the faeces of each of the group was measured over time.
Follow-up study was performed on one (mouse C5-Amp #5) of the Amp-treated mice. The cecal contents were collected and cultured in vitro using various media under anaerobic conditions. Subsequently, 432 different colonies were isolated, and the base sequence of the 16S rRNA gene was analyzed. As a result, the cells were subdivided into 35 unique strains. These 35 strains widely covered the bacterial species colonizing the C5-Amp #5 mouse. When the mixture of the 35 strains of the isolated bacteria (35-mix) was administered to GF mice (GF+35-mix), the faecal trypsin activity was significantly decreased. The level of this decrease was the same as the decrease in the mice colonized by the microbiota of the original donor C.
Subsequently, in order to narrow down the bacteria that serve as effectors, the 16S rRNA gene sequence of the faecal samples obtained in the above-mentioned antibiotic administration test was analyzed. A Spearman's rank correlation test was performed to evaluate the relativity between the relative abundance of each of the 35 strains and a decrease in the trypsin activity. As a result, 14 strains out of the 35 strains showed a negative correlation with the trypsin activity (p≤−0.3).
Subsequently, gnotobiotic mice were produced to compare the effects of the 14 bacteria and the remaining 21 bacteria. As a result, in GF+14-mix mice, the same level of a potent decrease in the trypsin activity in the faeces was shown as in the GF+35-mix mice, whereas GF+21-mix mice did not decrease the activity. Subsequently, 9 strains significantly associated with a decrease in trypsin activity were further selected from this 14-mix (p≤−0.5, p<0.05).
The colonization of the GF mice by 9-mix showed the same level of a potent decrease in the trypsin activity in the faeces as in the mice colonized by the 14-mix. Finally, the 9-mix was divided into 3-mix consisting of three Bacteroidales species, and 6-mix consisting of non-Bacteroidales species.
It was clarified that the 3-mix consisting of Paraprevotella clara (P. clara, strain ID: 1C4), Bacteroides uniformis (B. uniformis, strain ID: 3H3), and Parabacteroides merdae (P. merdae, strain ID: 1D4) was sufficient to decrease the faecal trypsin activity in vivo, and that the non-Bacteroidales mix with 6 species was completely ineffective in decreasing the faecal trypsin activity.
In order to specify the bacteria that decompose trypsin, each of the strains of the 9-mix was mixed with recombinant mouse trypsin (to which rmPRSS2 and C-terminal His-tag were added) and incubated to measure the decomposition of trypsin by western blot.
Based on the above description, it was determined that, contrary to the initial hypothesis that microbiota communities are required for decreasing trypsin activity, only P. clara (1C4) is required for decreasing trypsin activity.
Subsequently, recombinant human trypsin isoforms PRSS1, PRSS2, and PRSS3 (rhPRSS1-3) were mixed with P. clara (1C4) and incubated to analyze the decomposition of human trypsin by western blot.
Subsequently, whether or not the effect of decreasing trypsin activity by P. clara was strain-specific was examined. Specifically, recombinant mouse PRSS2 (rmPRSS2) was incubated in vitro with the strain of the genus Paraprevotella and the strain of the genus Prevotella to analyze the decomposition of rmPRSS2 by western blot. P. clara strains (JCM 14859, P237E3b, P322B5) and P. xylaniphila strains (JCM 14860, 82A6) were used as the strain of the genus Paraprevotella. Prevotella copri, Prevotella denticola, Prevotella stercorea, and Prevotella oulorum were used as the strain of the genus Prevotella.
Paraprevotella is a recently identified genus of Prevotellaceae and includes only two types of P. clara and Paraprevotella xylaniphila. Accordingly, the P. xylaniphila (JCM 14860) strain and the P. xylaniphila (82A6) strain which were isolated from healthy human faecal samples were examined.
As a result, it was clarified that three other P. clara strains (JCM 14859, P237E3b, P322B5) isolated from the faecal samples collected from healthy subjects including non-Japanese donors had the effect of reducing trypsin in common. In addition, it was clarified that both the P. xylaniphila (JCM 14860) strain and the P. xylaniphila (82A6) strain exhibit a potent trypsin-reducing ability similar to that of the P. clara strains.
On the other hand, all of the bacteria of the genus Prevotella (Prevotella copri, Prevotella denticola, Prevotella stercorea, and Prevotella oulorum), which have a close lineage with Paraprevotella, did not decrease the trypsin activity.
These results indicate that bacteria belonging to the genus Paraprevotella are a representative constituent element of human microbiota having a trypsin-decomposing ability.
The substrate specificity of P. clara was examined. Specifically, the cecal contents of the GF mice containing a large amount of trypsin together with various proteins were subjected to ex vivo incubation together with P. clara (1C4). Subsequently, the abundance of peptides derived from each protein was examined by peptidome analysis by LC-MS. As a result, among 7614 peptides derived from 276 proteins, trypsin was only one protein that exhibited a pattern of a clear increase in peptide concentration over time in the presence of P. clara.
These results indicate that P. clara has a narrow substrate specificity and high activity with respect to trypsin. In addition, when the reaction rate of trypsin decomposition by P. clara was examined, it was clarified that the reaction gradually occurs stoichiometrically.
In addition, the P. clara-mediated trypsin decomposition occurred only in the presence of a sufficient amount of divalent cations (for example, Ca2+). Accordingly, it was clarified that this decomposition is mediated by an enzyme (protease). However, this decomposition was not observed when trypsin was incubated with the culture supernatant of P. clara. Furthermore, when the live bacteria of P. clara or the filtered supernatant of P. clara was incubated together with a protease substrate (fluorescein-labeled casein), no proteolytic activity was detected.
Subsequently, for the purpose of elucidating the mechanism by which P. clara promotes the autolysis of trypsin, trypsin was labeled with Alexa Fluor 488, and the interaction of trypsin and P. clara was visualized.
As a result, the fluorescent-labeled trypsin was observed to accumulate on the surface of P. clara within minutes. On the other hand, no accumulation of trypsin was observed in Prevotella denticola and Prevotella oulorum. Based on these results, it was thought that the trypsin decomposition occurred on the surface of P. clara via trypsin-binding surface molecules, suggesting that the trypsin-binding surface molecules promoted the accumulation and autolysis of trypsin.
Subsequently, in order to identify the trypsin-binding surface molecules of P. clara, a treatment was performed with disuccinimidyl sulfoxide (DSSO) as a chemical crosslinking agent that captures the complex of trypsin (His-tag rmPRSS2) and P. clara-derived molecules. Subsequently, the complex formed by crosslinking of rmPRSS2 and P. clara-derived molecules was analyzed by western blot.
As a result, by the DSSO treatment, anew band of a high-molecular-weight amount (up to 250 kDa) that was blotted with an anti-His-tag antibody appeared. This band indicates the presence of a high-molecular-weight complex containing trypsin.
Although mass spectrometry was not sensitive enough to detect crosslinked peptides derived from this complex, a band around 250 kDa that was smeared in size indicated that trypsin was interacting with heterogeneous molecules.
In Bacteroidetes including Paraprevotella, it is known that glycan complexes are modified on the cell surface. For this reason, it was thought that glycan-containing molecules present on the surface of P. clara are involved in the binding and decomposition of trypsin.
In order to examine this hypothesis, inhibitors targeting the sugar chain synthesis of P. clara were used. First, P. clara was treated with tunicamycin which is an inhibitor targeting a WecA-like transferase that mediates the first step in O-glycan formation of bacterial lipopolysaccharides (LPS).
T9SS (type IX secretion system) is a bacterial mechanism that transports a protein having a conserved C-terminal domain to the surface across the outer membrane in cooperation with the Sec system. T9SS plays a role of removing the C-terminal domain by protease activity such as sortase and binding the protein transported to the outside of the cell to a surface polysaccharide.
The inventors of the present invention performed the following examination while presuming that the cell surface protein secreted by T9SS is responsible for the recruitment and decomposition of trypsin. First, a gene sequence presumed to be contained in T9SS was identified in the genomes of P. clara and P. xylaniphila species.
Subsequently, a mutant P. clara (JCM 14859) strain from which the expression of PorU, which is a basic constituent factor of T9SS, was deleted was produced by homologous recombination of the plasmid sequence.
Subsequently, in order to identify a cell surface protein having an action effect on trypsin decomposition, a proteome analysis of the culture supernatant of P. clara was performed in the presence or absence of tunicamycin. As a result, 20 bacteria-derived proteins were found in the culture supernatant of P. clara subjected to the tunicamycin treatment.
Therefore, by introducing a plasmid sequence into each of the 20 target loci or by removing gene clusters (Δ03048 to 03053), the series of mutant P. clara strains in which the synthesis of tunicamycin-sensitive proteins was inhibited were produced.
As a result, by disrupting the gene encoding the 00502 protein (UniProtKB ID: G5SNC9, Omp28-related extracellular membrane protein) or the 00509 protein (UniProtKB ID: G5SNC1, protein of unknown function), it was clarified that trypsin decomposition was lost in vitro as in the case of PorU or WecA (target factor of tunicamycin)-deficient mutant strains.
Subsequently, P. clara strains (Δ00502 and Δ00509) from which the 00502 protein or the 00509 protein was deleted were produced in place of the insertion mutant, and the same examination was performed.
Subsequently, the genome sequence of Paraprevotella strains was analyzed.
In addition, between the Paraprevotella strains, 00503 to 00508 genes were conserved at sites distant from the locus of the 00502 gene and the 00509 gene.
Recombinant 00502 protein and 00509 protein were produced. Escherichia coli into which an expression vector of 00502 protein or 00509 protein had been incorporated were treated with isopropyl-β-thiogalactopyranoside (IPTG) to induce the expression of the recombinant protein, and the expressed recombinant 00502 protein or 00509 protein was purified from a cell lysate with magnetic agarose beads.
As a result, it was clarified that the free-form 00502 protein and 00509 protein did not exhibit a trypsin-decomposing activity. On the other hand, the recombinant 00502 protein bound to microbeads exhibited effective recruitment and decomposition of trypsin.
These results support the model of the inventors of the present invention in which the 00502 protein acts as a scaffold for trypsin binding and promotes the self-decomposition of trypsin. Furthermore, it was clarified that although trypsin was effectively bound to the recombinant 00509 protein bound to microbeads, trypsin decomposition did not occur.
From these results, it was suggested that the 00502 protein is a major constituent factor exerting the effect of the recruitment and autolysis of trypsin, whereas the 00509 protein has an action of assisting the recruitment of trypsin.
As shown in
The exact mechanism by which the 00502 protein, rather than the 00509 protein, promotes the autolysis of trypsin is still unclear, but it is presumed that the binding of trypsin to the 00502 protein may change the structure of trypsin, thereby making the autolysis easy.
Colonization by P. clara Strain Contributes to Maintenance of IgA
In order to confirm the contribution of the 00502 and 00509 proteins in vivo to trypsin decomposition, GF mice which had been colonized by three P. clara (JCM 14859) strains (a wild-type (WT), Δ00502, or Δ00509) were analyzed.
Since the P. clara strains as a single strain did not colonize the mice, the mice were inoculated with the P. clara strain together with two strains not decomposing trypsin (2-mix: Bacteroides uniformis 3H3 and Parabacteroides merdae 1D4). By inoculation together with the 2-mix, all of the three P. clara strains effectively colonized the intestines of the mice.
Subsequently, the importance of 00502 was examined under conditions in which more complex microbiota communities were present.
Based on the above results, it was confirmed that the essential role of the 00502 protein is to promote the trypsin decomposition in vivo.
Subsequently, the influence of the regulation of intestinal trypsin levels by the wild-type (WT) type P. clara and the mutant type P. clara on important intestinal defense factors such as IgA and antimicrobial peptides was examined.
As a result, in the colonization of the mice by the wild-type P. clara strain, a larger amount of the IgA heavy chain (a chain) in the faeces was detected than in the colonization of the mice by the Δ00502 or Δ00509 P. clara strain. In contrast, because the κ light chain is resistant to trypsin, the κ light chain levels in the faeces were similar between the mice colonized by various types of the P. clara strains. In addition, Reg3p, which is an antimicrobial peptide against gram-negative bacteria, was resistant to trypsin decomposition as in the case of the κ light chain.
These results show that the colonization by the P. clara strains protects IgA from being decomposed by trypsin in vivo.
As a result, it was shown from the results of the first lane that in the faeces of the GF mice, trypsin was contained but the IgA heavy chain was decomposed. In addition, it was shown from the results of the second lane that in the faeces of the GF mice inoculated with the wild-type P. clara strain together with the 2-mix, trypsin was reduced but the IgA heavy chain remained. In addition, it was shown from the results of the third lane that the IgA heavy chain remaining in the faeces of the GF mice inoculated with the wild-type P. clara strain together with the 2-mix was decomposed by the trypsin remaining in the faeces of the GF mice. In addition, it was shown from the results of the fourth lane that the addition of the trypsin inhibitor inhibited the decomposition of the IgA heavy chain in the lane 3. In addition, it was shown from the results of the third and fourth lanes that the IgA heavy chain is more easily decomposed by trypsin than the κ light chain.
Examination of Influence of Colonization by P. clara Strain on Infection with Enteric Pathogenic Bacteria
Whether the P. clara strain-mediated trypsin decomposition could maintain an IgA amount even under conditions of infection with enteric pathogenic bacteria was examined.
GF mice were inoculated with the 2-mix or with the wild-type (WT) or Δ00502 P. clara strain in addition to the 2-mix, and after 14 days, the mice were infected with the Citrobacter rodentium (C. rodentium) which is a pathogen of mice mainly infecting the large intestine.
The upper part of
As a result, in the 2-mix group, rapid body weight loss and severe cecal inflammation were shown after the infection with C. rodentium. On the other hand, in the mice colonized by P. clara, body weight loss was milder, and cecal inflammation was also milder. This was surprisingly the same in both the 2-mix+WT group and the 2-mix+Δ00502 group. These results suggest that the P. clara strains protect against the infection with C. rodentium by the mechanism independent of trypsin decomposition.
As a result, it was clarified that in the mice in the 2-mix+WT group, a high total IgA amount was maintained regardless of the degree of the cecal inflammation, and as compared to the 2-mix+Δ00502 group, fairly large amounts of the C. rodentium-specific IgA were present from as early as 7 days after the infection with C. rodentium.
Based on the above description, it was shown that the P. clara strains protect pathogen-specific IgA, thereby enhancing an adaptive immune response against the pathogen, in addition to the protective effect by an unknown mechanism against the infection with C. rodentium.
P. clara Strain-Mediated Trypsin Decomposition Enhances Vaccine Effect Against Infection with C. rodentium
The P. clara strain-mediated trypsin decomposition and the consequent protection of pathogen-specific IgA may enhance the oral vaccine effect against intestinal pathogens, thereby further increasing resistance to re-exposure to the same pathogen.
To confirm this hypothesis, the following examination was performed.
Subsequently, the cecal patch and the luminal contents were recovered from the mice 14 days after the infection with C. rodentium, and the CFU of C. rodentium was measured.
As a result, in both the 2-mix+WT group and the 2-mix+Δ00502 group, although the abundance of C. rodentium in the cecum was similar, the infiltration of C. rodentium into the cecal tissue was inhibited in the 2-mix+WT group.
As a result, in the cecum of the mice colonized by the wild-type (WT) P. clara strain, a significantly high total IgA amount and a significantly high amount of C. rodentium-specific IgA were detected. Based on this result, it was thought that the effect of the vaccine was great as the excellent protective effect against the invasion of C. rodentium to the 2-mix+WT group.
The above results support the view of the inventors of the present invention in which delivery of the P. clara strains makes it possible to more effectively correspond to intestinal pathogens that a host has previously encountered.
P. clara Strain Protects Mice from Coronavirus Infection
Trypsin-like proteases such as trypsin and a type II transmembrane serine protease (TMPRSS2) are known to be involved in the proteolytic activation of the spike protein of coronaviruses and in the fusion of virus and host cell membranes.
TMPRSS2, which is expressed as a transmembrane protein in lung and intestinal epithelial cells, is capable of releasing the protease domain by undergoing self-cleaving. Interestingly, the inventors of the present invention found that in Experimental Example 5 described above, the amount of TMPRSS2 in the faeces was reduced in the GF mice engrafted by the wild-type P. clara strain. This suggests that the P. clara strain has an effect similar to the release of the TMPRSS2 active form in vivo. This indicates that in the intestines, free-form TMPRSS2 may promote coronavirus infection together with trypsin.
A probability of the P. clara strain protecting against intestinal infection of coronavirus by decomposition of trypsin and TMPRSS2 was examined. Specifically, the influence of the colonization by the P. clara strains on the infection with mouse hepatitis virus (MHV) (mice tropic coronavirus) was examined.
As a result, it was clarified that the colonization by the wild-type P. clara strain significantly prevented the spread of the virus in the liver and the brain. In addition, the number of the virus particles excreted in the faeces was significantly smaller in the mice in the 2-mix+WT group than in the mice in the 2-mix+Δ00502 group.
The above result indicates that upon infection with coronavirus, the carrier of the P. clara strain brings a protective effect to the host.
The abundance and the prevalence of the genus Paraprevotella, and trypsin-related 00502 genes and 00509 genes were analyzed by newly creating an intestinal bacterial gene catalog consisting of approximately 6 million non-redundant complete genes acquired from six geographically distinct cohorts.
As a result of performing a homology search at the USEARCH ublast (protein level) on the non-redundant intestinal bacterial gene catalog having 5,929,528 genes, the minimum e-value was aggregated to 0.1 hits.
As a result, first, whether or not the 00502 gene of the P. clara strain and the homologue of the 00502 gene of the P. xylanphila strain were the same as or almost the same as each other was confirmed. Similarly, whether or not the 00509 gene of the P. clara strain and the homologue of the 00509 gene of the P. xylanphila strain were the same as or almost the same as each other was confirmed. Furthermore, also regarding the 00503 to 00508 genes positioned between the 00502 gene and the 00509 gene, whether or not the gene of the P. clara strain and the homologue of the gene of the P. xylanphila strain were the same as or almost the same as each other was confirmed.
In addition, two metagenome species (MSP 0303 and MSP 0335) having the homologues of the 00502 to 00509 genes were identified, and it was confirmed that they may belong to the genus Paraprevotella.
Two metagenome species (MSP 0303 and MSP 0335) annotated to the genus Paraprevotella encoded all or almost all homologues of the 00502 to 00509 genes of the P. clara strain. The five MSPs (MSP 0081, MSP 0224, MSP 0288, MSP 0410, and MSP 0435) annotated with Bacteroidetes encoded the homologues of the 00502 gene and the 00509 gene, but lacked the homologues of the 00503 to 00508 genes.
In addition, it was thought that Prevotella muris did not have the homologue of the 00509 gene.
In the metagenomics analysis, the genus Paraprevotella showed a relative abundance of up to 3% on average, the abundance of which varies greatly from cohort-to-cohort (7% to 50% of samples). The P. clara strain is the most abundant species, followed by P. xylaniphilla.
These data suggest that Paraprevotella species make up a significant portion of the microbiota in humans, which may be associated with individual differences in susceptibility to intestinal pathogens for infection.
It is thought that human trypsin concentrations are regulated by a bacterial species that decomposes trypsin, such as Paraprevotella, as in the case of mice. It has been reported that the faecal trypsin concentration is increased in patients suffering from inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). Therefore, the trypsin activity in the faeces of patients in a non-IBD control group (healthy subjects) from a Japanese cohort, an ulcerative colitis (UC) group, and a Crohn's disease (CD) group was measured.
Subsequently, the Paraprevotella carriage rate in the non-IBD control group of two IBD cohorts (PRISM and HMP2), the UC group, and the CD group was analyzed.
These results suggest that the Paraprevotella carriage is associated with an intestinal health state.
According to the present invention, a technique for regulating the activity of proteases can be provided.
The present invention relates to a composition for decomposition of trypsin or TMPRSS2. More specifically, the present invention relates to a composition for decomposition of trypsin or TMPRSS2, a diagnostic drug for diseases caused by trypsin or TMPRSS2, and a quasi-drug for diseases caused by trypsin or TMPRSS2. Priority is claimed on United States Provisional Patent Application No. U.S. 63/138,798, filed Jan. 19, 2021, and United States Provisional Patent Application No. U.S. 63/229,077 filed Aug. 4, 2021, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/001494 | 1/18/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63138798 | Jan 2021 | US | |
| 63229077 | Aug 2021 | US |
