Patent Application
20210322492
The present invention pertains to a probiotic comprising a generally recognized as safe (GRAS) microbiological organism, which GRAS microbiological organism comprises a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, wherein said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof, as further defined in the claims. Methods for treating a disease in a patient, comprising oral administration of the probiotic as defined herein are also described, as well as methods of preparing the probiotic disclosed herein.
The contents of the electronic sequence listing (227-361_Sequence_Listing.txt; Size: 49,937 bytes; and Date of Creation: Apr. 15, 2020) is herein incorporated by reference in its entirety.
In 2001, the World Health organization (WHO) defined in a report probiotics as live microorganisms that, “when administered in adequate amounts, confer a health benefit on the host.” Following this definition, a working group of the Food and Agriculture Organization (FAO)/WHO issued the Guidelines for the Evaluation of Probiotics in Food in 2002. A consensus definition of the term probiotics, based on available information and scientific evidence, was adopted after the aforementioned joint expert consultation between the FAO of the United Nations and the WHO.
The National Center for Complementary and Integrative Health describe probiotics as live microorganisms that are intended to have health benefits when consumed or applied to the body. They are usually provided in form of yoghurt and other fermented foods, dietary supplements, and beauty products. Some bacteria are considered to help in digesting food, destroy disease-causing cells, or produce vitamins. Administration of probiotics is intended to induce changes in the microbiome in the gut, often in order to promote growth of microorganisms which are considered beneficial over those which are considered detrimental. Another mode of action of probiotics is considered by interactions between the probiotic microorganism and the host.
Ottman et al. (PLOS ONE (2017), 12(3): e0173004; doi:10.1371/journal.phone.0173004) disclose that the gut symbiont Akkermansia muciniphilia is positively correlated with a lean physiology, reduced body weight gain, amelioration or metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. The authors identified some of these beneficial effects to be due to an outer membrane pili-like protein named Amuc_1100. When expressed in a non food-grade expression vector as a purification-tagged protein in the non-GRAS microorganism E. coli, and following its purification, the purified protein was found to be a strong TLR2 activator and inducer of inter alia IL-10. Ottman et al. finally suggest the use of gram-negative Akkermansia muciniphilia as a probiotic.
Similarly, Plovier et al. (Nature Medicine 2016; doi: 10.10387 nm.4236) report that a purified His-tagged form of the membrane protein Amuc_1100 from Akkermansia muciniphila (expressed in E. coli) or the pasteurized Akkermansia muciniphila bacterium improves metabolism in obese and diabetic mice. Plovier et al. conclude that either live or pasteurized A. muciniphila (i.e. the bacterium) grown on synthetic medium are a promising therapeutic tool in the management of metabolic syndrome.
Toll-like receptor 2 (TLR2), also designated as CD282, is a receptor of the Toll-like receptor (TLR) family, which plays a fundamental role in the recognition of pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents. Upon activation, TLRs mediate the production of cytokines necessary for modulating the immune response. TLR2 is expressed most abundantly in peripheral blood leukocytes, and mediates host response to mainly gram-positive bacteria, and yeast via stimulation of NF-κB. However, TLR2 recognizes many bacterial, viral and fungal compounds, as well as certain endogenous substances. In the intestine, TLR2 regulates the expression of CYP1A1, an enzyme which is key in detoxication of certain carcinogenic substances. Recently, it was found that TLR2 is involved in the activation of regulatory T cells (Tregs), that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. TLR2 is also expressed by intestinal epithelial cells and subsets of lamina propria mononuclear cells in the gastrointestinal tract. TLR2 has been observed downregulated in human papillomavirus-positive neoplastic keratocytres derived from uterine cervical preneoplastic lesions. Thus, TLR2 is assumed to be associated with tumorigenesis.
Often the microorganisms in probiotic foods are the same or similar to the ones naturally abundant in the human body. In contrast thereto, prebiotics are non-digestable food components that selectively stimulate the growth or activity of certain microorganisms. The term synbiotics commonly refers to products that combine probiotics and prebiotics.
Nguyen et al. (J. Agric. Food Chem. 2011, 59, 5617-5624) discloses a food-grade system for inducible gene expression in Lactobacillus plantarum.
In 2015, the global retail market value for probiotics was US$41 billion, including sales of probiotic dietary supplements, fermented dairy products, and yoghurt, the latter accounting for 75% of total consumption. In 2015 supplements produced US$4 billion and their growth is projected to be as high as 37% globally by 2020. At the same time, consumption of probiotic yoghurt in China has increased by 20% per year since 2014.
There is an existing need in the art for new useful probiotics, which exhibit and combine beneficial health effects. Such probiotics may suitably be applied in the treatment of diseases, including obesity and diabetes.
The aforementioned need is addressed by the present invention, which is characterized by improving the health benefit of a generally recognized as safe (GRAS) microbiological organism, by incorporating into said GRAS microbiological organism a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, such that the GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.
The invention is particularly advantageous for embodiments, wherein the GRAS microbiological organism is selected from the group of organisms consisting of a gram-positive bacteria and a gram-negative bacteria. This is because it is expected that the beneficial effects reported for Amuc_1100, in particular its Toll-like receptor 2 (TLR-2) agonistic activity, will further improve the beneficial health effects which are ascribed to the induction of TLR-2 by PAMPs found in the membrane of these microorganisms. A particular advantageous benefit is to be expected in embodiments, wherein the GRAS microbiological organism is a gram-positive bacteria belonging to the order of lactic acid bacteria.
To the Applicant's best knowledge, there is no suggestion in the prior art to express a soluble form of Amuc_1100, or a functionally equivalent soluble fragment thereof, in a probiotic GRAS microbiological organism, let alone in a GRAS microorganism of the embodiments described herein. Rather, prior to the present invention, it was suggested to use live or pasteurized Akkermansia muciniphila. However, in the context of the invention, the GRAS microorganism is not Akkermansia muciniphila. In the alternative, a His-tagged Amuc_1100 protein was produced in E. coli and used in purified form for research purposes. In contrast, in embodiments of the present invention, said soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100 does not need to comprise such a purification tag, and need not to be purified.
Moreover, while food-grade expression systems are disclosed for primary use in organisms of the genus Lactobacillus, in embodiments these expression systems are used in genus other than Lactobacillus, where these food-grade expression vectors are also functional. In this context, in embodiments said food-grade expression vector carries the SH71rep replicon, which has a broad functionality. Usually, said food-grade expression vector may carry a food-grade selection marker, which provides prototrophy to an otherwise auxotroph GRAS microbiological organism. In embodiments, the marker is alanine racemase (alr).
In embodiments, the nucleic acid sequence in said food-grade expression vector encodes a soluble form of Amuc_1100 having an amino acid sequence with at least 80% identity to SEQ ID NO: 2 (Amuc_1100). In embodiments, said nucleic acid sequence encodes for a fragment of said soluble form of Amuc_1100, which has a length of at least 100 and up to 286 amino acids. Said nucleic acid sequence may also be optimized for expression in the genus selected from the group of Bifidobacterium, Bacillus, Brevibacillus, Lactococcus and Saccharomyces. Hence, in embodiments said nucleic acid sequence has at least 70% identity to SEQ ID NO: 1 (Amuc_1100). One useful example of said food-grade expression vector is p3050alrAmuc_1100-sh71 (SEQ ID NO: 9) or p3050Alr_Amuc1100-sh71 with 5′UTR, 3′UTR and terminator (SEQ ID NO: 15).
In a further aspect, the present invention also pertains to a method of treating a disease in a patient, comprising the step of administering orally a probiotic of the present invention. In embodiments, the disease is selected from the group consisting of obesity, diabetes, hypercholesterolemia, and/or the patient is a human patient.
Further provided is a method for preparing a prebiotic according to the present invention, wherein the method comprises the step of introducing a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, into a GRAS microbiological organism, such that said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.
Other objectives, aspects, embodiments, details and advantages of the present invention will become apparent from the following figures, detailed description, examples, and dependent claims.
The present invention provides a probiotic comprising a GRAS microbiological organism, which GRAS microbiological organism comprises a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100, wherein said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.
The term “probiotic” as used herein in the context of the present invention is defined as live microorganism, which when administered in adequate amounts, confers health benefit on the host. The probiotic may be in the form of a fermented dairy food product, a fermented non-dairy product, or a probiotic food supplement. Examples of a fermented dairy food product comprise yoghurt, yoghurt drinks, kefir, buttermilk, sour cream, viili, fil, and creme fraiche. Often dairy products are fermented are with lactic acid bacteria such as Lactococcus, Lactobacillus and Leuconostoc. However, in particular cheese may comprise bacteria and molds from other genera. Examples of fermented non-dairy products comprise pickled vegetables, sauerkraut, kimchi, pao cai, soy products including miso, tempeh, and soy sauce. Probiotic food supplements may be in the form of capsules, microcapsules, tablets, powders, and sachets, and may optionally be formulated to deliver the probiotic bacteria through the acidic environment of the stomach.
Generally recognized as safe (GRAS) is a designation of the United States Food and Drug Administration (FDA) designating that a chemical or substance added to food is considered safe by experts, and so is exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements. The term “GRAS microbiological organism” as used herein in the context of the present invention is intended to mean that the microorganism is known or is found to be suitable for consumption by a host, in particular a human, without causing a state of disease. Indeed, any organism causing a state of disease, i.e. a deterioration in health, would also not be considered as a probiotic. For example, Escherichia coli is not a GRAS microbiological organism. Thus, the terms “GRAS microbiological organism” and “probiotic” are intended to complement each other.
Microorganisms which are intended to fulfill both requirements of a “probiotic” and a “GRAS microbiological organism” are exemplified in the review article of Fijan, “Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature” Int. J. Environ. Res. Public Health 2014, 11, 4745-4765, the content of which is incorporated herein by reference. In embodiments, the GRAS microbiological organism may be selected from the group of organisms consisting of a gram-positive bacteria, a gram-negative bacteria, and a yeast. In embodiments, the GRAS microbiological organism is selected from the group consisting of organisms of the genus Lactobacillus, Bifidobacterium, Brevibacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, Bacteroides, Prevotella, Parabacteroides, Ruminococcacaeae, Corynebacterium, Neisseria, Planococcaceae, Rothia, Ruminococcus, Veilonella, Coprococcus, Alistsipes, Clostridium, Lachnospiraceae, Faecalibacterium, Rikenellaceae, Comamonas, Dialister, Blautia, Roseburia, Turicibacter, and Saccharomyces. In embodiments, the GRAS microbiological organism is selected from the group consisting of organisms of the species Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevies, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus reuteri, Bifidobacterium infantis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Enterococcus durans, Enterocococcus faecium, Streptococcus thermophilus, Pediococcus acidilactici, Leuconostoc mesentoroides, Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Saccharomyces boulardi. Preferably, the GRAS microbiological organism is not of the genus Akkermansia, in particular not Akkermansia muciniphila.
The invention is particularly advantageous for embodiments, wherein the GRAS microbiological organism is selected from the group of organisms consisting of a gram-positive bacteria and a gram-negative bacteria. This is because it is expected that the beneficial effects reported for Amuc_1100, in particular its Toll-like receptor 2 (TLR-2) agonistic activity, will further improve the beneficial health effects which are ascribed to the induction of TLR-2 by PAMPs found in the membrane of these microorganisms. A particular high expression of Amuc_1100 has been found in embodiments, wherein the GRAS microbiological organism is a gram-positive bacteria belonging to the order of lactic acid bacteria.
As noted above, said GRAS microbiological organism comprises a food-grade expression vector. Several food-grade expression vectors are described in the art. Food-grade expression vectors are characterized by containing only the DNA from homologous hosts or generally considered as safe organisms, and by not being dependent antibiotic markers. Consequently, said food-grade expression vector may carry a food-grade selection marker, which provides prototrophy to an otherwise auxotroph GRAS microbiological organism. Suitable vectors for lactic acid bacteria are reviewed by Landete, Critical Review in Biotechnology, 2017, 37(3): 296-308, the content of which is incorporated herein by reference. These vectors can also be used for identifying building blocks, which can be combined.
The various components of the food-grade expression vector are comprised in the vector in functional linkage. The expression “in functional linkage” as used herein, is intended to mean that the respective component of the food-grade expression vector is arranged within said vector, such that they can bring about their intended function. A marker gene is in functional linkage in case the gene is expressed such that its gene product provides the selection advantage. A replicon is in functional linkage in case the vector or plasmid is reproduced and maintained in the host cell due to the effect of said replicon. In the context of the nucleic acid encoding Amuc_1100, or a fragment thereof, said nucleic acid encoding Amuc_1100 or a fragment thereof is in functional linkage in case its gene product is expressed, such that its translated gene product is secreted into the host cells supernatant.
The food grade selection marker may be, for example, a marker selected from the group of alanine racemase (alr), thymidylate snynthase (thyA), lactose phosphotransferase (lacF), and phospho-β-galactosidase (lacG). In one particular embodiment, the marker is alanine racemase (alr), such as the alanine racemase (alr) marker encoded by SEQ ID NO: 8. The alr marker, and a food-grade expression vector using same is described in further detail in Nguyen et al., J. Agric. Food Chem. 2011, 59: 5617-5624; and Bron et al. Appl. Environ. Microbiol. 2002, 68(11): 5663-5670; each the content of which is incorporated herein by reference. In embodiments, the food-grade expression vector carries the SH71rep replicon, which has a broad functionality. The SH71rep replicon is further described by Karlskas et al., PLOS One 2014, 9(3): e91125, the content of which is incorporated herein by reference. Other suitable replicons may be employed as well. An additional 5′UTR ‘AGGAGGT’ (SEQ ID NO: 13) sequence may be optionally inserted directly upstream of the Amuc-protein sequence and 3′UTR sequence ‘TACTTGAA’ (SEQ ID NO: 14) directly downstream of the Amuc-protein sequence followed by a terminator, for example iGEM-part BBa_B1006 (SEQ ID NO: 12).
Signal sequences steering the gene of interest to the secretion pathway are known to the skilled person. For example, Dieye et al. J. Bacteriol. 2001, 183(14): 4157, the content of which is incorporated herein by reference, disclose the M6 preprotein and the Usp45 preprotein signal peptide sequence, which provides secretion when fused to the gene product of interest. Whether a gene product of interest has been expressed and secreted into the supernatant of the host cell can be tested for by assays generally known in the art, including SDS-PAGE followed by Coomassie Blue Staining, or any immunological method including dot blots, ouchterlony assays, western blots, or ELISA techniques.
In any case, the food-grade expression vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100. In embodiments, the nucleic acid sequence in said food-grade expression vector encodes a soluble form of Amuc_1100 having an amino acid sequence with at least 80% identity to SEQ ID NO: 2 (Amuc_1100), such as with at least 82% identity to SEQ ID NO: 2, such as with at least 84% identity to SEQ ID NO: 2, such as with at least 86% identity to SEQ ID NO: 2, such as with at least 88% identity to SEQ ID NO: 2, such as with at least 90% identity to SEQ ID NO: 2, such as with at least 92% identity to SEQ ID NO: 2, such as with at least 94% identity to SEQ ID NO: 2, such as with at least 96% identity to SEQ ID NO: 2, such as with at least 98% identity to SEQ ID NO: 2, for example with at least 99% identity to SEQ ID NO: 2. For example, the Amuc_1100 encoded by the nucleic acid sequence comprised in functional linkage in said food-grade expression vector may comprise one or more conservative or semi-conservative substitutions, as generally known in the art, or it may be a homolog or an allelic variant to Amuc_1100 of SEQ DI NO: 2.
In one embodiment, the nucleic acid sequence in said food-grade expression vector encodes a soluble form of Amuc_1100 having an amino acid sequence as set out in SEQ ID NO: 2. A protein sequence comparison can be conducted using a sequence comparison and alignment tool, such as the publicly available program BLASTp, wherein sequence identity is intended to mean the identity of two amino acids at the same position, when both sequences are aligned, and over the total length of SEQ ID NO: 2 (287 amino acids).
In embodiments, said nucleic acid sequence may also encodes for a fragment of said soluble form of Amuc_1100, which has a length of at least 100 and up to 286 amino acids. These fragments may, for example, be N- or C-terminally truncated fragments. Alternatively, these fragments may arise from internal deletion(s). For example, said fragment may have a length of up to 285 amino acids, up to 284 amino acids, up to 283 amino acids, up to 282 amino acids, up to 281 amino acids, up to 280 amino acids, up to 275 amino acids, up to 270 amino acids, up to 265 amino acids, up to 260 amino acids, up to 255 amino acids, up to 250 amino acids, up to 240 amino acids, up to 230 amino acids, up to 220 amino acids, up to 210 amino acids, up to 200 amino acids; and/or at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids, at least 230 amino acids, at least 240 amino acids, at least 250 amino acids, at least 260 amino acids, at least 270 amino acids, or at least 280 amino acids.
In any case, the soluble Amuc_1100 protein or the fragment thereof must be selected such that it maintains at least in part the functional properties observed for Amuc_1100 of SEQ ID NO: 2. The term “functionally equivalent” or “functional properties” as used herein is intended to mean that the candidate protein maintains at least in part the property to increase the transepithelial electrical resistance (TEER), and/or the TLR-2 agonistic activity, observed for Amuc_1100 of SEQ ID NO: 2.
TLR-2 agonistic activity of the full length Amuc_1100 of SEQ ID NO: 2. TLR-2 agonistic activity can be determined using methods as described in the prior art, for example as described in Ottman et al. PLOS One 12(3): e0173004. Briefly, HEK-Blue hTLR2 cells (Invivogen, CA, USA) are grown and subcultured up to 70-80% of confluency using DMEM supplemented with 4.5 g/I D-glucose, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin, 2 mM L-glutamine, and 10% (v/v) of heat-inactivated FBS. For the experiment, cells are seeded in 180 μl in flat bottom 96-well plates and stimulated by addition of Amuc_1100 (fragment) protein to a final concentration of 5 μg/ml. Pam3CSK4 (10 ng/ml) are used as positive control, and culture medium is used as negative control. The 96-well plates are incubated for 20-24 hours at 37° C. in a 5% CO2 incubator. Stimulation of the hTLR2 receptor activates NF-κB and AP-1, which induces the production of secreted embryonic alkaline phosphatase (SEAP), the levels of which are measured spectrophotometrically. SEAP secretion is detected by measuring the OD600 at 1 hour after addition of 180 μl of QUANTI-Blue (Invivogen) to 20 μl of induced HEK-Blue hTLR2 supernatant. Experiments are performed in triplicate. The candidate soluble Amuc_1100 or the fragment thereof are considered to have or maintain TLR-2 agonistic activity in case its TLR-2 signalling activity, as determined using the foregoing assay, is at least 50% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 60% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 70% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 75% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, such as at least 80% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2, for example at least 85% of the TLR-2 signalling activity of Amuc_1100 of SEQ ID NO: 2 as measured in the above-described assay.
In addition, or alternatively, the property to increase the development of transepithelial electrical resistance can be tested for using the transepithelial electrical resistance (TEER) assay, as described in Ottman et al. PLOS One 12(3): e0173004. Briefly, 5×104 Caco-2 cells/insert are seeded in Millicell culture inserts with a 3 μm pore size (Merck Millipore) and grown for 8 days, whereas the growth conditions are as described in Kainulainen et al. BMC microbiology, 2015, 15(1): 4, incorporated herein by reference. Transepithelial resistance is determined using a Millicell ERS-2 TEER meter (Merck Millipore) from Caco-2 cell cultures at 0 h, and 24 h after addition of 0.5 μg/ml of Amuc_1100 protein. The candidate soluble Amuc_1100 or the fragment thereof are considered to have or maintain the property to increase the development of transepithelial electrical resistance (TEER) in case its increase in TEER compared to medium control, as determined using the foregoing assay, is at least 50% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 60% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 70% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 75% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, such as at least 80% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2, for example at least 85% of the increase in TEER observed for Amuc_1100 of SEQ ID NO: 2 as measured in the above-described assay.
Due to the degeneration of the genetic code, one and the same amino acid sequence can be encoded by different nucleic acid sequences. Indeed, different microorganisms have different preferences for encoding a particular amino acid. Depending on the abundance of the respective tRNAs in said microorganisms, expression of a gene product can be further improved by optimizing the nucleic acid sequence to the codon usage of the respective host. Thus, in embodiments, said nucleic acid sequence encoding for Amuc_1100 or a fragment thereof can be optimized for expression in a genus selected from the group of Bifidobacterium, Bacillus, Brevibacillus, Lactococcus and Saccharomyces. For example, said nucleic acid sequence may have a sequence selected from SEQ ID NO: 3 to SEQ ID NO: 7.
Within this context, said nucleic acid sequence encoding for Amuc_1100 or a fragment thereof has at least 70% sequence identity to SEQ ID NO: 1 (Amuc_1100), such as at least 72% sequence identity to SEQ ID NO: 1, such as at least 74% sequence identity to SEQ ID NO: 1, such as at least 76% sequence identity to SEQ ID NO: 1, such as at least 78% sequence identity to SEQ ID NO: 1, such as at least 80% sequence identity to SEQ ID NO: 1, such as at least 82% sequence identity to SEQ ID NO: 1, such as at least 84% sequence identity to SEQ ID NO: 1, such as at least 86% sequence identity to SEQ ID NO: 1, such as at least 88% sequence identity to SEQ ID NO: 1, such as at least 90% sequence identity to SEQ ID NO: 1, such as at least 92% sequence identity to SEQ ID NO: 1, such as at least 94% sequence identity to SEQ ID NO: 1, such as at least 96% sequence identity to SEQ ID NO: 1, such as at least 97% sequence identity to SEQ ID NO: 1, such as at least 98% sequence identity to SEQ ID NO: 1, or at least 99% sequence identity to SEQ ID NO: 1. A nucleic acid sequence comparison can be conducted using a sequence comparison and alignment tool, such as the publicly available program BLASTn, wherein sequence identity is intended to mean the identity of two nucleotides at the same position, when both sequences are aligned, and over the total length of SEQ ID NO: 1 (864 nucleotides).
In embodiments of the present invention, said soluble form of Amuc_1100 or a functionally equivalent fragment of said soluble form of Amuc_1100 does not need to comprise such a purification tag, as it is not required nor intended to purify Amuc_1100.
Moreover, while food-grade expression systems are disclosed for primary use in organisms of the genus Lactobacillus, in embodiments these expression systems are used in genera other than Lactobacillus, in which these food-grade expression vectors are also functional.
One useful example of said food-grade expression vector is p3050alrAmuc1100-sh71 (SEQ ID NO: 9) or p3050Alr_Amuc1100-sh71 with 5′UTR, 3′UTR and terminator (SEQ ID NO: 15). Many (shuttle) vectors for gram positive bacteria or for yeasts may be used, this particular vector is however the highest yielding.
In a further optional embodiment, the food-grade expression vector has an additional ethanol inducible promoter AlcA followed by human aldehyde dehydrogenase 1B1 (UniProt P30837; SEQ ID NO: 10). A corresponding food-grade expression vector is exemplified in SEQ ID NO: 11. Said vector is able to additionally express aldehyde dehydrogenase following the consumption of potable ethanol. Acetaldehyde, a metabolite of ethanol, is carcinogenic and the expression vector enables providing aldehyde dehydrogenase locally to colon, so to turn acetaldehyde into acetic acid. At the same time, it is reported that aldehyde dehydrogenase 1 expression is significantly higher in lean mice than in obese mice (Singh et al., Biochem Biophys Res Commun. 2015; 463(4): 768-773; and Yasmeen et al., Diabetes 2013; 62: 124-136; each of which is incorporated herein by reference).
Further disclosed is a method of preparing a probiotic as disclosed herein above, wherein the method comprises the step of introducing a food-grade expression vector, which vector comprises in functional linkage a nucleic acid sequence encoding for a soluble form of Amuc_1100 or a fragment of said soluble form of Amuc_1100, into a GRAS microbiological organism, such that said GRAS microbiological organism is capable of expressing and secreting said soluble form of Amuc_1100 or said fragment thereof.
Methods for introducing the vector into the GRAS microbiological organism are known in the art, and include, for example, electroporation techniques, or heat-shock techniques.
The link between gut microbiota and health is well-recognized and described, and biotherapeutic strategies evolved in the recent years, including fecal microbiota transplant (FMT), as also reviewed in Hage et al. Frontiers in Microbiology 2017, 8: article 1889, the content of which is incorporated by reference. Moreover, Plovier et al. (Nature Medicine 2016, doi: 10.1038/nm.4236, the content of which is incorporated herein by reference) and Ottman et al. (PLOS One 2017, 12(3): e0173004, the content of which is incorporated herein by reference) demonstrate that Akkermansia muciniphila or the pasteurized bacterium improve metabolism in obese and diabetic mice. It was furthermore shown that these beneficial health effects are due to a membrane protein, Amuc_1100. When added as a His-tagged purified protein in soluble form, the following beneficial health effects were observed: a reduction in body weight gain, a reduction in fat mass gain, a decrease in intestinal energy absorption, normalization of plasma LPS concentration, normalizing/reducing plasma cholesterol (in particular HDL-levels), normalizing/reducing plasma triglyceride levels, and normalizing/reducing plasma glucose levels, and improving the intestinal barrier function (as can be followed, for example, by an increase in the development of transepithelial electrical resistance).
In addition, it was demonstrated Ottman et al. (PLOS One 2017, 12(3): e0173004, the content of which is incorporated herein by reference) that the soluble, His-tagged Amuc_1100 purified protein has TLR-2 agonistic activity, and is thus considered to be involved with cross-talk with the host. In the intestine, TLR-2 regulates the expression of CYP1A1, an enzyme which is key in detoxication of certain carcinogenic substances. Recently, it was found that TLR-2 is involved in the activation of regulatory T cells (Tregs), that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. TLR-2 is also expressed by intestinal epithelial cells and subsets of lamina propria mononuclear cells in the gastrointestinal tract. TLR-2 has been observed downregulated in human papillomavirus-positive neoplastic keratocytres derived from uterine cervical preneoplastic lesions. Thus, TLR-2 is assumed to be associated with tumorigenesis.
Thus, in a further aspect, the above-described probiotic is for use in medicine for therapeutic purposes. Likewise, disclosed is the use of a probiotic as defined herein above for the manufacture of a medicament. Accordingly, also provided is a method of treatment of a patient, comprising the step of orally administering a probiotic as defined herein above to said patient. The patient may be a mammal, in particular a dog, cat, rat, or mouse. Preferably, the patient is a human patient. Dosages (cfu) will vary based on the formulation, the indication, and the physical state of the patient (for example dependent on the age and/or weight), but are commonly in the range of 109 to 1010 CFU/day. Suitable dosages can be determined by a person skilled in the art.
More specifically, the probiotic is for use in the treatment of obesity, diabetes, and/or hypercholesterolemia. Hence, the probiotic may be used for the manufacture of a medicament for the treatment of obesity, diabetes, and/or hypercholesterolemia.
Provided is thus a method for treating obesity, diabetes, and/or hypercholesterolemia in a patient, such as a human patient, comprising the step of orally administering a probiotic as defined herein above to said patient. Similarly, also provided is a method for (i) reducing body weight gain, (ii) reducing fat mass gain, (iii) decreasing intestinal energy absorption, (iv) normalizing plasma LPS concentration, (v) normalizing/reducing plasma cholesterol (in particular HDL-levels), (vi) normalizing/reducing plasma triglyceride levels, and (vii) normalizing/reducing plasma glucose levels, and (viii) improving the intestinal barrier function in a patient, such as a human patient, comprising the step of orally administering a probiotic as defined herein above to said patient.
As used herein, the term “or” has the meaning of both “and” and “or” (i.e. “and/or”). Furthermore, the meaning of a singular noun includes that of a plural noun and thus a singular term, unless otherwise specified, may also carry the meaning of its plural form. In other words, the term “a” or “an” may mean one or more.
It is apparent to the skilled reader that, as technology develops, the basic idea of the invention can be accomplished in many different ways. The invention and its embodiments are therefore not confined to the examples described above, but may vary in the framework of patent requirements and the below claims.
If not otherwise stated, the following example uses routine methods of molecular biology, as also described in reference textbooks in the art, in particular with regard to techniques concerning molecular cloning, polymerase chain reaction, and gel electrophoresis. See, for example, ‘Molecular Cloning: A Laboratory Manual’ by Michael Green and Joseph Sambrook, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
In order to construct a food-grade expression and secretion vector comprising a nucleic acid sequence encoding for a soluble form of Amuc_1100, the plasmid p3050sNucA-sh71 was selected as the starting point. The plasmid p3050sNucA-sh71 is based on pSIP411, described in Sørvig et al. Microbiology 2005, 151(7): 2439-2449 (the disclosure of which is incorporated herewith by reference), which is also the source of the sh71 replicon. The plasmid p3050sNucA-sh71 and its construction is described in Mathiesen et al. BMC Genomics 2009, 10: 425; and Karlskas et al. PLoS One, 2014, 9(3): e91125, the respective disclosure of which is hereby incorporated by reference. The plasmid p3050sNucA-sh71 (see FIG. 1 in Karlskås et al.) was first linearized by digestion with 4 restriction enzymes (BamH I, Afl III, Sal I, Hind III) yielding following bands in an agarose gel: 2852 bp (AflIII-SalI), 1962 bp (AflIII-BamHI), 1100 bp (BamHI-AflIII), 307 bp (SalI-HindIII), 178 bp (HindIII-HindIII), 17 bp (HindIII-AflIII), (linear: 2727(AflIII-End), 1962(AflIII-BamHI), 1100(BamHI-AflIII), 307(SalI-HindIII), 178(HindIII-HindIII), 125(Start-SalI), 17(HindIII-AflIII)).
The bands containing the erythromycin resistance marker gene at 1.1 kb and NucA fragments at 0.3 kb and 0.2 kb were discarded, and the DNA was cleaned.
The sh71-replicon (2 kb band) was ligated back to the backbone leaving BamHI-AflIII and SalI-HindIII restriction site pairs open to which alanine racemase and Amuc_1100 inserts were then ligated.
The food-grade alanine racemase (alr) marker gene and its isolation is described in Nguyen et al. J. Agric. Food Chem. 2011, 59, 5617-5624, the content of which is incorporated herein by reference. The following is the sequence of the alr marker gene in 5′ to 3′-direction:
alanine racemase (alr) (SEQ ID NO: 8):
For introducing same into the backbone vector, the alr selection marker was PCR-amplified with 5′ BamHI and 3′ AflIII restriction sites.
The complete nucleic acid sequence encoding for Amuc_1100 is publicly available from the KEGG GENOME Database under reference ID T00376. Isolation of Amuc_1100 from Akkermansia muciniphila is also described in Plovier et al. Nature Medicine, doi: 10.1038/nm.4236, the disclosure of which is incorporated herein by reference. The nucleic acid sequence encoding a soluble form of Amuc_1100 (i.e. an Amuc_1100 encoding gene insert lacking it's signal sequence in the N-terminal residues 1-30) was synthesized with 5′ SalI and 3′ HindIII-sites, and cloned into the above-mentioned vector backbone.
The following is the nucleic acid sequence encoding the soluble form of Amuc_1100, which lacks the first 30 N-terminal residues (in 5′ to 3′ direction): p3050Alr_Amuc1100_sh71 (SEQ ID NO: 9):
The construct, p3050Alr_Amuc1100_sh71 (SEQ ID NO: 9), was then verified by DNA-sequencing and electrotransformed into the following competent probiotic strains:
Every recombinant strain secreted the protein Amuc_1100, when running the supernatant on a SDS-PAGE, and stained with Coomassie Blue.
