Patent Application
20200354727
This instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 23, 2020, is named 06245-006US1_Sequence_Listing and is 50 KB in size.
The present invention relates to modified bacterial spores, and particularly, although not exclusively, to modified bacterial spores which comprise one or more heterologous genes, but without the introduction of antibiotic resistance genes. The invention extends to methods for producing such modified bacterial spores, vectors and kits for introducing heterologous genes in a spore-forming bacterium.
Bacillus endospores, or spores, are dormant entities with well-known resistance properties including heat stability, exposure to noxious chemicals and desiccation (1). As such, spores offer a number of applications. First and foremost has been their use as mucosal vaccines where antigens can be displayed on the spore surface by fusion to a coat protein anchor (2). Immunization of recombinant spores by a mucosal route (oral, sub-lingual or nasal) has shown promising results and in some cases levels of protection that could be efficacious in humans or animals (3-5). In the case of the latter, the ability to incorporate a vaccine in feed is particularly attractive and for some animals, arguably, the only way to vaccinate. For example, vaccinating farmed shrimp against viral pathogens, for example, White Spot Syndrome Virus (WSSV) where recombinant spores expressing the WSSV VP26 and VP28 envelope proteins have been shown to confer protection in shrimps (6-9).
A number of Bacillus species are considered ‘food grade’, that is, safe for human consumption and are designated as QPS (Qualified Presumption of Safety as defined by EFSA, European Food Safety Authority) (10). In the USA, some strains carry GRAS (Generally Recognized As Safe as defined by the Food and Drug Administration) status. QPS and GRAS status has supported the use of a number of Bacillus strains, including those of B. subtilis, as probiotics in both human and animal feeds (11). The concept of recombinant probiotics (12) is conceptually, a logical next step forward in exploiting the beneficial properties of Bacillus (13).
In addition to vaccines, spores have been shown to facilitate expression of enzymes on the spore surface, for example the animal feed enzyme phytase (14). The ability to use probiotic bacteria that deliver enzymes negates the need to purify enzymes and could offer significant advantages to industry. Finally, streptavidin has been expressed on B. subtilis spores enabling monoclonal antibodies to be conjugated to the spore surface and the targeting of spores loaded with anti-cancer drugs to cancer cells (15).
Despite the potential utility of Bacillus spores for industrial applications, there remain a number of obstacles regarding the deliberate release of genetically modified Bacillus. First, is the use of antibiotic resistance (AbR) genes used in the genetic engineering of stable recombinant strains. Regarding AbR the majority of procedures requiring insertion of heterologous DNA into B. subtilis require ectopic insertion using an AbR gene for positive selection (16). Plasmid vectors carrying chloramphenicol or erythromycin resistance genes are typically used for ectopic insertion at a genetic locus that is redundant for cell growth, typically the alpha amylase (amyE) or threonine C (thrC) genes (17). The potential risk of AbR gene transfer following release is recognised and at least two systems in B. subtilis have now been described that enable insertion of heterologous genes without introduction of an AbR gene (18,19).
The second, and most challenging, hurdle is the ultimate fate of recombinant spores once released, since spores have been shown to survive for millions of years as viable entities (20). The soil is normally enriched with dormant spores (21) and their intrinsic robustness makes it difficult to argue that they would not persist after deliberate release. One approach might be to construct germination deficient spores but, at best, the germination rate can be reduced to 0.0015% (22) which is unlikely to satisfy regulatory authorities.
Therefore, there is a need for improved methods of introducing heterologous genes into spore-forming bacteria without the introduction of antibiotic resistance genes.
The inventors have adopted the approach of thymine starvation resulting in a so-called ‘thymine-less death’ (23-25). The majority of prokaryotes are known to carry a single thymidylate synthase enzyme that is encoded by the thyA gene and prokaryotes carrying a mutated thymidylate synthase gene are unable to grow in low concentrations of thymidine or thymine resulting in cell death. Thymine starvation therefore is different from the biostatic effects commonly found with deprivation of other nutrients. Thymine-less death has been documented for B. subtilis (26) and the bacterium is unique in that it carries two thymidylate synthase enzymes encoded by the thyA and thyB genes (27).
However, the concept of thymine-less death, despite being known since 1954, has never been successfully applied to spore-forming bacteria for the purposes of introducing heterologous genes without the introduction of antibiotic resistance genes.
The inventors have successfully adopted the principles of a ‘thymine-less death’ to exploit spore-forming bacteria, such as Bacillus, for the introduction of heterologous genes without the introduction of antibiotic resistance genes. As recombinant spores, should they germinate, they will die immediately and fail to survive or proliferate (i.e., grow) in the environment due to the lack of (or only low concentrations of) thymine or thymidine. The approach developed is particularly novel since it is a two-step gene integration process enabling positive selection on successively higher levels of trimethoprim, but without the introduction of any antibiotic resistance genes.
Thus, according to a first aspect of the invention, there is provided a process for introducing at least one heterologous gene into a spore-forming bacterium and rendering the bacterium unable to proliferate in the absence of thymine or thymidine, the process comprising:
In a preferred embodiment, and advantageously, the process operates without the introduction of an antibiotic resistance gene, and therefore overcomes the problems discussed above.
In a preferred embodiment, the method further comprises:
It will be appreciated that the bacterial spore may or may not germinate, but if it does germinate, it is unable to grow out of that germinated spore and proliferate.
Advantageously, the inventor has devised an elegant way to inactivate all thymidylate synthases in a bacterium, for example in embodiments in which the bacterium carries two or more thy genes, or where the bacterium carries a thyA and a thyB gene (e.g., Bacillus spp.), or where the inactivation of thyA is unable to affect the growth of the bacterium under normal physiological conditions.
Hence, in a preferred embodiment, one or more heterologous genes is introduced into each thymidylate synthase gene present within the bacterium. For instance, in one embodiment, the spore-forming bacterium may be a Bacillus Spp. (which is unique in that it is known to have two thymidylate synthase genes, thyA and thyB). Preferably, a heterologous gene is introduced into each of the two thymidylate synthase genes, for example thyA and thyB of Bacillus spp. The Bacillus spp. bacterium may be B. subtilis, B. cereus, B. pumilus, B. amyloliquefaeceans, B. coagulans, B. clausii, B. licheniformis or B. megaterium. Most preferably, however, the Bacillus spp. bacterium is B. subtilis.
It should be appreciated that inactivation of thyA only in Bacillus will not induce a thymineless death, and will not damage or prevent growth under normal physiological conditions.
In another embodiment, the spore-forming bacterium may be Clostridium Spp. (which is known to have one thymidylate synthase gene). Preferably, in this embodiment, a heterologous gene is introduced into the thymidylate synthase gene. The Clostridium spp. bacterium may be C. difficile, C. perfringens, C. tetani, C. botulinum, C. acetobutylicum, C. cellulolyticum, C. novyi or C. thermocellum. Most preferably, the Clostridium spp. bacterium is Clostridium difficile.
In preferred embodiments of the process, the spore-forming bacterium is present in a bacterial culture.
Step (ii) of the process may be performed using any suitable vessel, such as a Petri dish or glass culture flask, containing any growth medium as disclosed herein. The growth medium may comprise trimethoprim at a concentration of 0.001 μg/ml to 20 μg/ml, 0.01 μg/ml to 15 μg/ml, 0.1 μg/ml to 10 μg/ml, 1 μg/ml to 5 μg/ml, 2 μg/ml to 4 μg/ml, or preferably approximately 3 μg/ml.
In a further embodiment, during step (ii), the bacterium may be grown in a medium comprising yeast extract (YE) at a concentration lower than 2 mg/ml, or any concentration disclosed herein. In an embodiment, the medium contains no YE.
In an embodiment, during step (ii), the bacterium may be grown in a medium comprising thymine or thymidine at any concentration disclosed herein, for instance approximately 50 μg/ml.
In an embodiment, during step (ii), the bacterium may be grown in a medium comprising trimethoprim at any concentration disclosed herein, for instance approximately 3 μg/ml.
In an embodiment, during step (ii), the bacterium may be grown in a medium comprising casamino acids (CAA) at any concentration disclosed herein, for instance approximately 0.2% (w/v).
In an embodiment, during step (ii), the bacterium may be grown in a medium supplemented with at least one amino acid or any amino acid mixture as disclosed herein.
In an embodiment, cells carrying a gene insertion may be verified by the inability to grow unless supplemented with thymine or thymidine. A further or alternative verification is to amplify, by PCR, the presence of the chimeric genes from transformants.
In a most preferred embodiment, the invention provides a cloning system suitable for biological containment of genetically modified Bacillus Spp., such as B. subtilis.
Hence, according to this embodiment of the invention, there is provided a process for introducing at least one heterologous gene into a Bacillus Spp. and rendering the bacterium unable to proliferate in the absence of thymine or thymidine, the process comprising:
In a preferred embodiment, the heterologous gene is introduced into one of the thy genes, and then subsequently into the other thy gene. Preferably, therefore, there is provided a process for introducing at least one heterologous gene into a Bacillus Spp. and rendering the bacterium unable to proliferate in the absence of thymine or thymidine, the process comprising:
In a preferred embodiment, the process further comprises:
It will be appreciated that the bacterial spore may or may not germinate, but if it does germinate, it is unable to grow out of that germinated spore and proliferate. In a preferred embodiment, the process operates without the introduction of an antibiotic resistance gene. In a preferred embodiment, step (i) is the introduction of a heterologous gene into the thyA gene in a Bacillus Spp. and step (iii) is the introduction of a heterologous gene into the thyB gene in a Bacillus Spp.
In a preferred embodiment, the Bacillus Spp. is B. subtilis. In preferred embodiments, the Bacillus Spp. or B. subtilis is present in a bacterial culture.
In one embodiment, the open reading frame of thyA may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 1, as follows:
Preferably, therefore, the process comprises introducing a heterologous gene into a nucleic acid sequence substantially as set out in SEQ ID NO: 1, or a fragment or variant thereof, within Bacillus Spp. In an alternative embodiment, the processes may comprise introducing a heterologous gene into Bacillus Spp. not into SEQ ID NO: 1 or a fragment or variant thereof, but such that the expression of SEQ ID NO: 1 is disrupted or reduced.
In one embodiment, the open reading frame of thyA may be encoded by a nucleic acid sequence which encodes an amino acid sequence provided herein as SEQ ID NO: 18, as follows:
SEQ ID NO: 1 and 18 are examples from B. subtilis, and thus variants of these sequences include the relevant thyA genes from all Bacillus Spp. It is important to note that the invention is applicable to any thyA of a Bacillus Spp. and that SEQ ID NO: 1 and 18 are intended to be illustrative and not limiting.
In one embodiment, the open reading frame of thyB may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 2, as follows:
Preferably, therefore, the process comprises introducing a heterologous gene into a nucleic acid sequence substantially as set out in SEQ ID NO: 2, or a fragment or variant thereof, within Bacillus Spp. The processes may comprise introducing a heterologous gene into Bacillus Spp. not into SEQ ID NO: 2 or a fragment or variant thereof, but such that the expression from SEQ ID NO: 2 is disrupted or reduced.
In one embodiment, the open reading frame of thyB may be encoded by a nucleic acid sequence which encodes an amino acid sequence provided herein as SEQ ID NO: 19, as follows:
SEQ ID NO: 2 and 19 are examples from B. subtilis, and thus variants of these sequences include the relevant thyB genes from all Bacillus Spp. It is important to note that the invention is applicable to any thyB of a Bacillus Spp. and that SEQ ID NO: 2 and 19 are intended to be illustrative and not limiting.
In an embodiment, step (i) may be performed by the transformation of Bacillus Spp. with a plasmid, which may be linearised, which may comprise at least a portion of the left arm of a Bacillus Spp. thyA gene, a heterologous gene, and at least a portion of the right arm of a Bacillus Spp. thyA gene. Step (i) may be performed with any vector as disclosed herein. Transformation may be carried out using electroporation.
Step (ii) and/or Step (iv) may be performed using any suitable vessel, such as a Petri dish or a cell culture flask, containing any growth medium as disclosed herein. In an embodiment, during step (ii) and/or step (iv), the Bacillus Spp. may be grown in a medium comprising yeast extract (YE) at a concentration lower than 2 mg/ml. In a preferred embodiment, the medium has YE at a concentration lower than 1.5 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml, or 0 mg/ml. In an embodiment, the medium in which the Bacillus Spp. is grown contains no YE.
In an embodiment, during step (ii) and/or step (iv), the Bacillus Spp. may be grown in a medium comprising thymine or thymidine. In a preferred embodiment, the thymine or thymidine may be present between 0.1 μg/ml and 1000 μg/ml, 1 μg/ml and 500 μg/ml, less than or equal to 300 μg/ml, 10 μg/ml and 200 μg/ml, 20 μg/ml and 100 μg/ml, 30 μg/ml and 80 μg/ml, 35 μg/ml and 65 μg/ml, 45 μg/ml and 55 μg/ml, or preferably approximately 50 μg/ml.
In an embodiment, during step (ii) and/or step (iv), the growth medium is any suitable growth medium, for instance Spizizen's Minimal Medium (SMM).
In an embodiment, the amount of trimethoprim present in step (iv) is higher than the amount of trimethoprim present in step (ii).
In an embodiment, during step (ii), the medium may comprise trimethoprim at a concentration of 0.001 μg/ml to 20 μg/ml, 0.01 μg/ml to 15 μg/ml, 0.1 μg/ml to 10 μg/ml, 1 μg/ml to 5 μg/ml, 2 μg/ml to 4 μg/ml, or preferably approximately 3 μg/ml.
In a preferred embodiment, the concentration of trimethoprim is lower than the concentration of trimethoprim in step (iv).
In an embodiment, step (ii) may comprise or consist of 12 hours to 168 hours, 24 hours to 144 hours, 48 hours to 120 hours, 60 hours to 108 hours, or 72 hours to 96 hours of growth.
In an embodiment, cells carrying an insertion at the thyA locus may be verified by the ability to grow at 37° C. with or without thymine or thymidine but the inability to grow at 46° C. unless supplemented with thymine or thymidine. A further, or alternative, verification is to amplify, by PCR, the presence of the chimeric gene from transformants using primers annealing to the thyA sequences. In some embodiments, the colonies may be screened and separated into two types: i) large opaque colonies (2-3 mm after 72 h-96 h of growth), ii) translucent, smaller colonies (1 mm after 72 h-96 h of growth). In some embodiments, type (i) colonies may be selected.
In an embodiment, step (iii) may be performed by the transformation of Bacillus Spp. with a plasmid, which may be linearised, which may comprise at least a portion of the left arm of a Bacillus Spp. thyB gene, a heterologous gene, and at least a portion of the right arm of a Bacillus Spp. thyB gene. Step (iii) may be performed with any vector as disclosed herein. Transformation may be carried out using electroporation.
In an embodiment, the Bacillus Spp. may be prepared or made electro-competent using a method that involves culturing Bacillus Spp. in medium that contains yeast extract (YE) at a concentration lower than 2 mg/ml. In a preferred embodiment, the medium has YE at a concentration lower than 1.5 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml, or 0 mg/ml. In an embodiment, the medium in which the Bacillus Spp. is grown contains no YE. In a most preferred embodiment, the Bacillus Spp. is a strain carrying a thyA insertion as generated by steps (i) and (ii).
In an embodiment, YE is present at a concentration lower than 2 mg/ml, any concentration disclosed herein, or preferably not present in the media used at any stage of the process.
In an embodiment, step (iv) may be performed using medium which may comprise trimethoprim at a concentration of less than or equal to 80 μg/ml, less than or equal to 40 μg/ml, less than or equal to 30 μg/ml, 0.01 μg/ml to 30 μg/ml, 0.1 μg/ml to 20 μg/ml, 1 μg/ml to 15 μg/ml, 2 μg/ml to 10 μg/ml, 5 μg/ml to 7 μg/ml, or preferably approximately 6 μg/ml. In a preferred embodiment the concentration of trimethoprim is higher than the concentration of trimethoprim in step (ii).
Casamino acids (CAA) are produced by the acid hydrolysis of casein. The typical composition is as follows, although these numbers are approximate and variations in composition are acceptable for use with the invention: Ala 2.8%, Arg 3.6%, Asp 6.3%, Cystine 0.3%, Glu 21.1%, Gly 2.2%, His 2.7%, Ile 5.6%, Leu 8.4%, Lys 7.5%, Met 2.7%, Phe 4.6%, Pro 9.9%, Ser 5.6%, Thr 4.2%, Trp 1.1%, Tyr 6.1%, Val 5.0%. CAA may not contain tryptophan.
In an embodiment, during step (iv), the Bacillus Spp. may be grown in a medium comprising casamino acids (CAA). The medium may comprise CAA at 0.0001% to 5%, 0.001% to 2%, 0.01% to 1%, 0.05% to 0.5%, 0.1% to 0.3%, or preferably approximately 0.2% (w/v).
In an embodiment, during step (iv), the Bacillus Spp. may be grown in a medium supplemented with at least one amino acid. In other embodiments the medium may comprise any naturally occurring amino acid or amino acids, and optionally all naturally occurring amino acids or all naturally occurring amino acids except tryptophan. The medium may comprise serine, glycine, cysteine, methionine, any combination thereof, or all four amino acids. The medium may comprise any individual amino acid at a concentration equivalent to medium containing CAA at 0.0001% to 5%, 0.001% to 2%, 0.01% to 1%, 0.05% to 0.5%, 0.1% to 0.3%, or preferably approximately 0.2% (w/v). Thus, in a preferred embodiment, the medium may comprise serine, glycine, cysteine, and/or methionine at a concentration equivalent to medium containing CAA at approximately 0.2% (w/v). In another preferred embodiment, the medium may comprise the same amino acids as medium containing CAA at approximately 0.2% (w/v).
In an embodiment, step (iv) may comprise or consist of 6 hours to 144 hours, 12 hours to 120 hours, 24 hours to 96 hours, 36 hours to 72 hours, or approximately 48 hours of growth.
In an embodiment, cells carrying two insertions may be verified by the inability to grow at 37° C. or 46° C. unless supplemented with thymine or thymidine. A further or alternative verification is to amplify, by PCR, the presence of the chimeric genes from transformants using primers annealing to the thyA and thyB sequences.
Any heterologous gene may be used for the gene insertion into the or each thymidylate synthase gene. In particular embodiments, the heterologous genes are fused to genes encoding spore coat proteins, for instance CotA, CotB, CotC, CotD, CotE, CotF, or CotG of B. subtilis. The spore protein used may be chosen depending on the particular bacterium being modified, and the appropriate gene used. Appropriate spore coat proteins for use with Clostridium Spp. include CotA, CotB, CotC, CotD, CotE, CotF, CdeC, BcA1, BcA2, or BcA3 (53, 54, 55).
In some embodiments, a heterologous gene may be inserted into the thyA gene and the same heterologous gene may be inserted into the thyB gene. In other embodiments, a different heterologous gene may be inserted into the thyA gene from that inserted into the thyB gene.
In some embodiments, the resultant spore produced by the process may carry a heterologous protein for use in vaccination. The resultant spore may carry a heterologous protein which is displayed as an active enzyme. The resultant spore may carry a heterologous protein allowing the conjugation of proteins to the spore surface.
After generation and selection of a strain comprising the heterologous genes that are unable to proliferate in the absence of thymine or thymidine, the process may comprise cultivating the strain. The cultivation may be on any media comprising thymine or thymidine, YE at a concentration less than 2 mg/ml or absent, and at least one amino acid or any mixture as disclosed herein. The concentrations may be any as disclosed herein.
Thus, according to a particular embodiment of the invention, there is provided a process for introducing at least one heterologous gene into B. subtilis and rendering the B. subtilis unable to proliferate in the absence of thymine or thymidine, preferably wherein the process does not involve the introduction of an antibiotic resistance gene, comprising:
In a preferred embodiment, YE is present at a concentration lower than 2 mg/ml, preferably 0 mg/ml, during all steps following step (i). In a preferred embodiment, thymine or thymidine is present for all cultures following step (i). In a preferred embodiment, a heterologous gene is introduced into the thyA gene during step (i) and a heterologous gene is introduced into the thyB gene during step (iii).
Thus, according to a particular embodiment of the invention, there is provided a process for introducing at least one heterologous gene into B. subtilis and rendering the B. subtilis unable to proliferate in the absence of thymine or thymidine, preferably wherein the process does not involve the introduction of an antibiotic resistance gene, comprising:
In a preferred embodiment, thymine or thymidine is present for all cultures following step (i). In a preferred embodiment, a heterologous gene is introduced into the thyA gene during step (i) and a heterologous gene is introduced into the thyB gene during step (iii).
According to a second aspect of the invention, there is provided a bacterial spore obtained or obtainable by the process of the first aspect.
Preferably, the bacterial spore is Bacillus spp. and comprises non-functional thyA and thyB genes, preferably due to the introduction of heterologous genes therein.
In a third aspect, there is provided a bacterial spore comprising at least one heterologous gene and unable to proliferate in the absence of thymine or thymidine, wherein the bacterial spore is produced by a process comprising:
In a preferred embodiment, the bacterial spore does not comprise an antibiotic resistance gene.
In a preferred embodiment, the bacterial spore may be Bacillus Spp. The Bacillus spp. bacterium may be B. subtilis, B. cereus, B. pumilus, B. amyloliquefaeceans, B. coagulans, B. clausii, B. licheniformis or B. megaterium. Most preferably, the Bacillus spp. bacterium is B. subtilis. In another preferred embodiment, the bacterial spore may be Clostridium Spp. The Clostridium spp. bacterium may be C. difficile, C. perfringens, C. tetani, C. botulinum, C. acetobutylicum, C. cellulolyticum, C. novyi or C. thermocellum. Most preferably, the Clostridium spp. bacterium is Clostridium difficile.
In an embodiment, there is provided a bacterial spore comprising at least one heterologous gene and unable to proliferate in the absence of thymine or thymidine, wherein the bacterial spore is produced by any process as disclosed herein, or using any culture or conditions as disclosed.
According to a particular embodiment of the invention, there is provided a Bacillus Spp. spore comprising at least one heterologous gene and unable to proliferate in the absence of thymine or thymidine, wherein the Bacillus Spp. spore is produced by a process comprising:
In a preferred embodiment, a heterologous gene is introduced into the thyA gene during step (i) and a heterologous gene is introduced into the thyB gene during step (iii).
According to a fourth aspect of the invention, there is provided a vector for use in introducing a heterologous gene into the thyA gene within a spore-forming bacterium.
In an embodiment, the vector may comprise at least a portion of the 5′ portion of a Bacillus Spp. thyA gene, a heterologous gene, and at least a portion of the 3′ portion of a Bacillus Spp. thyA gene.
In an embodiment, the vector may comprise the left and right arms of a Bacillus Spp. thyA gene. The arms may flank a multiple cloning site. For instance, the vector may include the left (e.g. 900 bp) and right (e.g. 950 bp) arms of the B. subtilis thyA gene.
The length of the flanking arms may be adjusted for the length of the heterologous sequence to be inserted. Longer flanking arms (e.g. ˜1500 bp) may be necessary for large inserts.
The vectors may comprise a heterologous gene inserted at the multiple cloning site.
The heterologous gene may be a chimeric gene comprising a spore coat protein fused to a heterologous gene. For instance, the spore coat protein may be CotB or CotC of B. subtilis. The vectors may comprise: the left arm of a Bacillus Spp. thyA gene, a spore coat protein, a multiple cloning site allowing insertion of a heterologous gene such that it is fused to the spore coat protein, and the right arm of a Bacillus Spp. thyA gene.
In one purely illustrative embodiment, the left arm of thyA may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 3 as follows:
CAATTCGATAAACAATACAATTCAATTATAAAGGATATTATCAATA
ATGGAATCTCAGACGAAGAGTTTGATGTAAGAACCAAGTGGGACTC
AGATGGAACACCGGCACATACTCTAAGTGTAATCAGTAAGCAAATG
AGATTCGACAACTCAGAGGTTCCGATTTTAACGACAAAAAAGGTTG
CCTGGAAAACAGCCATTAAAGAGTTGCTCTGGATTTGGCAGCTGAA
ATCTAATGATGTTAATGATTTAAACATGATGGGCGTCCATATTTGG
GATCAGTGGAAACAAGAAGACGGAACCATCGGACATGCATATGGAT
TTCAGCTGGGGAAGAAAAACAGAAGTCTAAATGGAGAAAAAGTGGA
TCAGGTAGACTATCTTCTTCATCAAT
The bold sequence represents the thyA coding sequence, the non-bold sequence represents the upstream region. Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 3, or a fragment or variant thereof.
In one purely illustrative embodiment, the right arm of thyA may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 4 as follows:
TGAAGAACAATCCATCTTCACGCAGACACATTACAATGCTGTGGAA
TCCTGATGAATTAGACGCAATGGCCTTAACGCCATGTGTATACGAG
ACACAATGGTACGTTAAACATGGGAAACTCCACCTTGAGGTAAGAG
CACGGAGCAATGATATGGCATTGGGAAATCCATTCAATGTATTCCA
GTATAATGTGTTGCAGCGCATGATTGCTCAAGTGACTGGTTATGAG
CTTGGTGAATATATCTTTAACATTGGGGATTGCCATGTGTACACAC
GTCATATAGACAATTTGAAAATTCAAATGGAAAGAGAACAGTTTGA
AGCACCTGAACTATGGATCAATCCTGAAGTGAAAGATTTTTATGAC
TTTACCATTGATGATTTCAAGTTAATCAACTATAAACATGGGGACA
AGCTTTTATTTGAGGTAGCGGTTTAATGCTGCCTTTTTATTGTGCA
The bold sequence represents the thyA coding sequence, the non-bold sequence represents the downstream region. Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 4 or a fragment or variant thereof.
Any suitable multiple cloning site could be used in a vector of the invention. For instance, in an embodiment, the multiple cloning site may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 5 as follows:
Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 5 or a fragment or variant thereof.
In one purely illustrative embodiment, the thyA vector may comprise a nucleic acid sequence which may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 6 as follows:
TC
AGGTAGACTATCTTCTTCATCAATAAGCTTGCATGCCTGCAGGT
Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 6, or a fragment or variant thereof. Example primer annealing sites, which could be used to verify insertion of the heterologous gene, are shown in bold and underlined.
According to a fifth aspect of the invention, there is provided a vector for use in introducing a heterologous gene into the thyB gene within a spore-forming bacterium.
In an embodiment, the vector may comprise at least a portion of the 5′ portion of a Bacillus Spp. thyB gene, a heterologous gene, and at least a portion of the 3′ portion of a Bacillus Spp. thyB gene.
In an embodiment, the vector may comprise the left and right arms of a Bacillus Spp. thyB gene. The arms may flank a multiple cloning site. For instance, the vector may include the left (900 bp) and right (1.1 kb) arms of the B. subtilis thyB gene.
The length of the flanking arms may be adjusted for the length of the heterologous sequence to be inserted. Longer flanking arms (e.g. ˜1500 bp) may be necessary for large inserts.
The vectors may include a heterologous gene inserted at the multiple cloning site. The heterologous gene may be a chimeric gene comprising a spore coat protein fused to a heterologous gene. For instance, the spore coat protein may be CotB or CotC of B. subtilis. The vectors may comprise: the left arm of a Bacillus Spp. thyB gene, a spore coat protein, a multiple cloning site allowing insertion of a heterologous gene such that it is fused to the spore coat protein, and the right arm of a Bacillus Spp. thyB gene.
In a purely illustrative embodiment, the left arm of thyB may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 7, as follows:
CAGTATAAGGATTTCTGCAGACATGTTTTAGAGCATGGTGAGAAAA
AGGGAGACCGGACTGGGACCGGAACAATCAGCACTTTCGGATATCA
AATGAGATTTAATTTACGGGAAGGCTTTCCGATGCTCACCACTAAA
AAACTCCACTTTAAATCAATTGCGCATGAACTGCTGTGGTTCTTAA
AAGGAGATACGAATGTACGCTATCTGCAGGAAAACGGAGTGCGAAT
CTGGAATGAGTGGGCTGATGAAAACGGTGAACTTGGACCTGTATAT
GGCTCCCAATGGCGTTCTTGGCGGGGAGCTGATGGAGAAACCATTG
ATCAAATTTCCCGTCTTATTGAAGATATTAAAACAAATCCGAACTC
CAGACGCTTAATCGTCAGCGCCTGGA
The bold sequence represents the thyB coding sequence, the non-bold sequence represents the upstream region. Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 7, or a fragment or variant thereof.
In one purely illustrative embodiment, the right arm of thyB may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 8, as follows:
ATGTTGGTGAAATTGATAAAATGGCGTTGCCGCCGTGCCATTGCCT
GTTCCAATTCTATGTGTCTGACGGCAAGCTGTCCTGTCAGCTGTAT
CAGCGCTCTGCCGATGTTTTCTTAGGTGTGCCGTTTAATATTGCAT
CTTATGCCCTCCTAACCATGATCATTGCTCATGTGACTGGGCTTGA
ACCGGGCGAGTTCATCCATACGTTTGGTGATGTTCATATTTACCAA
AATCATATTGAACAAGTCAATTTGCAGCTGGAAAGAGATGTTAGAC
CGCTTCCGCAGCTTCGTTTCGCCAGAAAGGTTGATTCTATTTTTAA
CTTTGCATTTGAGGACTTTATCATCGAGGATTATGATCCGCATCCT
CATATAAAAGGGGCGGTCAGCGTATGATTTCATTCATTTTTGCGAT
The bold sequence represents the thyB coding sequence, the non-bold sequence represents the downstream region. Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 8, or a fragment or variant thereof.
The above illustrative embodiments of both thyA and thyB sequences are intended as an example. As the skilled person would be aware, the sequences will vary between species, in particular there is a great deal of variability between the upstream and downstream sequences.
Any suitable multiple cloning site could be used in a vector of the invention. For instance, in an embodiment, the multiple cloning site may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 9, as follows:
Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 9, or a fragment or variant thereof.
In one embodiment, the thyB vector may comprise a nucleic acid sequence which may be encoded by the nucleic acid sequence provided herein as SEQ ID NO: 10, as follows:
CAATTCTATGTGTCTGAC
GGCAAGCTGTCCTGTCAGCTGTATCAGC
Preferably, therefore, the vector comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 10, or a fragment or variant thereof. Example primer annealing sites, which could be used to verify insertion of the heterologous gene, are shown in bold and underlined.
According to a sixth aspect of the invention, there is provided a kit comprising reagents for use with the processes of the invention.
In an embodiment, the kit may comprise any medium as disclosed herein, for instance medium comprising YE at a concentration lower than 2 mg/ml, or comprising no YE. The kit may comprise trimethoprim. The kit may comprise medium comprising trimethoprim, optionally two mediums one with a higher level of trimethoprim than the other. The kit may comprise CAA. The kit may comprise one or more amino acids, or any combination or concentration as disclosed herein. The kit may comprise instructions describing the use of the kit or describing any process as disclosed herein. The kit may comprise at least one container for performing a process as disclosed herein, for instance a petri dish.
In an embodiment, the kit may comprise one or more vectors of the invention. The kit may comprise a vector for use in introducing a heterologous gene into the thyA gene within a spore-forming bacterium and/or a vector for use in introducing a heterologous gene into the thyB gene within a spore-forming bacterium. The kit may comprise a nucleic acid sequence including a heterologous gene, such as a gene for a vaccine, for a conjugation protein, or an enzyme. The kit may comprise a nucleic acid sequence including a spore coat protein. The spore coat protein may be configured to be easily attachable to a heterologous gene, for instance by proximity to a multiple cloning site.
It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms “substantially the amino acid/nucleotide/peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-19.
Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:—(i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g., functional form and constants.
Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity. For protein alignments: Gap Open Penalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.
Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:—Sequence Identity=(N/T)*100.
Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridises to the sequences shown or their complements, under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3× sodium chloride/sodium citrate (SSC) at approximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:—
Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores coupled with their desiccation resistance make them suitable for delivery to humans or to animals by the oral route. Despite these attributes one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. The inventors have addressed the biological containment of spore GMOs by utilizing the concept of a ‘thymine-less death’, a phenomenon first reported six decades ago but which has never been successfully put into practice for introducing heterologous genes into spore-forming bacteria. Using B. subtilis the inventors have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB using a two-step process. Insertion first at thyA followed by thyB where insertion generates resistance to trimethoprim enabling selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant bacteria or vegetative cells have a strict dependence on thymine or thymidine and in their absence cells lyse and die. Insertions are stable and no evidence for suppression or reversion was evident. Using this system the inventors have successfully created a number of spore vaccines as well as spores displaying active enzymes.
PY79 is a prototrophic strain of B. subtilis derived from the type strain 168 (28). PP108 (amyE::cotC-tcdA26-39 thrC::cotB-tcdA26-39) has been described elsewhere (4). SH250 is a prototrophic derivative of PY79 carrying the cat gene (encoding resistance to chloramphenicol) inserted at the amyE locus. General methods for work with B. subtilis including the ‘two-step transformation procedure’ were performed as described previously (29). DSM (Difco Sporulation Medium) is a standard medium for growth and sporulation of B. subtilis (30). For western blotting purified spores were prepared and 2×108 suspended in 40 μl Bolt LDS buffer (Life Tech.) and incubated at 95° C. for 10 min. The spore suspension was centrifuged (10 min. 18,000 g) and 20 μl of supernatant run on a 12% SDS-PAGE gel.
pThy Vectors
pThyA (4,274 bp) carried a 1,910 bp segment comprising the left (900 bp) and right (950 bp) arms of the B. subtilis thyA gene surrounding a multiple cloning site (MCS) cloned into pMA-RQ (2,556 bp; Genscript, USA). Both arms carried additional proximal and distal DNA sequences adjacent to thyA. Similarly, pThyB1(4,973 bp) carried a 2,057 bp segment comprising the left (900 bp) and right (1.1 kb) arms of the B. subtilis thyB gene surrounding a MCS cloned into pBluescript SK(+) (2,958 bp). Plasmids carried the bla gene and the nucleotide sequences of the thyA and thyB segments are given in SEQ ID NO: 6 and SEQ ID NO: 10 (respectively) and shown schematically in
Construction of B. subtilis strains carrying thyA and thyB insertions The procedure developed here consisted of two steps. In the first stage cells of a wild-type recipient strain (in the work described here the prototrophic strain PY79 was used) were made competent using a ‘two-step transformation’ procedure described by Dubnau and Davidoff-Abelson (33) and in common use in Bacillus labs (33, 34). pThyA plasmids carrying the chimeric gene were linearised with either ApaLI or ScaI digestion and cells plated on SMM (Spizizen's Minimal Medium (SMM) (29) agar supplemented with thymine (50 μg/ml) and trimethoprim (3 μg/ml). After 72 h-96 h of growth single colonies were colony purified and assessed for growth at 37° C. and 46° C. on SMM agar±thymine (50 μg/ml)+trimethoprim (3 μg/ml). Cells carrying an insertion at the thyA locus would grow at 37° C. with or without thymine but were unable to grow at 46° C. unless supplemented with thymine. A further verification was to amplify, by PCR, the presence of the chimeric gene from transformants using primers annealing to the thyA sequences. Typically transformation frequencies were about 2×103/μg of competent cells with about 15-20% of colonies carrying the correct insertion (see later for explanation). In the second stage, a linearised (ApaL1 or Sca1) pThyB plasmid carrying a chimeric gene was introduced into cells of the thyA insertion strain by electroporation. Electroporated cells were plated on SMM+CAA (SMM containing 0.2% (w/v) casamino acids (CAA)) containing thymine (50 μg/ml) and trimethoprim (6 μg/ml) and incubated at 37° C. for 48 h. To confirm the presence of both a thyA and thyB insertion colonies were streaked on SMM+CAA agar±thymine (50 μg/ml) and grown at 37° C. Cells carrying two insertions were unable to grow at both 37° C. and 46° C. unless supplemented with thymine. A final verification was made using PCR primers that amplified the two insertions. Using electroporation, integration frequencies were about 1×103/μg of linear DNA with ˜20% of trimethoprim resistance colonies carrying two insertions (thyA and thyB).
The procedure used here was modified from established methods (35) for electroporation in Bacillus primarily with the use of SOC2 medium that contained no yeast extract. SOC2 was tryptone (2% w/v), NaCl (10 mM), KCl (2.5 mM), MgCl2 (5 mM), MgSO4.7H2O (5 mM) and glucose (20 mM). An overnight culture of the strain carrying a thyA insertion was sub-cultured in 25 ml of SOC2 medium (supplemented with 0.5M sorbitol) to give a starting OD600 of 0.2. The culture was grown at 37° C. to an OD600 of 1.4, cooled on ice for 10 min. and then harvested by centrifugation at 4° C. (5,000×g, 5 min.). Cells were washed 4-times in ice-cold electroporation solution (0.5M sorbitol, 0.5M mannitol, 10% (v/v) glycerol) and suspended in 1.6 ml of the same ice-cold solution. The cells were now electro-competent and ready for immediate use. Cells were kept on ice and used within 30 min. although aliquots could be stored at −80° C. 1 μl (˜50 ng) of linearised plasmid DNA was added to 60 μl of electro-competent cells and the mixture transferred to a pre-chilled cuvette (1 mm gap width) and incubated for 1.5 min. on ice. The cuvette was then placed inside the electroporator (BioRad GenePulser Xcell) and the following parameters used for electroporation: Voltage 2, 100V, resistance 200 W, time, 5 milliseconds and number of pulses, 1. After electroporation 1 ml of recovery medium (SOC2 medium containing 0.5M sorbitol and 0.38M mannitol) was added to the cuvette and the mixture transferred to a 2 ml Eppendorf and incubated for 3 h in 37° C. after which cells were serially diluted and plated on appropriate selective media.
An ELISA method was used to detect surface exposed proteins as described previously (36). Microplate wells were coated with 50 μl of a suspension of pure spores (2×108 spores/well) which left overnight at 4° C. Plates were blocked with 2% (w/v) BSA for 1 h at 37° C. Rabbit polyclonal antibodies recognizing either the heterologous antigen expressed on the spore surface or the whole B. subtilis spore were used as primaries with incubation for 2 h at RT. Anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (1:5,000 in PBS plus 0.05% Tween-20) was used as a secondary with 1 h incubation at RT. TMB (3, 3′, 5, 5′-tetramethylbenzidine) was used as substrate.
Polyclonal antibodies to VP28 and TcdA26-39 were raised in rabbits using four sub-cutaneous injections (1 mg/dose, every 14 days). Recombinant proteins were complexed with Freund's adjuvant and serum purified using protein A chromatography. VP26 polyclonals were raised in mice using purified rVP26 protein (4 intra-peritoneal doses at 14-day intervals, 10 μg/dose, with Freund's adjuvant).
For immunogenicity studies mice (C57 Black 6, female, age 7 weeks) were dosed with preparations of pure spores of PY79 or SH14. Immune responses to TcdA26-39 in serum and faecal samples were made as described previously (4). For longevity studies Balb/c mice (female, age 7-8 weeks) were used. Mice (n=5) were administered a single dose of pure spores (2×109) of SH14 or SH250 by oral gavage. At times thereafter freshly voided faeces was collected (3-4 pellets), homogenised and for SH14 faeces serial dilutions plated on (i) DSM and (iii) DSM+trimethoprim (6 μg/ml)+thymine (50 μg/ml) agar plates. SH250 faecal dilutions were plated on DSM plates containing chloramphenicol (5 μg/ml). Individual SH14 colonies were randomly checked for the presence of the thy insertions using PCR.
Polyclonal antibodies (rabbit; 100 μg) raised to rTcdA26-39 protein were biotinylated using the Lightning-Link Rapid biotin conjugation kit type A (Innova Biosciences). Purified spores (1×109) of strain PY79 or spores expressing CotB-SA (SH16) in 200 μl of PBS were mixed with 1 μg of biotinylated antibody and incubated overnight at 4° C. Spores were then washed four-times with PBS and suspended in 1 ml of PBS. ˜3×108 of conjugated spores were used to coat microplate wells which were then probed with an anti-rabbit IgG-horseradish conjugate (1:5,000 in PBS plus 0.05% Tween-20) with 1 h incubation at RT followed by three washes before TMB (3,3′,5,5′-tetramethylbenzidine) colour development. As a control PY79 spores were taken through the same procedure. For immunoflourescence 5×106 of treated spores of SH16 or PY79 were added to microscope slides and allowed to air dry. After three washes with PBS slides were blocked with PBS containing 2% (w/v) BSA plus 0.05% (v/v) Tween-20 for 45 min. at 37° C. Biotinylated anti-TdA26-39 antibodies (1:300 diln.; 200 μl) were added to slides, incubated for 30 min. at RT and then washed 3-times with PBS+0.05% (v/v) Tween-20. Rabbit FITC serum (Sigma F0382 at 1:200 diln.) was added and slides incubated for 30 min. at RT. Image analysis was done with an EVOS fl LED microscope.
C. difficile strain RT176 (tcdA+ tcdB+) was grown in TY broth (3% (w/v) tryptose, 2% (w/v) yeast extract and 0.1% (w/v) sodium thiodlycolate) for 24 h at 37° C. The cell free supernatant was filter-sterilised and kept at 4° C. till assay. The minimum lethal concentration (MLC) of supernatant required to cause 100% toxicity to HT29 cells was determined using 2-fold dilution and addition of the diluted lysate to HT29 cells followed by assessment of toxicity using a cell rounding assay (4). For the assay 109 pure spores of SH16 spores conjugated to TcdA26-39 polyclonal antibodies (see above) were added to 200 μl of 2% McCoy's medium containing the MLC of toxins (typically a 1/4000 dilution). The mixture was incubated for 5 min. at RT and then cytotoxicity assessed using HT29 cells and incubation for 24 h. As a control PY79 spores that had been mixed with TcdA26-39 antibodies (see above) were used in parallel.
Protease activity was determined using the Universal Protease Assay described by Sigma Aldrich on their web site using casein as a substrate. Casein agar was 1% (w/v) casein, 1% (w/v) skimmed milk, 1% (w/v) and 1.2% (w/v) agar technical No. 2, Oxoid). Amylase activity in liquid was measured as described (37). Production of active amylase was tested by applying suspensions of spores (volume of 20 μl) to agar plates carrying only soluble starch (1% w/v) and beef extract (0.3% w/v) and three antibiotics (trimethoprim 10 μg/ml, chloramphenicol 30 μg/ml and erythromycin 30 μg/ml). Antibiotics were used to prevent any bacterial growth on the plates ensuring activity arose from dormant spores only. Plates were incubated 48 h at 37° C. after which the plate was flooded with Lugol solution (Sigma) for 2 min. to reveal zones of starch degradation.
An absolute requirement for thymine in B. subtilis requires two thymidylate synthases (TSase) encoded by the unlinked thyA (TSaseA) and thyB (TSaseB) genes (27). These genes are linked to the folate pathway and provide pyrimidines for cell growth (FIG. 14). TSaseB is thermo-sensitive and retains only ˜5-8% activity at a restrictive temperature of 46° C. Thus, inactivation of the thyA locus requires supplementation with thymine (or thymidine) for growth at 46° C. TSaseA is not thermo-sensitive and so inactivation of thyB allows cells to grow at an elevated temperature. Inactivation of both thyA and thyB however, produces an absolute requirement on thymine for growth at both 37° C. and 46° C. As shown by Neuhard et al (27) inactivation of the thy genes produces resistance to the anti-folate drug trimethoprim (or aminopterin) since the need for dihydrofolate reductase (DHFR), the target for trimethoprim, is dispensed with. However, the level of resistance differs, with insertion at thyA producing a lower level of resistance than that found in a thyA thyB mutant (27). This attribute of differential resistance to trimethoprim enables a novel, two-step, ectopic cloning system to be designed (
To demonstrate proof of concept for ectopic cloning at the thy loci the inventors chose a number of heterologous genes whose products had previously been expressed on the spore surface. In each case expression had been achieved by fusion to a B. subtilis gene encoding a surface expressed spore coat protein (either CotB or CotC). The proteins chosen were VP26 and VP28, both envelope proteins of the shrimp virus WSSV (White Spot Syndrome Virus). When displayed on B. subtilis spores and incorporated in feed these recombinant spores have been shown to confer protection to shrimps challenged with WSSV (7, 8, 9). (ii) TcdA26-39 encoding a C-terminal domain of Clostridium difficile toxin A (TcdA) expressed on the spore surface has been shown to confer protection to C. difficile infection (CDI) in hamsters (4, 38) dosed orally with these recombinant spores. (iii) Streptavidin (SA) when expressed on spores can be conjugated to the monoclonal antibody Cetuximab enabling targeting to colon cancer cells (15). (iv) Finally, two enzymes, subtilisin E (AprE), an alkaline protease and alpha amylase (AmyE) which are both enzymes of industrial importance and commonly incorporated in animal feed (32, 39).
Two plasmids, pThyA and pThyB, were designed and synthesised that carry the complete thyA or thyB genes interrupted midpoint with a multiple cloning site (MCS) for insertion of heterologous DNA (
The inventors examined growth of the insertions in minimal SMM medium at 37° C. and
In addition, using whole genome sequencing the inventors confirmed that strain SH12 (thyA::cotC-vp26 thyB::cotB-vp28) and SH14 (thyA::cotB-tcdA26-39thyA::cotC-tcdA26-39) carried the specific insertions at the thy loci without any chromosomal abberations (not shown). Expression of chimeric proteins was confirmed by Western blotting of proteins extracted from purified spores (
Both thymine and thymidine could be used for growth of strains carrying insertions in the thyA and thyB loci. Using SH14 the inventors showed that for optimal growth 15 μg/ml of thymine or 20 μg/ml of thymidine was sufficient (
1growth (+) or no growth (−) on SMM agar with or without thymine (50 μg/ml)
2minimal inhibitory concentration (MIC; μg/ml) of trimethoprim (Tm) determined using a microdilution method (52).
3% of heat-resistant spores in cultures grown overnight in DSM medium (+thymine) at 37° C. TC, total count cfu/ml;
Finally, using this cloning system the inventors were able to introduce insertions that were of the same or opposed transcriptional polarity to the respective thy gene (
In rich medium such as LB and DSM the single thyA insertion strain was able to grow at 37° C. without thymine supplementation while the thyA thyB double insertion showed no growth however (
Since the thyA thyB insertion strain could grow, albeit with reduced fitness, in minimal medium supplemented with thymine (see
The inventors grew strains (thy+ and thyA thyB) in LB+thymine with varying levels of YE and found that YE concentrations of ≥2 mg/ml inhibited growth of strains carrying two thy (thyA+thyB) insertions (
As mentioned earlier DNA-mediated transformation of competent cells could be used to introduce plasmid DNA into B. subtilis cells. However, for introducing DNA into thyA cells in the 2nd step, electroporation using a modified medium lacking YE yielded higher frequencies of integration.
The inventors found that following the 1st genetic cross two colony types, in equal proportion, were apparent on SMM agar supplemented with thymine and trimethoprim (3 μg/ml) (
A second important finding was that for the 2nd genetic transfer recombinants could only be selected on SMM minimal media supplemented with thymine, trimethoprim (6 μg/ml) and CAA. If plated directly onto agar lacking CAA small (<1 mm), slow growing colonies would result but after reculture these were found to have lost the thyB insertion as determined by colony PCR. Even in the presence of CAA all colonies were small and only about 20% of those growing on trimethoprim (6 μg/ml) carried a stable thyB insertion. Work in E. coli as well as B. subtilis has shown that disruption of the folate pathway can lead to depletion of key amino acids as well as purines and pyrimidines (43, 44). Trimethoprim-mediated inactivation of DHFR would deplete intracellular levels of THF, MTHF as well as DHF. In turn this would affect the reversible interconversion of serine and glycine with tetrahydrofolic acid (THF), a vital reaction in the synthesis of purines and catalysed by a serine hydroxymethyltransferase (SHMT or GlyA, (45, 46)). MTHF is also utilised in the final step of the biosynthetic pathways of cysteine and methionine (47) and disruption of the pathway by the thyA thyB alleles could introduce a requirement for methionine.
The inventors measured the growth of a thyA thyB insertion strain SH14 in SMM supplemented with CAA, adenine and thymine (
Therefore, for the second genetic transfer step (i.e., for construction of the double, thyA thyB, insertion) selective media may preferably provide all amino acids. However, once constructed the use of trimethoprim is no longer required and strains can be cultivated on any media so long as three criteria are met, first, that the media contains thymine or thymidine, second, YE is either absent or at a concentration less than 2 mg/ml, and third, amino acids are provided in the growth medium.
For expression of heterologous proteins on the spore surface the coat proteins CotB and CotC can be used for both mono or divalent expression. As expected, when using two different spore coat anchors expression of a protein was higher than using expression from only one. So, as shown in
An insertion generated by a double-crossover recombinational event should be inherently stable yet there exists the possibility of acquisition of a compensatory suppressor or bypass mutation. To address this the inventors conducted an experiment to determine whether upon repeated culture in the absence of any selective pressure the thymine dependence could be lost. As shown in Table 2 repeated culture and reculture of SH14 (thyA::cotB-tcdA26-39 thyA::cotC-tcdA26-39) yielded no loss of the thymine dependency showing the insertions were stable and suggesting that the acquisition of compensatory mutations, if they were to occur, must be an extremely rare event.
1Two 25 ml cultures of SH14 (thyA::cotB-tcdA26-39 thyA::cotC-tcdA26-39) were made in DSM + thymine (50 μg/ml). The culture was grown for 24 h at 37° C. and a sample analyzed for CFU on either DSM agar with or without thymine (50 μg/ml). A sample was also used to subculture fresh medium (25 ml) and the process repeated four times. CFU/ml for each reculture are shown.
In Vivo Fate of thyA− thyB− Spores in the GI-Tract
It has been demonstrated that for E. coli to colonise the murine GI-tract synthesis of purines and pyrimidines is necessary (48). This implies that the low levels of purines and pyrimidines that might result from digested food or from spurious lysis of resident gut microbiota would not be sufficient to permit growth of a B. subtilis thyA thyB mutant. In humans the intestinal concentration of thymidine is estimated as 0.075 μM and in pigs ˜1.0 μM (49). The inventors gave mice a single oral dose of 2×109 spores of SH250 (thyA+ thyB+ CmR) or SH14 (thyA::cotB-tcdA26-39 thyA::cotC-tcdA26-39) spores by intra-gastric gavage. Subsequent shedding of heat-resistant spores in freshly voided faeces was determined. For SH250 the strain carried a silent insertion of the cat gene (chloramphenicol acetyltransferase) so after serial dilution heat resistant CFU was determined on chloramphenicol plates. SH14 spores were shed in the faeces and could only be detected on agar plates supplemented with thymine but when plated on plates lacking thymine no CFU could be detected (
The inventors used three examplars to demonstrate that spores carrying insertions at the thyA and thyB loci were suitable for applied purposes, as vaccine delivery vehicles, for conjugation of proteins to the spore surface and finally for display of active enzymes. As vaccines the inventors used SH14 spores that express the TcdA26-39 antigen of C. difficile fused to two spore coat protein anchors, CotB and CotC. SH14 is equivalent to strain PP108 that has previously been shown to confer protection to C. difficile infection (CDI) in murine and hamster models of infection (4, 38). The inventors immunised mice with spores of SH14 and PP108 using oral administration and after a total of four doses measured TcdA26-39-specific IgG (
For conjugation of proteins to streptavidin displayed on the spore surface the inventors first biotinylated a rabbit polyclonal TdA26-39-specific antibody and demonstrated that the antibody conjugated to SH16 spores using immunofluorescence detection (
1Cytoxicity in HT29 cells was measured using crude toxins from C. difficile or from the same toxins pretreated with SH16 spores conjugated with a polyclonal antibody to TcdA26-39 or PY79 treated spores.
Enzymes are commonly included in animal feeds where they improve digestion and nutrition. Proteases and amylases are pertinent examples used here to show that it is possible to display an enzyme that retains activity on the spore surface. SH18 spores expressing amylase were found able to express active amylase on their surface (
The inventors have described a straightforward method to contain genetically modified bacterial spores. Their approach expands yet differs from those described for Lactobacillus acidophilus (51) and Lactococcus lactis (Steidler et al., 2003) that rely on the indigenous suicide resulting from a ‘thymine-less death’. First described in 1954 (23) thymine dependence differs from other auxotrophies in that the absence of thymine is bactericidal and so bacteria carrying defects in the thymidylate synthase genes cannot accumulate in the environment. Bacillus species carry two thymidylate synthase genes (thyA and thyB) requiring inactivation of both loci to achieve complete dependence on thymine. The inventors have shown here that this is possible and have identified a two-step cloning procedure requiring insertional inactivation of first thyA and then thyB loci. Their approach does not require the introduction of antibiotic resistance markers for selection but rather the development of increasing levels of resistance to trimethoprim that arise from successive disruption of the folate pathway. Coupled with the temperature sensitive phenotype of insertions at the thyA and thyB loci this provides a useful method for both selection and screening of insertion although technically there are a number of constraints that must be considered. The inventors show that the absence of thymine is bactericidal and observe no evidence for reversion or suppression despite repeated passage of these strains. Of course, the purpose of the cloning system is to construct strains able to express proteins for applied purposes, for example, for expression of heterologous antigens or enzymes. The inventors have used examples here showing that chimeric proteins comprised of a foreign protein fused to a spore coat protein can be displayed on the spore surface. This has included the delivery of two enzymes (subtilisin E and α-amylase), putative vaccine protective antigens as well as streptavidin which was used to conjugate to a polyclonal antibody. It is clear though that this system could equally be used for expression of proteins in, or secretion from, the vegetative cell.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1718133.0 | Nov 2017 | GB | national |
This application is a 371 National Stage filing and claims the benefit under 35 U.S.C. § 120 to International Application No. PCT/GB2018/053178, filed Nov. 1, 2018, which claims priority to Great Britain Application No. 1718133.0, filed Nov. 2, 2017, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/053178 | 11/1/2018 | WO | 00 |
