MODIFIED BACTERIAL SPORES
20200354727 ยท 2020-11-12
Inventors
Cpc classification
International classification
Abstract
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.
Claims
1. 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: (i) introducing a heterologous gene into a thymidylate synthase gene within a spore-forming bacterium; and (ii) growing said spore-forming bacterium in the presence of trimethoprim.
2. The process of claim 1, wherein the spore-forming bacterium is (i) Bacillus Spp., preferably B. subtilis, or (ii) Clostridium Spp., preferably C. difficile.
3. The process claim 1, wherein: step (i) is the introduction of a heterologous gene into the thyA gene and the thyB gene in a Bacillus Spp.
4. The process of claim 1, wherein: step i) is the introduction of a heterologous gene into either the thyA gene or the thyB gene in a Bacillus Spp. and preferably further comprising: (iii) introducing a heterologous gene into the other of the thyA gene or the thyB gene in said Bacillus Spp.; and (iv) growing said Bacillus Spp. in the presence of trimethoprim.
5. (canceled)
6. The process of claim 4, wherein: 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.
7. The process of claim 1, wherein: during step (ii), the spore-forming bacterium is grown in a medium comprising yeast extract (YE) at a concentration lower than 2 mg/ml, 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, preferably the medium in which the spore-forming bacterium is grown contains no YE.
8. The process of claim 4, wherein: during step (iv), the Bacillus Spp. is grown in a medium comprising yeast extract (YE) at a concentration lower than 2 mg/ml, 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, preferably the medium in which the Bacillus Spp. is grown contains no YE.
9. The process of claim 1, wherein: during step (ii), the spore-forming bacterium is grown in a medium comprising thymine or thymidine.
10. The process of claim 4, wherein: during step (iv), the Bacillus Spp. is grown in a medium comprising thymine or thymidine.
11. The process of claim 4, wherein: the amount of trimethoprim present in step (iv) is higher than the amount of trimethoprim present in step (ii).
12. The process of claim 5, wherein cells carrying an insertion at the thyA locus are 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.
13. The process of claim 1, wherein: YE is present at a concentration lower than 2 mg/ml, or preferably not present, in the media used at any stage of the process.
14. The process of claim 4, wherein: during step (iv), the Bacillus Spp. is grown in a medium supplemented with at least one amino acid, preferably wherein: during step (iv), the Bacillus Spp. is grown in a medium comprising casamino acids (CAA).
15. (canceled)
16. The process of claim 2, wherein: cells carrying two insertions are verified by the inability to grow at 37 C. or 46 C. unless supplemented with thymine or thymidine.
17. (canceled)
18. The process of claim 1, further comprising: forming a bacterial spore from said spore-forming bacterium.
19. A bacterial spore obtained or obtainable by the process of claim 1, preferably wherein the bacterial spore is Bacillus spp. and comprises non-functional thyA and thyB genes, preferably due to the introduction of heterologous genes therein.
20. (canceled)
21. 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: (i) introducing a heterologous gene into a thymidylate synthase gene within a spore-forming bacterium; (ii) growing said spore-forming bacterium in the presence of trimethoprim; and (iii) forming a bacterial spore from said spore-forming bacterium.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
Description
[0144] 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:
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EXAMPLES
[0164] 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.
Materials and Methods
Strains, Media and General Methods
[0165] PY79 is a prototrophic strain of B. subtilis derived from the type strain 168 (28). PP108 (amyE::cotC-tcdA.sub.26-39 thrC::cotB-tcdA.sub.26-39) 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 210.sup.8 suspended in 40 l 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 l of supernatant run on a 12% SDS-PAGE gel.
pThy Vectors
[0166] 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
[0167] 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 g/ml) and trimethoprim (3 g/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 agarthymine (50 g/ml)+trimethoprim (3 g/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 210.sup.3/g 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 g/ml) and trimethoprim (6 g/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 agarthymine (50 g/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 110.sup.3/g of linear DNA with 20% of trimethoprim resistance colonies carrying two insertions (thyA and thyB).
Electroporation
[0168] 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), MgCl.sub.2 (5 mM), MgSO.sub.4.7H.sub.2O (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 OD.sub.600 of 0.2. The culture was grown at 37 C. to an OD.sub.600 of 1.4, cooled on ice for 10 min. and then harvested by centrifugation at 4 C. (5,000g, 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 l (50 ng) of linearised plasmid DNA was added to 60 l 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.
Whole-Spore ELISA
[0169] An ELISA method was used to detect surface exposed proteins as described previously (36). Microplate wells were coated with 50 l of a suspension of pure spores (210.sup.8 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.
Antibodies
[0170] Polyclonal antibodies to VP28 and TcdA.sub.26-39 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 g/dose, with Freund's adjuvant).
Animal Studies
[0171] 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 TcdA.sub.26-39 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 (210.sup.9) 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 g/ml)+thymine (50 g/ml) agar plates. SH250 faecal dilutions were plated on DSM plates containing chloramphenicol (5 g/ml). Individual SH14 colonies were randomly checked for the presence of the thy insertions using PCR.
Conjugation to Streptavidin
[0172] Polyclonal antibodies (rabbit; 100 g) raised to rTcdA.sub.26-39 protein were biotinylated using the Lightning-Link Rapid biotin conjugation kit type A (Innova Biosciences). Purified spores (110.sup.9) of strain PY79 or spores expressing CotB-SA (SH16) in 200 l of PBS were mixed with 1 g of biotinylated antibody and incubated overnight at 4 C. Spores were then washed four-times with PBS and suspended in 1 ml of PBS. 310.sup.8 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 510.sup.6 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-TdA.sub.26-39 antibodies (1:300 diln.; 200 l) 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.
Toxin Subtraction Assays
[0173] C. difficile strain RT176 (tcdA.sup.+ tcdB.sup.+) 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 10.sup.9 pure spores of SH16 spores conjugated to TcdA.sub.26-39 polyclonal antibodies (see above) were added to 200 l 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 TcdA.sub.26-39 antibodies (see above) were used in parallel.
Enzyme Activity
[0174] 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 l) to agar plates carrying only soluble starch (1% w/v) and beef extract (0.3% w/v) and three antibiotics (trimethoprim 10 g/ml, chloramphenicol 30 g/ml and erythromycin 30 g/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.
Example 1
Rationale
[0175] 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 (
[0176] 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) TcdA.sub.26-39 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).
A Two-Step Method for Ectopic Gene Insertion
[0177] 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 (
[0178] The inventors examined growth of the insertions in minimal SMM medium at 37 C. and
[0179] In addition, using whole genome sequencing the inventors confirmed that strain SH12 (thyA::cotC-vp26 thyB::cotB-vp28) and SH14 (thyA::cotB-tcdA.sub.26-39thyA::cotC-tcdA.sub.26-39) 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 (
[0180] 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 g/ml of thymine or 20 g/ml of thymidine was sufficient (
TABLE-US-00013 TABLE 1 Phenotypes of B. subtilis recombinant strains 37 C..sup.1 46 C..sup.1 Tm Sporulation.sup.3 Strain Genotype thy +thy thy +thy MIC.sup.2 TC Spores % PY.sub.79 thyA.sup.+ thyB.sup.+ + + + + 0.25 2.8 10.sup.8 2.4 10.sup.8 85.7 SH.sub.11 thyA::cotC-vp26 + + + 16 1.9 10.sup.8 1.6 10.sup.8 82 SH.sub.12 thyA::cotC-vp26 thyB::cotB-vp28 + + >64 2 10.sup.8 1.5 10.sup.8 75 AC.sub.01 thyA::cotB-vp28 + + + 16 2.6 10.sup.8 2.2 10.sup.8 85 AC.sub.02 thyA::cotB-vp28 thyB::cotB-vp28 + + >64 1.9 10.sup.8 1.5 10.sup.8 78.6 SH.sub.13 thyA::COth-tedA.sub.26-39 + + + 16 2.2 10.sup.8 1.8 10.sup.8 85 SH.sub.14 thyA::cotB-tcdA.sub.26-39 thyB::cotC-tcdA.sub.26-39 + + >64 2.5 10.sup.8 1.9 10.sup.8 77.2 SH.sub.15 thyA::cotB-SA + + + 16 2.8 10.sup.8 2.3 10.sup.8 82 SH.sub.16 thyA::cotB-SA thyB::cotB-SA + + >64 3.1 10.sup.8 2.4 10.sup.8 77.4 SH.sub.17 thyA::cotB-amyE + + + SH.sub.18 thyA::cotB-amyE thyB::MCS + + SH.sub.19 thyA::cotB-aprE + + + 16 8.7 10.sup.8 5.5 10.sup.8 90 SH.sub.20 thyA::cotB-aprE thyB::MCS + + >64 8.1 10.sup.7 6.8 10.sup.7 83.9 .sup.1growth (+) or no growth () on SMM agar with or without thymine (50 g/ml) .sup.2minimal inhibitory concentration (MIC; g/ml) of trimethoprim (Tm) determined using a microdilution method (52). .sup.3% of heat-resistant spores in cultures grown overnight in DSM medium (+thymine) at 37 C. TC, total count cfu/ml; Spores, heat-resistant spore count cfu/ml.
[0181] 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 (
Example 2
Experimental Considerations for Use of Ectopic Insertion at the Thy Loci
a) Growth in Rich Media
[0182] 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 (
[0183] Since the thyA thyB insertion strain could grow, albeit with reduced fitness, in minimal medium supplemented with thymine (see
[0184] The inventors grew strains (thy.sup.+ 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 (
b) Gene Transfer
[0185] 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 2.sup.nd step, electroporation using a modified medium lacking YE yielded higher frequencies of integration.
[0186] The inventors found that following the 1.sup.st genetic cross two colony types, in equal proportion, were apparent on SMM agar supplemented with thymine and trimethoprim (3 g/ml) (
[0187] A second important finding was that for the 2.sup.nd genetic transfer recombinants could only be selected on SMM minimal media supplemented with thymine, trimethoprim (6 g/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 g/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.
[0188] The inventors measured the growth of a thyA thyB insertion strain SH14 in SMM supplemented with CAA, adenine and thymine (
[0189] 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.
c) Choice of One Coat Protein Anchor
[0190] 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
Example 3
Reversion
[0191] 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-tcdA.sub.26-39 thyA::cotC-tcdA.sub.26-39) 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.
TABLE-US-00014 TABLE 2 Reversion.sup.1 CFU Culture 1 CFU Culture 2 DSM + DSM + Culture thymine DSM thymine DSM 1st 2.8 10.sup.8 0 2.1 10.sup.8 0 2nd 1.8 10.sup.8 0 3.4 10.sup.8 0 3rd 1.85 10.sup.8 0 1.73 10.sup.8 0 4th 1.82 10.sup.8 0 1.6 10.sup.8 0 5th 1.54 10.sup.8 0 2.4 10.sup.8 0 .sup.1Two 25 ml cultures of SH14 (thyA::cotB-tcdA.sub.26-39 thyA::cotC-tcdA.sub.26-39) were made in DSM + thymine (50 g/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 g/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.
Example 4
[0192] In Vivo Fate of thyA.sup. thyB.sup. Spores in the GI-Tract
[0193] 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 M and in pigs 1.0 M (49). The inventors gave mice a single oral dose of 210.sup.9 spores of SH250 (thyA.sup.+ thyB.sup.+ Cm.sup.R) or SH14 (thyA::cotB-tcdA.sub.26-39 thyA::cotC-tcdA.sub.26-39) 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 (
Example 5
Utility of Spore Display
[0194] 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 TcdA.sub.26-39 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 TcdA.sub.26-39-specific IgG (
[0195] For conjugation of proteins to streptavidin displayed on the spore surface the inventors first biotinylated a rabbit polyclonal TdA.sub.26-39-specific antibody and demonstrated that the antibody conjugated to SH16 spores using immunofluorescence detection (
TABLE-US-00015 TABLE 3 Subtraction of C. difficile toxins.sup.1 Mixture Cytotoxicity (%) Medium only 0 Toxins only 100 PY79 treated spores + toxins 80-90 SH16 conjugated spores + toxin 10 .sup.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 TcdA.sub.26-39 or PY79 treated spores.
[0196] 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 (
Summary of Examples 1 to 5
[0197] 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 -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.
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