Modified bacteriophage

Abstract

Provided is a modified bacteriophage capable of infecting a target bacterium, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacterium, wherein the SASP gene is under the control of a constitutive promoter which is foreign to the bacteriophage and the SASP gene.

Claims

1. A process for the production of a modified bacteriophage comprising growing a bacterial host comprising genetic material encoding a modified bacteriophage, wherein said modified bacteriophage is able to replicate in said bacterial host; causing the bacteriophage to replicate in the bacterial host; and harvesting the bacteriophage, wherein said modified bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the bacterial host, wherein the SASP gene is inserted into a lysis gene of said bacteriophage, wherein the SASP gene is under the control of a constitutive bacterial promoter which is foreign to the bacteriophage and the SASP gene and wherein the phage comprises a single copy of the SASP gene linked to said constitutive bacterial promoter.

2. The process according to claim 1, wherein the SASP comprises Bacillus megaterium SASP-C.

3. The process according to claim 1, wherein said bacteriophage comprises a modified Staphylococcus aureus bacteriophage.

4. The process according to claim 3, wherein the Staphylococcus aureus bacteriophage is a ϕ11 bacteriophage.

5. The process according to claim 1, wherein the promoter is selected from pdhA, rpsB, pgi, and fbaA.

6. The process according to claim 5, wherein the bacterial fbaA promoter is from S. aureus.

7. The process according to claim 1, wherein said bacteriophage is non-lytic.

8. The process according to claim 7, wherein said bacteriophage is holin.sup.-.

9. The process of claim 1, wherein said promoter drives production of toxic levels of SASP when present in multiple copies in a target bacterium.

10. The process according to claim 1, wherein said bacteriophage further comprises a non-antibiotic resistance marker.

11. The process according to claim 10, wherein the non-antibiotic resistance marker is a cadmium resistance marker.

12. The process of claim 1, further comprising admixing said modified bacteriophage with a carrier.

13. A process for the production of a modified bacteriophage comprising growing a bacterial host comprising genetic material encoding a modified bacteriophage, wherein said modified bacteriophage is able to replicate in said bacterial host; causing the bacteriophage to replicate in the bacterial host; and harvesting the bacteriophage, wherein said bacteriophage comprises a ϕ11 bacteriophage having a holin gene into which is inserted a gene encoding Bacillus megaterium SASP-C under the control of an fbaA constitutive promoter.

14. The process according to claim 13, wherein the bacterial fbaA promoter is from S. aureus.

15. The process according to claim 13, wherein said bacteriophage is non-lytic.

16. The process of claim 13, wherein said promoter drives production of toxic levels of SASP when present in multiple copies in a target bacterium and wherein the bacteriophage contains a single copy of the SASP gene functionally linked to said fbaA constitutive bacterial promoter.

17. The process according to claim 13, wherein said bacteriophage further comprises a non-antibiotic resistance marker.

18. The process according to claim 17, wherein the non-antibiotic resistance marker is a cadmium resistance marker.

19. The process of claim 13, further comprising admixing said modified bacteriophage with a carrier.

20. The process of claim 1, wherein the constitutive promoter is a bacterial promoter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Region of S. aureus phage ϕ11, showing the holin and amidase genes, and the priming sites for amplification of the genes and flanking DNA

(2) FIG. 2. Diagram of pSA1, showing the cloned region and the location of the priming sites for inverse PCR of pSA1.

(3) FIG. 3. Diagram of pSA4, showing the cloned promoter-saspC region with the cadmium resistance (Cd.sup.R) gene and the flanking ϕ11 DNA, together with the location of relevant priming sites.

(4) FIG. 4. Arrangement of DNA within the genome of PTL1003, showing the replacement of the holin gene with foreign genes. The arrangement of genes in the wild-type ϕ11 genome is shown for comparison.

(5) FIG. 5. An example of a kill curve showing efficacy of PTSA1.2/A against an S. aureus strain.

(6) FIG. 6. A kill curve comparing the killing ability of PTSA1.2/A with the same phage minus the SASP gene.

(7) FIG. 7. A kill curve of PTSA1.2/A infecting an S. aureus strain.

SUMMARY OF CONSTRUCTION OF A GENETICALLY ALTERED BACTERIOPHAGE CARRYING SASP-C UNDER CONTROL OF A FRUCTOSE BISPHOSPHATE ALDOLASE HOMOLOGUE (FBAA) PROMOTER

(8) Genes can be removed and added to the phage genome using homologous recombination. There are several ways in which phages carrying foreign genes and promoters can be constructed and the following is an example of such methods.

(9) For the construction of a ϕ11 derivative it is shown how, using an E. coli/S. aureus shuttle vector, as an example only, the phage holin gene has been replaced with the gene for SASP-C, under the control of a S. aureus fructose bisphosphate promoter homologue (fbaA is used from this point on to denote the fructose bisphosphate aldolase promoter). Genes for resistance to the heavy metal Cadmium (referred to henceforth as Cd.sup.R) are used as a non-antibiotic resistance marker.

(10) The fbaA-SASP-C and Cd.sup.R regions were cloned between two regions of ϕ11 DNA which flank the ϕ11 holin gene. Subsequently, this plasmid was introduced into cells and double recombinants were selected for, where the holin was replaced with the fbaA-SASP-C and Cd.sup.R region.

EXPERIMENTAL PROCEDURES

(11) All PCR reactions were performed using Expand High Fidelity PCR system and stringent conditions, depending upon the melting temperatures (T.sub.m) of the primers, according to the manufacturers instructions. Unless otherwise stated, general molecular biology techniques, such as restriction enzyme digestion, agarose gel electrophoresis, T4 DNA ligase-dependent ligations, competent cell preparation and transformation were based upon methods described in Sambrook et al. (1989). DNA was purified from enzyme reactions and prepared from cells using Qiagen DNA purification kits. S. aureus cells were transformed with plasmid DNA by electroporation, using methods such as those described by Schenck and Ladagga (1992).

(12) Primers were obtained from Sigma Genosys. Where primers include recognition sequences for restriction enzymes, an extra 2-6 nucleotides was added at the 5′ end to ensure digestion of amplified PCR DNA.

(13) All clonings, unless otherwise stated, are achieved by ligating DNAs overnight with T4 DNA ligase and then transforming them into E. coli cloning strains, such as DH5α or XL1-Blue, with isolation on selective medium, as described elsewhere (Sambrook et al., 1989)

(14) An E. coli/S. aureus shuttle vector, designated pSM198 was used to transfer to genes between E. coli and S. aureus. Plasmid pSM198 was previously produced by combining E. coli cloning vector pUC18 and the tetracycline resistance and replication regions of S. aureus plasmid pT181. The plasmid carries resistance markers that can be selected for in E. coli and S. aureus. This plasmid retains the pUC18 multiple cloning site (MCS), although not all the sites remain as unique sites. The remaining unique sites in the MCS of pSM198 are: PstI, SalI, BamHI, SacI and EcoRI.

(15) Construction of a Plasmid for Targeted Replacement of the ϕ11 Holin Gene with fbaA-SASP-C/Cd.sup.R

(16) 1. Plasmid pSA1, comprising pBluescript SK+ containing a 1.8 kb fragment of ϕ11 spanning the lytic genes, was constructed as follows. FIG. 1 shows the priming sites for the oligonucleotides described below for amplification of regions from the ϕ11 genome.

(17) PCR amplification of ϕ11 DNA using primers B1001 and B1002, was carried out and yielded a 1.8 kb fragment which was cleaned and digested with XbaI and PstI. After digestion, the DNA was cleaned and cloned into XbaI and PstI digested pBluescript SK+, yielding pSA1.

(18) Primer B1001 (SEQ ID NO: 1) comprises a 5′ PstI site (underlined) followed by sequence of ϕ11 (Genbank: AF424781) from base 39779 to base 39798, (see FIG. 1). Primer B1002 (SEQ ID NO: 2) comprises an XbaI site (underlined) followed by the reverse and complement of sequence of ϕ11 from base 41537 to base 41556 (see FIG. 1).

(19) TABLE-US-00001 B1001 (SEQ ID NO: 1) 5′-AACTGCAGGTGTATTGCAACAGATTGGCTC-3′ B1002 (SEQ ID NO: 2) 5′-GCTCTAGACTTTGCTCCCTGCGTCGTTG-3′

(20) 2. Inverse PCR was carried out on pSA1 as the template, using primers B1003 (SEQ ID NO: 3) and B1004 (SEQ ID NO: 4) (see FIG. 2).

(21) Primer B1003 comprises a 5′ BamHI site (underlined) followed by the reverse and complement sequence of ϕ11 from base 40454 to base 40469 (see FIG. 1). Primer B1004 comprises a 5′ SpeI site (underlined), followed by sequence of ϕ11 from base 40891 to base 40911 (see FIG. 2).

(22) TABLE-US-00002 B1003 (SEQ ID NO. 3) 5′-CGGGATCCGACTAAAAATTAGTCG-3′ B1004 (SEQ ID NO. 4) 5′-GGACTAGTGAATGAGTATCATCATGGAGG-3′

(23) This PCR reaction yielded an ˜4.2 kb fragment which constituted: ϕ11 left arm, the entire pBluescript SK+ plasmid, and the ϕ11 right arm. This fragment was digested with BamHI and SpeI, cleaned, and subsequently used as a vector to clone in the following fragment.

(24) 3. The cadmium resistance region from pI258 was amplified by PCR using primers B1005 and B1006, yielding an ˜2.8 kb fragment. The PCR product was cleaned and digested with BamHI and XbaI. The digested PCR product was cleaned and cloned into pSA1 (PCR amplified and digested, above), making pSA2.

(25) Primer B1005 (SEQ ID NO: 5) is complementary to DNA 308 bp upstream from the ATG for the putative cadmium-responsive regulatory protein gene cadC from pI258 (Genbank: J04551), the 3′ end being nearest the ATG (see FIG. 3). The 5′ end of the primer carries a non-complementary tail with a BamHI site (underlined) to aid cloning.

(26) Primer B1006 (SEQ ID NO: 6) is complementary to DNA at the 3′ end of the cadA gene for the cadmium resistance protein from plasmid pI258, such that the last 3 complementary nucleotides are complementary to the stop codon TAG of the cadA gene (see FIG. 3). The 5′ end carries a non-complementary XbaI site (underlined) to aid cloning.

(27) TABLE-US-00003 B1005 (SEQ ID NO: 5) 5′-CGATGGATCCTCTCATTTATAAGGTTAAATAATTC-3′ B1006 (SEQ ID NO: 6) 5′-GCAGACCGCGGCTATTTATCCTTCACTCTCATC-3′

(28) 4. The DNA containing the ϕ11 left and right arms and Cd.sup.R were cut out of pSA2 using PstI and SacI, and gel purified away from the vector. This fragment was cloned into shuttle vector pSM198 which was also cut PstI and SacI. Clones were screened for the restriction fragment and candidates were sent for sequencing. A correct plasmid construct was identified and named pSA3. This plasmid was used to clone in the following fragments.

(29) 5. PCR amplification of the fbaA promoter using B1007 and B1008 yielded an approximately 300 bp fragment which was cleaned and subsequently digested with NcoI, then re-cleaned.

(30) The fbaA PCR fragment was ligated to the SASP-C coding sequence from B. megaterium. The amplification and preparation of the SASP-C gene is described below.

(31) Primer B1007 (SEQ ID NO: 7) comprises a 5′ sequence tail which includes a BamHI site, followed by the reverse complement of bases 2189404 to 2189427 from the S. aureus NCTC 8325 genome (Genbank: CP000253) (see FIG. 3).

(32) Oligonucleotide B1008 (SEQ ID NO: 8) comprises a sequence tail which includes an NcoI site, then the sequence of bases 2189214 to 2189232 from the S. aureus NCTC 8325 genome (see FIG. 3). When a PCR product is made using this primer, the NcoI site incorporated into the primer at the ATG of the gene results in the change of the base 2 nucleotides upstream of the ATG from T>C.

(33) TABLE-US-00004 B1007 (SEQ ID NO: 7) 5′-CTACGGATCCTTTATCCTCCAATCTACTTATAAA-3′ B1008 (SEQ ID NO: 8) 5′-CATGCCATGGAAGTTCCTCCTTGAGTGCT-3′

(34) 6. The SASP-C gene from B. megaterium strain KM (ATCC 13632) was amplified by PCR with primers B1009 and B1010 and yielded an ˜300 bp fragment. The PCR product was cleaned and digested with NcoI. The digested PCR product was cleaned and used in a ligation with the fbaA PCR fragment, as described below.

(35) Oligonucleotide B1009 (SEQ ID NO: 9) comprises a 5′ tail containing an NcoI site and is complementary to the first 20 nucleotides of SASP-C (accession no. K01833), starting at the ATG, from B. megaterium strain KM (see FIG. 3). The NcoI site at the beginning of the oligonucleotide incorporates the ATG of the SASP-C gene.

(36) TABLE-US-00005 B1009 (SEQ ID NO: 9) 5′-CGATCCATGGCAAATTATCAAAACGC-3′

(37) Oligonucleotide B1010 (SEQ ID NO: 10) comprises a BglII site (underlined), and an EcoRI site (double underlined), followed by the reverse complement of DNA starting 59 bases downstream of the stop codon to 74 bases downstream of the stop codon of the SASP-C gene (see FIG. 3).

(38) TABLE-US-00006 B1010 (SEQ ID NO: 10) 5′-AGTGAGATCTGAATTCGCTGATTAAAAGAAAC-3′

(39) 7. The fbaA and the SASP-C PCR fragments (both cut NcoI) were ligated together using T4 DNA ligase. The ligated DNAs were used as a template for PCR, to amplify the joined fbaA and SASP-C DNAs. PCR was performed using primers B1007 and B1010. The main PCR product of ˜500 bp was gel purified. The PCR product was digested with BamHI and BglII and cleaned. This fragment was cloned into pSA3 which was prepared as follows. The plasmid was cut with BamHI, and the ends were dephosphorylated using calf intestinal alkaline phosphatase (CIAP). The DNA was cleaned again.

(40) Plasmids were screened so that the end of the SASP-C gene was adjacent to the “left arm” region of ϕ11, and so the start of the fbaA promoter was adjacent to the cadmium chloride resistance region. The resulting plasmid, carrying fbaA-SASP-C, was named pSA4.

(41) Replacement of the Holin Gene from S. aureus Phage ϕ11 with fbaA-SASP-C and the Cd.sup.R Marker

(42) 1. pSA4 was transformed into S. aureus strain PTL47. PTL47 is a monolysogen of ϕ11 in RN4220.

(43) 2. Cells which had undergone a double crossover, where the DNA contained between the ϕ11 left and right arms of pSA4 have replaced the DNA between the ϕ11 left and right arms in the phage genome (ie the holin gene) gave rise to colonies with the following phenotype: CdCl.sub.2 (0.1 mM) resistant, tetracycline (5 μg/ml) sensitive. Tetracycline resistance is carried by the shuttle vector pSM198. Loss of tetracycline resistance is indicative of loss of pSM198. Colonies which had the phentoype: CdCl.sub.2.sup.R, tetracycline.sup.S were screened further by colony PCR.

(44) 3. PCR reactions were performed to check that the holin gene was no longer present, and that the fbaA-SASP-C and the CdCl.sub.2.sup.R gene were present and correctly placed in the ϕ11 prophage genome. PCR fragments were sequenced to ensure that the isolate carried the expected sequence, especially in regions: fbaA and SASP-C.

(45) Verified prophage constructs were thus identified and a representative was picked and named PTL1001.

(46) 4. Phage was induced from a culture of strain PTL1001 by heat shock, and the cells were lysed with lysostaphin (0.25 μg/ml), and then filtered through a 0.2 μm filter, yielding a crude cell-free phage lysate.

(47) 5. This lysate was used to infect S. aureus strain 8325-4. The infection mixture was plated onto ϕVPB (vegetable peptone brothcontaining 10 g/l sodium chloride)+CdCl.sub.2 (0.1 mM) agar plates to select for lysogens after overnight growth at 37° C.

(48) 6. Lysogens were checked by colony PCR as described above. A verified lysogen was identified and named PTL1002.

(49) 7. PTL1002 was passaged 5 times on ϕVPB agar, picking a single colony and re-streaking to single colonies at each passage.

(50) 8. A single colony was picked and analysed again by PCR and sequencing. The verified isolate was named PTL1003. The phage carried by this lysogen strain is called PTSA1.2/A (see FIG. 4).

(51) SASPject vector PTSA1.2/A has been tested against a panel of S. aureus strains and clinical isolates, including methicillin sensitive S. aureus (MSSA) and MRSA strains belonging to each of the 5 recognised scc-mec types. An example of a kill curve showing efficacy of PTSA1.2/A against an S. aureus strain is given in FIG. 5.

(52) A kill curve comparing the killing ability of PTSA1.2/A versus the same phage minus the SASP gene (phage SA0/A) is given in FIG. 6, and confirms that the kill rate is due to presence of the SASP.

(53) A kill curve of PTSA1.2/A infecting an S. aureus strain which is a monolysogen of PTSA1.2/A is given in FIG. 7, and shows that superinfection immunity to the phage does not prevent SASP from inhibiting infected cells.

REFERENCES

(54) Donegan, N. 2006. Annual Meeting of the Soc. for Healthcare Epidemiology of America. Francesconi, S. C., MacAlister, T. J., Setlow, B., and Setlow, P. 1988. Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis. J. Bacteriol. 170: 5963-5967. Frenkiel-Krispin, D., Sack R., Englander, J., E. Shimoni, Eisenstein, M., Bullitt, Horowitz-Scherer, E. R., Hayes, C. S., Setlow, P., Minsky, A., and Wolf, S. G. 2004. Structure of the DNA-SspC Complex: Implications for DNA Packaging, Protection, and Repair in Bacterial Spores. J. Bacteriol. 186: 3525-3530. Mainous, A. G. III, Hueston, W. J., Everett, C. J., and Diaz V. A. 2006. Nasal Carriage of Staphylococcus aureus and Methicillin Resistant S. aureus in the US 2001-2002. Annals of Family Medicine 4:132-137. Nicholson, W. L., Setlow, B., and Setlow, P. 1990. Binding of DNA in vitro by a small, acid-soluble spore protein from Bacillus subtilis and the effect of this binding on DNA topology. J. Bacteriol. 172: 6900-6906. Noskin, G. A., Rubin, R. J., Schentag, J. J., Kluytmans, J., Hedblom, E. C., Smulders, M., Lapetina, E., and Gemmen, E. 2005. The Burden of Staphylococcus aureus Infections on Hospitals in the United States: An Analysis of the 2000 and 2001 Nationwide Inpatient Sample Database. Arch Intern Med 165: 1756-1761 Sambrook, J., Fritsch, E. F. and Maniatis, T. in Molecular Cloning, A Laboratory Manual 2nd edn (Cold Spring Harbor Press, New York, 1989). Schenk, S., and R. A. Laddaga. 1992. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. 73:133-138.

APPENDIX 1

(55) A list of common pathogens and some of their phages. (This list is representative but not exhaustive).

(56) Coliphages:

(57) TABLE-US-00007 Bacteriophage lambda Bacteriophage 933W (Escherichia coli O157:H7) Bacteriophage VT2-Sa (E. coli O157:H7) Coliphage 186 Coliphage P1 Coliphage P2 Coliphage N15 Bacteriophage T3 Bacteriophage T4 Bacteriophage T7 Bacteriophage KU1

(58) Bacteriophages of Salmonella spp

(59) TABLE-US-00008 Bacteriophage Felix Bacteriophage P22 Bacteriophage L Bacteriophage 102 Bacteriophage  31 Bacteriophage F0 Bacteriophage  14 Bacteriophage 163 Bacteriophage 175 Bacteriophage Vir Bacteriophage ViVI Bacteriophage  8 Bacteriophage  23 Bacteriophage  25 Bacteriophage  46 Bacteriophage E15 Bacteriophage E34 Bacteriophage 9B

(60) Bacteriophages of Shigella dysenteriae

(61) TABLE-US-00009 Bacteriophage ϕ80 Bacteriophage P2 Bacteriophage  2 Bacteriophage  37 Bacteriophage fs-2 Bacteriophage 138 Bacteriophage 145 Bacteriophage 149 Bacteriophage 163

(62) Bacteriophages of Vibrio cholerae

(63) Bacteriophages of Mycoplasma arthritidis

(64) TABLE-US-00010 Bacteriophage MAV1

(65) Bacteriophages of Streptococci

(66) TABLE-US-00011 Bacteriophage CP-1 Bacteriophage ϕXz40 Bacteriophage 1A Bacteriophage 1B Bacteriophage 12/12 Bacteriophage 113 Bacteriophage 120 Bacteriophage 124

(67) Bacteriophages of Pseudomonas aeruginosa

(68) TABLE-US-00012 Bacteriophage D3 Bacteriophage ϕCTX Bacteriophage PP7

(69) Bacteriophages of Haemophilus influenzae

(70) TABLE-US-00013 Bacteriophage S2 Bacteriophage HP1 Bacteriophage flu Bacteriophage Mu

(71) Bacteriophages of Staphylococcus aureus

(72) TABLE-US-00014 Bacteriophage Twort Bacteriophage tIII-29S Bacteriophage ϕPVL Bacteriophage ϕPV83 Bacteriophage ϕ11 Bacteriophage ϕ12 Bacteriophage ϕ13 Bacteriophage ϕ42 Bacteriophage ϕ812 Bacteriophage K Bacteriophage P3 Bacteriophage P14 Bacteriophage UC18 Bacteriophage  15 Bacteriophage  17 Bacteriophage  29 Bacteriophage 42d Bacteriophage  47 Bacteriophage  52 Bacteriophage  53 Bacteriophage  79 Bacteriophage  80 Bacteriophage  81 Bacteriophage  83 Bacteriophage  85 Bacteriophage  93 Bacteriophage  95 Bacteriophage 187

(73) Bacteriophages of Chlamydia

(74) TABLE-US-00015 Bacteriophage ϕCPAR39

(75) Mycobacteriophage

(76) TABLE-US-00016 Bacteriophage L5 Bacteriophage LG Bacteriophage D29 Bacteriophage Rvl Bacteriophage Rv2 Bacteriophage DSGA

(77) Bacteriophages of Listeria monocytogenes

(78) TABLE-US-00017 Bacteriophage A118 Bacteriophage  243 Bacteriophage A500 Bacteriophage A511 Bacteriophage   10 Bacteriophage  2685 Bacteriophage 12029 Bacteriophage   52 Bacteriophage  3274

(79) Bacteriophages of Klebsiella pneumoniae

(80) TABLE-US-00018 Bacteriophage 60 Bacteriophage 92

(81) Bacteriophages of Yersinia pestis

(82) TABLE-US-00019 Bacteriophage R Bacteriophage Y Bacteriophage P1