PRODUCTION OF LYTIC PHAGES

Abstract

The present invention concerns a production bacterial cell for producing lytic phage particles or lytic phage-derived delivery vehicles, said production bacterial cell stably comprising at least one phage structural genes and at least one phage DNA packaging genes, said phage structural gene(s) and phage DNA packaging gene(s) being derived from a lytic bacteriophage, wherein the expression of at least one of said phage structural genes and/or at least one of said phage DNA packaging gene(s) in said production bacterial cell is controlled by an induction mechanism.

Claims

1. Production bacterial cell for producing lytic phage particles or lytic phage-derived delivery vehicles, said production bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene, said phage structural gene(s) and phage DNA packaging gene(s) being derived from a lytic bacteriophage, wherein the expression of at least one of said phage structural genes and/or at least one of said phage DNA packaging gene(s) in said production bacterial cell is controlled by an induction mechanism.

2. The production bacterial cell according to claim 1, wherein said bacterial cell further comprises a payload to be packaged into said phage particles or phage-derived delivery vehicles.

3. The production bacterial cell according to claim 2, wherein said payload is a nucleic acid payload comprising a packaging site derived from said lytic bacteriophage.

4. The production bacterial cell according to claim 2, wherein said payload is to be delivered into targeted bacterial cells.

5. The production bacterial cell according to claim 4, wherein said payload is stably maintained in said targeted bacterial cells.

6. The production bacterial cell according to claim 4, wherein said payload does not replicate in said targeted bacterial cells.

7. The production bacterial cell according to claim 4, wherein said payload comprises a sequence of interest.

8. The production bacterial cell according to claim 7, wherein said sequence of interest only generates an effect in said targeted bacterial cells.

9. The production bacterial cell according to claim 8, wherein said targeted bacterial cells are from a species or strain different from the production bacterial cell.

10. The production bacterial cell according to claim 1, wherein the same induction mechanism controls the expression of the at least one of said phage structural gene(s) and the at least one of said phage DNA packaging gene(s).

11. The production bacterial cell according to claim 1, wherein the expression of the at least one of said phage structural gene(s) and the expression of the at least one of said phage DNA packaging gene(s) are controlled by different induction mechanisms.

12. The production bacterial cell according to claim 1, wherein the at least one induction mechanism controls the expression of all said phage structural gene(s).

13. The production bacterial cell according to claim 1, wherein the at least one induction mechanism controls the expression of all said phage DNA packaging gene(s).

14. The production bacterial cell according to claim 1, wherein said induction mechanism further controls the copy number of said at least one of said phage structural gene(s) and/or said at least one of said phage DNA packaging gene(s).

15. The production bacterial cell according to claim 2, wherein said at least one induction mechanism further controls the copy number of said payload in said production bacterial cell.

16. The production bacterial cell according to claim 2, wherein another induction mechanism controls the copy number of said payload in said production bacterial cell.

17. The production bacterial cell according to claim 1, wherein said phage structural gene(s) and phage DNA packaging gene(s) derived from said lytic bacteriophage are comprised in at least one plasmid, chromosome and/or helper phage.

18. The production bacterial cell according to claim 1, further comprising at least one gene, derived from said lytic bacteriophage, involved in phage regulation.

19. The production bacterial cell according to claim 1, wherein said production bacterial cell further comprises at least one gene, derived from a non-lytic bacteriophage, involved in phage excision/insertion, phage DNA replication, and/or phage regulation.

20. The production bacterial cell according to claim 7, wherein said production bacterial cell is from the same bacterial species or strain as the bacterial species or strain from which said non-lytic bacteriophage comes and/or that said non-lytic bacteriophage targets.

21. The production bacterial cell according to claim 1, wherein said production bacterial cell is an E. coli bacterial cell.

22. The production bacterial cell according to claim 1, wherein said production bacterial cell is a P. freudenreichii bacterial cell.

23. The production bacterial cell according to claim 22, wherein said phage structural gene(s) and phage DNA packaging gene(s) derive from a C. acnes bacteriophage.

24. A method for producing lytic phage particles or lytic phage-derived delivery vehicles, comprising: (a) providing a production bacterial cell for producing lytic phage particles or lytic phage-derived delivery vehicles, said production bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene, said phage structural gene(s) and phage DNA packaging gene(s) being derived from a lytic bacteriophage, wherein the expression of at least one of said phage structural genes and/or at least one of said phage DNA packaging gene(s) in said production bacterial cell is controlled by an induction mechanism, and (b) inducing, in said production bacterial cell, expression of said at least one of said phage structural gene(s) and said at least one of said phage DNA packaging gene(s), and assembly of the products expressed by said at least one phage structural gene(s) and said at least one phage DNA packaging gene(s), thereby producing lytic phage particles or lytic phage-derived delivery vehicles.

25. Hybrid helper phage system comprising: (i) at least one phage DNA packaging gene(s) derived from a lytic bacteriophage, (i′) at least one phage structural gene(s) derived from said lytic bacteriophage, (i″) optionally, at least one phage gene(s) involved in phage regulation derived from said lytic bacteriophage, and (ii) at least one gene, derived from a non-lytic bacteriophage, involved in phage excision/insertion, phage DNA replication, and/or phage regulation, wherein said genes (i), (i′), (i″) and (ii) are comprised in a unique nucleic acid molecule or in separate nucleic acid molecules, and wherein said hybrid helper phage system does not comprise any expressed phage structural gene derived from said non-lytic bacteriophage.

26. The hybrid helper phage system according to claim 25, wherein said genes (i), (i′), (i″) and (ii) are comprised in a bacterial chromosome.

27. The hybrid helper phage system according to claim 25, wherein said genes (i), (i′), (i″) and (ii) are comprised in separate plasmids.

28. The hybrid helper phage system according to claim 25, wherein said hybrid helper phage system consists of a hybrid helper phage comprising: (i) at least one phage DNA packaging gene(s), at least one phage structural gene(s) and optionally at least one phage gene(s) involved in phage regulation, derived from a lytic bacteriophage, and (ii) at least one gene, derived from a non-lytic bacteriophage, involved in phage excision/insertion, phage DNA replication, and/or phage regulation, wherein said hybrid helper phage does not comprise any phage structural gene derived from said non-lytic bacteriophage.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0380] FIG. 1: Lambda genome organization (packaged variant). The structural operon is marked with a red line as well as the antitermination protein Q that allows transcription of the late structural operon. Figure adapted from Rajagopala et al. BMC Microbiol 11, 213 (2011).

[0381] FIG. 2: Titration of T7 phagemids produced in a Lambda-T7 hybrid prophage system. Production strain CY-L7 contains the payload p1883. Left panel, titration on MG1655; right panel, titration on KEIO-waaG.

[0382] FIG. 3: Titration of T7 phagemids produced in a Lambda-T7 hybrid prophage system. Left panel, titration on MG1655. Right panel, titration on KEIO-waaG. From left to right: left) payload p1883+p1885 (T7 RNA polymerase with fast degradation); middle) payload p1883+p1884 (T7 RNA polymerase with medium strength degradation); right) payload p1883 only.

[0383] FIG. 4: Identification of P. freudenreichii phages with PCR. PCR on ORF3 and ORFS was performed on all phage suspensions. BW4 from plaques 1-3 give a band at the expected size for both orf3 and orf5. Ladder is GeneRuler 1 kb plus.

[0384] FIG. 5: Immunity to superinfection of lysogen Pf0s14253. Left panel: Top agar of Pf0s2841 with spots of 4 different BW-like phage suspensions. Right panel: Top agar of Pf0s14253 with spots of 4 different BW-like phage suspensions.

[0385] FIG. 6: High induction of BW4 phage after mitomycin C treatment. Left panel: Top agar of Pf0s2841 with spots of culture supernatant from Pf0s14253 without mitomycin C (MMC) induction (ND: non diluted to dilution 10.sup.−3). Right panel: Top agar of Pf0s2841 with spots of culture supernatant from Pf0s14253 with 0.5 pg/ml of mitomycin C induction (ND: non diluted to dilution 10.sup.−7)

[0386] FIG. 7: Genome organization of BW4 and PAC7 bacteriophages. BW4 and PAC7 genome organization is similar with both putative structural operons (represented by the arrows) containing the packaging, head, tail and lysis modules.

[0387] FIG. 8: Construction of chimeric BW4-PAC7 prophage. Transformation of the pAN514 suicide plasmid into strain Pf1s22499 containing the BW4 prophage. Selection on chloramphenicol was used to select for double crossover at the Left Homology Arm (LHA) and Right Homology Arm (RHA). The prophage obtained is a chimer containing a structural operon with first BW4 gp1 followed by gp1-gp14 of PAC7 and after the chloramphenicol selection cassette (CmR) the leftover of BW4 structural genes (gp15-gp25).

[0388] FIG. 9: Plasmid map of cosmid pAN594.

[0389] FIG. 10: Titration of PAC7 phage-derived particles. Left Panel: Titration from Pf1s22904 plated on erythromycin. Right Panel: Titration from control suspension of strain Pf1s22903 that does not carry any cosmid plated on erythromycin.

[0390] FIG. 11: Confirmation for 8 colonies streaked from phage-derived particles titration of Pf1s22904 production by PCR. Top Panel: SLTS PCR (Scholz 2014) on 8 colonies streaked from the phage derived titration assay. Expected size is 612 bp. Bottom Panel: pAN594 specific PCR on 8 colonies. Expected size is 769 bp. Ladder is generuler 1 kb plus.

EXAMPLES

Example 1: Exchange of the Structural Operon of Lambda for Elements of a Lytic Phage

[0391] The inventors considered that phages can be viewed as more or less large genetic circuits whose final output is the generation of more phage particles. To do this, no matter if the phage is lytic, temperate or chronic (for instance filamentous phages such as M13), the information encoded in their genomes can be roughly categorized depending on the function it performs: [0392] Genes devoted to insertion/excision (for temperate phages). [0393] Genes devoted to DNA replication, RNA transcription, etc. . . . . Some lytic phages encode their own RNA or DNA polymerases, for instance. Some genes modify the host's RNA polymerases to be able to work past terminators, and some other genes are involved in the segregation of the prophage sequence if it exists in a plasmid or linear plasmid form. [0394] Genes related to defense from host's anti-phage mechanisms, degradation/modification of host's elements to complete the lytic cycle, super-exclusion mechanisms or genes that are advantageous for the host. [0395] Genes devoted to DNA packaging: terminases and accessory proteins, ligases, etc. [0396] Structural genes devoted to building a protein capsid for the DNA: apart from strictly structural genes, such as capsid genes, tape measure, fibers, baseplate etc, many other genes are needed to assemble the components (chaperones, proteases) as well as proteins that can be packaged inside the capsid, be it as scaffold or as pilot proteins injected into the cell (for instance, the RNA polymerase of phage N4 or some minor pilot proteins in other phages).

[0397] The last two categories (DNA packaging and structural genes) are deeply connected, since the packaging machinery recognizes the pre-assembled heads and the DNA to be packaged, initiates and terminates DNA packaging.

[0398] The inventors hypothesized that by abstracting and differentiating all the modules defined above, in principle a system could be built that contains all excision/insertion, replication and regulation elements from one phage, in particular a non-lytic phage, and encodes the packaging/structural elements from another phage, in particular from a lytic phage, since, in principle, they could be viewed as independent genetic modules.

[0399] In the present example, it is referred to “structural elements” for proteins needed for DNA packaging and structural proteins needed to assemble a mature virion.

[0400] Such a “hybrid structural phage” could be very advantageous for different approaches, because: [0401] a species which is more amenable for laboratory work/large scale production/safer could be used to produce such particles where the structural genes come from another species; [0402] pure phagemid producing strains could be constructed using the regulatory elements of a well-characterized phage (for instance, Lambda) driving the production of capsids of a different phage, etc., and [0403] and finally, structural hybrid prophages (i.e. carried in the genome) driving the production of lytic phage capsids could be constructed.

[0404] This is the approach that was developed herein. Using a production strain encoding a system to generate pure Lambda phagemids, its structural operon has been exchanged (from the small terminase to the STF gene, about 23 kb) with the structural elements of a strictly lytic E. coli phage, T7. A schematic diagram shows the lambda genome organization (FIG. 1).

[0405] In this system, the thermolabile version of the prophage Lambda contains all regulatory elements needed to excise the prophage, replicate the circularized excised genome and drive the expression of the long, late operon, including the presence of the antitermination protein Q. This should drive the assembly and packaging of pure phagemid particles completely based on other phages when supplemented with a plasmid containing the correct packaging signals (LTR for T7)

Construction of the Hybrid

[0406] The Lambda prophage structural operon (SEQ ID NO: 7) was exchanged with the structural “operon” of the lytic phage T7, from gp6.5 to gp19.5 (not strictly an operon since the T7 RNA polymerase drives the transcription of different mRNAs within this region), using the lambda red recombineering system, starting from a production strain containing a Lambda prophage without the cos site (s1965). Several changes were further made: [0407] Removal of putative holin and lysis genes in T7 (gp17.5 and gp18.5) [0408] Recoding of the 3′ part of the gp19 DNA maturation protein and the intergenic region between this gp19 and the next one, gp19.5 (explained below) [0409] All T7 RNA polymerase promoters were left intact but no T7 RNA polymerase was added to the system.

[0410] The complete edited structural “operon” spans about 20 kb (SEQ ID NO: 8). The final production was named CY-L7 and was built without any specific remarks.

Production and Titrations

[0411] A payload was built that should be packaged by T7 as described in Auster et al. RNA Biol. 2019 April; 16(4):595-599, called pJ23115-GFP T7 cos 2.0 (p1883, SEQ ID NO: 9). This payload contains the 5′ LTR necessary to be efficiently packaged by T7. The putative packaging region of this plasmid contains the 3′ part of gp19 and the intergenic region between gp19 and gp19.5. It is for this reason that the 3′ part of gp19 was recoded before inserting it into the genome of the production strain, so recombination is prevented.

[0412] Next, the CY-L7 strain was transformed with the p1883 payload and productions carried out as described below.

[0413] Overnight cultures were diluted 1:6 in a final volume of LB+5 mM CaCl.sub.2 supplemented with chloramphenicol and grown for 30 min at 30° C. with shaking. After that, a 45-minute-long heat shock at 42° C. was performed. Finally the cultures were grown at 37° C. for 3 hours with shaking. After this period, cells were recovered by centrifugation and lysed using 3 mL of B-PER protein extraction reagent, 600 mg of detergent removal bio-beads were added and an incubation at room temperature with mild shaking performed for 1 hour. After that, the lysates were centrifuged for 10 min at 10,000 g and the supernatants filtered through a 0.2 micron pore-size membrane.

[0414] The lysates were titrated in E. coli MG1655 and KEIO-waaG (a derivative with a deletion of the waaG gene, which has been shown to be necessary for T7 binding, (Qimron et al. Proc Natl Acad Sci USA. (2006) 103(50):19039-19044)). If phagemids are produced, colonies should only be detected in the MG1655 strain, since the KEIO-waaG does not contain the receptor for T7.

[0415] As can be seen in FIG. 2, a small number of colonies could be detected only in the MG1655 columns. This result is the first proof that a strictly lytic phage can be “tamed” and its structural and packaging genes controlled by a lysogenic one (lambda) to yield pure phagemid particles based on T7.

[0416] The titers obtained were very low, although pure T7-based phagemids were produced. The inventors sought to improve the titers by applying different rational approaches. For instance, it is known that for T7 plasmid or genome packaging, transcription by the T7 RNA polymerase from a promoter within the 5′ LTR is needed (Chung et al. J Mol Biol. 1990 Dec. 20; 216(4):927-38). Additionally, the T7 genome is transcribed by its cognate RNA polymerase and many different T7 promoters are found, even within the region encoding the different structural elements (Dunn et al. J Mol Biol. 1983 Jun. 5; 166(4):477-535). This produces different mRNAs that are then processed by the E. coli RNAse III (Studier et al. “Processing of bacteriophage T7 RNAs by RNase III” Ed: Thomas R. Russell, Keith Brew, Harvey Faber, Julius Schultz, From Gene to Protein: Information Transfer in Normal and Abnormal Cells, Academic Press, 1979, p. 261-269). For these two reasons, the production strain was complemented with the T7 RNA polymerase in trans, in an inducible plasmid under the control of the PhlF repressor.

[0417] Initially, the transformation of the T7 RNA polymerase plasmid in the CY-L7 strain containing the p1883 payload gave no colonies, presumably due to toxicity coming from leakiness of the inducible pphlF promoter (data not shown). For this reason, two alternative plasmids encoding the T7 RNA polymerase with two different degradation tags of different strengths were built (p1884, SEQ ID NO: 10; and p1885, SEQ ID NO: 11). The sequences of the T7 RNA polymerase encoded in these two plasmids are disclosed (SEQ ID NO: 12 and SEQ ID NO: 13 for version AAV; SEQ ID NO: 14 and SEQ ID NO: 15 for version LVA). It has been demonstrated that by adding a degradation tag to a protein, the potential effects of leaky expression from a repressible promoter are improved (Fernandez-Rodriguez et al. Nucleic Acids Res. (2016) 44(13):6493-6502).

[0418] Productions were carried out from strain CY-L7 harboring the payload p1883 and supplemented with the T7 RNA polymerase variants encoded in plasmids p1884 or p1885, with the same protocol specified above. The lysates were then titrated on MG1655 or on KEIO-waaG.

[0419] As can be seen on FIG. 3, the introduction of the T7 RNA polymerase increases the titers obtained by a factor of 100× (for the medium degradation tag) or by 1000× (for the fast degradation tag) as compared to productions harboring the p1883 payload only. The titers obtained in this system are about 2×10.sup.6 TU/mL.

[0420] These experiments show that, for certain types of phages, a regulatory protein not belonging strictly to the structural categories defined above may be needed, in this case the T7 RNA polymerase, either to improve or promote the packaging reaction or to control the amount or processing of the mRNAs encoding the structural components.

Example 2: Production of Cutibacterium acnes Phage-Derived Particles

[0421] Cutibacterium acnes is one of the most prevalent and abundant species of the skin (Kashaf et al. Nat Microbiol 7, 169-179 (2022)) where it colonizes the pilosebaceous unit (PSU). Unlike on the stratum corneum, bacteria present in the PSU are surrounded by living cells notably keratinocytes, sebocytes and different immune cells (Kabashima et al. Nat Rev Immunol 19, 19-30 (2019)). Close contact between C. acnes and these cells might lead to either beneficial or detrimental interactions. (Bruggemann et al. Front Microbiol 12, 673845 (2021)). Being able to genetically modify C. acnes was notoriously challenging before the applicant's' new tools as disclosed in US applications US2022/135986 and US2022/135987. In these patent applications, the inventors described, for the first time, the production of C. acnes phage-derived particles using C. acnes as a production strain.

[0422] In the present example, the inventors used P. freudenreichii strain to produce C. acnes phage-derived particles by swapping the structural genes from a P. freudenreichii prophage for the structural genes of a C. acnes phage.

Results

Isolation of BW4 Phage

[0423] P. freudenreichii and associated bacteriophages are known to be present in some dairy products (Gautier et al. (1995) Lait 75:427-434; Gautier et al. (1995) Appl. Environ. Microbiol. 61:2572-2576; Cheng et al. (2018) BMC Microbiology 18:19). The inventors therefore screened for the presence of both Propionibacterium phages or P. freudenreichii lysogens in cheese samples.

[0424] Different types of cheese samples were grinded, resuspended in Reinforced Clostridial Medium (RCM) and incubated at 30° C. in anaerobic conditions for 2 days. After incubation, a dilution of the culture was performed in lithium glycerol broth, a media selective for Propionibacteria (WO1994017201), and incubated for 6 days at 30° C. A final dilution in RCM+mitomycin C was incubated for 1 day at 30° C. in order to induce potential prophages. The induced cultures were filtered (0.2 pm) and spotted on different indicator strains. One of the samples led to turbid plaque formation on top agar of the P. freudenreichii strain Pf0s2841. Three individual plaques were isolated by two successive picking and streaking on Pf0s2841 and amplification was performed on top agar of Pf0s2841. For the three different plaques, amplification led to phage suspension ˜10.sup.10 PFU/mL.

[0425] Two clusters of temperate dsDNA P. freudenreichii phages (BW and BV) have been previously identified (Cheng et al. (2018) BMC Microbiology 18:19). Using PCRs, designed on BW genome from Doucette phage (KX620751), two different fragments were extracted: [0426] ORF3 with AD1334 (SEQ ID NO: 16)/AD1335 (SEQ ID NO: 17) [0427] ORF5 with AD1336 (SEQ ID NO: 18)/AD1337 (SEQ ID NO: 19).
The inventors could classify the isolated phages as BW-like (FIG. 4). Sequencing of ORF5 revealed that all phages were most probably identical and therefore were coming from the same BW-like phage that was named BW4.

Isolation of Pf0s2841 Lysogen Carrying the BW4 Phage

[0428] The inventors then isolated P. freudenreichii lysogen carrying the BW4 phage as a prophage. For that, BW4 phage suspension was spotted on strain Pf0s2841 and incubated for 3 days. Turbid plaques were picked, resuspended and streaked. After 5 days, single colonies were obtained, several colonies were streaked and incubated a second and third time and presence of the phage genes was checked, at each streaking, by PCR, after DNAse treatment, across the cohesive ends (AD1322 (SEQ ID NO: 20)/AD1323 (SEQ ID NO: 21)) to ensure presence of the phage but absence of phage particles.

[0429] After the third streak, colonies were grown as a top agar and a spot of non diluted BW-like phages suspensions were spotted on the putative lysogenic strain (Pf0s14253) and on the ancestor strain (Pf0s2841). After incubation, clearance was observed for both strains for BW13 and BW14 spots whereas clearance was only observed for Pf0s2841 in the case of BW4 spot (FIG. 5). This indicates that the strain Pf0s14253 is immune to BW4 phage superinfection and carries the BW4 prophage. The absence of immunity for BW14 and BW13 indicates that these phages have likely a different immunity repressor.

BW4 Prophage Induction

[0430] In order to use the BW4 lysogen strain as a production strain for phage-derived particles the inventors first had to test the ability to produce high concentration of the BW4 phage upon induction of the lytic cycle. To do so, Pf0s14253 was grown in absence or presence of mitomycin C (MMC), an antibiotic known to induce prophages, and the culture supernatant was titered for the presence of BW4 phage particles on the indicator strain Pf0s2841. A high amount of BW4 phage particles was observed in the condition supplemented with mitomycin C (FIG. 6) with 7.4×10.sup.7 PFU/μL against 3.0×10.sup.3 PFU/μL for the condition without mitomycin C. This indicates a high dynamic range between lytic and lysogenic cycle for BW4 prophage under such conditions and confirmed the potential of BW4 for the production of phage-derived particles.

Sequencing and Annotation of BW4 Phage

[0431] To engineer the BW4 prophage towards production of C. acnes phage-derived particles, the BW4 phage was sequenced. DNA isolation (Promega Wizard DNA Clean-Up System) followed by Illumina sequencing was performed on BW4 phage suspension. Raw reads were assembled into a single contig using Spades and termini were corrected by sanger sequencing (SEQ ID NO: 22). Annotation was performed using Phaster and manually curated based on homologies with other BW-like phages (Cheng et al. (2018) BMC Microbiology 18:19).

[0432] As described in Cheng et al. (2018) BMC Microbiology 18:19, BW-like phages have typical genomic architecture of other temperate phages with a large putative structural operon (also called lytic operon) organized in different functional modules with, in order of transcription: packaging, head, tail, and lysis module. Surprisingly, the first gene of the putative operon (gp1) appears to be related to DNA replication based on HHpred as it contains a domain similar to bifunctional primase and polymerase proteins. Other parts of the BW4 phage genome contain the genes necessary for prophage integration/excision, DNA replication, DNA recombination, regulation of the lytic/lysogenic cycle and other accessory proteins. This modular architecture confirms the possibility to swap the genes necessary for the production of BW4 phage capsid and the packaging of the phage genome by their equivalent from a C. acnes phage genome.

Isolation of C. acnes PAC7 Phage

[0433] C. acnes phages were isolated from skin of healthy volunteers. Briefly a patch (Biore) was applied to the nose allowing to extract comedones that were resuspended in RCM, plated on MRS and incubated at 37° C. in anaerobic conditions. For some of the plates, plaques could be observed in the dense lawn of C. acnes. DPBS (Dulbecco's Phosphate Buffered Saline) was poured on the plate to resuspend potential phages and filtered to remove bacteria. This phage suspension was streaked on plate and a top agar of strain Ca0s2345 was added. Plates were incubated for 2 days and plaques were reisolated by three successive picking, streaking and top agar plating. Finally a plaque was amplified on top agar with Ca0s2345 strain and the resulting phage suspension was PEG precipitated. High titer (>10.sup.6 PFU/μL) phage suspension was obtained when titered on Ca0s2345.

Sequencing and Annotation of PAC7 Phage

[0434] DNA isolation (Promega Wizard DNA Clean-Up System) followed by Illumina sequencing was performed on PAC7 phage suspension. Raw reads were assembled into a single contig using Spades and termini were corrected by sanger sequencing (SEQ ID NO: 23). Annotation was performed using Phaster and manually curated based on homologies with other C. acnes phages (Marinelli et al. (2012) mBio 3:e00279-12). Similar to the P. freudenreichii BW4 phage, a structural operon comprising modules for packaging, head and tail assembly and cell lysis was identified (FIG. 7). An HNH endonuclease was identified as the last gene of the phage (gp45). Such endonuclease has already been shown to be essential for efficient packaging (Quiles-Puchalt et al. (2014) Proc Nat. Acad. Sci. 111:6016-6021).

Construction of Lysogen Strain with a Chimeric BW4-PAC7 Prophage

[0435] The genes in the structural operon of BW4 prophage, from the small terminase gp2 to the tape-measure protein gp16 included, were replaced by the structural PAC7 genes from gp1 to gp14 (FIG. 8). This was performed by homologous recombination using plasmid pAN514 (SEQ ID NO: 24), a P. freudenreichii suicide vector that was cloned in E. coli DH10B. After transformation of the vector, a double crossing over event was selected in P. freudenreichii (Pf1s22499) by selection on chloramphenicol. The chimeric BW4-PAC7 structural operon integrity was globally confirmed by PCR and sanger sequencing of the entire chimeric structural operon.

Production and Titration of PAC7 Derived Particles from a Lysogen Strain Carrying a Chimeric BW4-PAC7 Propage

[0436] In order to produce C. acnes phage-derived particles from a P. freudenreichii BW4-PAC7 chimeric lysogen, the pAN594 cosmid (FIG. 9) containing the packaging signal of the PAC7 phage (SEQ ID NO: 25), an operon expressing five genes of the PAC7 tail module (gp15-gp19) and the gp45 endonuclease (SEQ ID NO: 26) and an origin of replication functional in P. freudenreichii and C. acnes (as disclosed in US applications US2022/135986 and US2022/135987) were transformed into Pf1s22903. Transformants were streaked and grown in presence of both chloramphenicol (1 pg/ml) to select for the presence of the prophage and erythromycin (2.5 pg/ml) to select for the presence of pAN594. At OD.sub.600nm˜0.4, culture was supplemented with 0.5 pg/ml of mitomycin C and grown overnight at 30° C. in anaerobic conditions. After incubation, cells were collected by centrifugation, lysed by bead beating (2×20 min at 30 Hz with 0.1 mm glass beads), supernatant was filtered and the presence of phage derived particles was titered on C. acnes Ca0s2258.

[0437] Up to ˜10.sup.2 potential transductants per μL were obtained (FIG. 10). 8 colonies were streaked on Brain Heart Infusion (BHI) erythromycin (5 μg/mL) and confirmed to be C. acnes and transductants carrying pAN594 using PCR (FIG. 11).

[0438] The inventors thus demonstrated for the first time that C. acnes phage-derived particles able to deliver DNA into Cutibacterium acnes can be produced by swapping structural genes of a P. freudenreichii prophage for the structural genes of a Cutibacterium acnes phage.

Material and Methods

Strain Used and Generated

[0439]

TABLE-US-00002 TABLE 1 Strains used and generated Eligo ID Description Pf0s2841 Indicator strain for P. freudenreichii BW4 phage (CIRM-BIA 509, TL110 belonging to INRAE) Pf0s14253 Strain Pf0s2841 with a BW4 prophage Pf1s22499 Strain Pf0s14253 with the packaging signal of BW4 deleted Pf1s22903 Strain Pf1s22499 with the BW4 genes gp2-gp16 replaced by PAC7 gp1-gp14 Pf1s22904 Strain Pf1s22903 with pAN594 Ca0s2345 Indicator strain for C. acnes PAC7 phage Ca0s2258 Cutibacterium acnes ATCC 11828

Culture Conditions

[0440] All incubations of P. freudenreichii strains were performed at 30° C. in anaerobic conditions (Thermo Scientific™ Sachet Oxoid™ AnaeroGen).

[0441] All incubations of C. acnes strains were performed at 37° C. in anaerobic chamber.

Construction of Strain Pf1s22499

[0442] Deletion of the packaging signal from BW4 prophage was performed by homologous recombination and CRISPR-Cas selection of the recombinant using the pAN241 P. freudenreichii vector that was cloned in E. coli and then transformed into Pf0s14253 strain. The pAN241 vector contains a template for homologous recombination (SEQ ID NO: 27) and a FnCpf1 transcriptional cassette with a crRNA targeting the cos of the BW4 prophage.

Transformation Protocol for P. freudenreichii

[0443] Transformation of P. freudenreichii was adapted from Brede, D. A. et al. Appl Environ Microb 71, 8077-8084 (2005), replacing SLB (sodium lactate broth) media for BHI.

Phage-Derived Particles Titration

[0444] Strain Ca0s2258 was streaked on BHI agar plate. Once dense growth on plate was obtained, a liquid culture was set up in BHI. After overnight incubation, the turbid culture was concentrated 10× in BHI. 90 μl of cells were mixed with pure, diluted 1/10 and diluted 1/100 solutions of 10 μL of phage-derived particles produced from either Pf1s22904 or Pf1s22903 as negative control. Samples were incubated 2 hours at room temperature and then 1/10 serial dilutions were performed in BHI, samples were incubated 2 h at 37° C. in anaerobic conditions before spotting 4 μL on BHI+5 μg/mL erythromycin. Plates were incubated for 7 days at 37° C. in anaerobic conditions.