Field of the Invention

The invention relates to genetically modified bacterial vaccine carriers, methods of producing the same and their uses in the treatment or prevention of disease.

Background of the Invention

Live bacterial vectors (LBVs) have been used extensively in experiments to express and deliver recombinant protein vaccines and anti-cancer compounds to humans and other animals. LBVs are usually constructed by introducing into a host bacterium an expression cassette containing a promoter which regulates the expression of one or more genes encoding heterologous antigens from one or more pathogens. This cassette can either be chromosomally integrated in the host bacterial genome or carried by the host as an autonomously replicating multi-copy plasmid (Kochi et at. 2003, Expert Rev. Vaccines 2:31-43). Post delivery of the LBV to a subject to be treated, the expressed heterologous antigen is delivered and presented to the subject’s immune system to promote a prophylactic or therapeutically effective immune response.

LBVs are usually delivered orally for vaccine applications, either in a liquid form as an oral suspension or as an enterically coated capsule. The LBVs then enter the small intestine where they invade the intestinal epithelium via microfold (M) cells into Peyer’s patches from where they are subsequently delivered to antigen-presenting cells (APCs) (Kotton & Hohmann 2004, Infect. Immun. 72: 5535-5547). Alternative delivery methods are available, and indeed often used, for example in anti-cancer applications, where the LBV may be injected systemically (e.g. intravenously) or directly into a tumour. The standard approach for preparing LBVs from a host bacterium is to introduce specific, attenuating mutations into pathogenic genes of the host bacterium. These attenuating mutations reduce the survival of the host bacterium within the subject to prevent LBV-mediated side-effects.

The field has numerous examples of animal studies using host bacteria comprising attenuating genetic mutations. In general, these LBVs are sufficient to induce a prophylactic response against the heterologous antigen, but are not safe enough to be used in humans. This provides a false picture of the potential utility of LBVs for recombinant vaccine delivery.

Most commonly, attenuating mutations are made in genes involved in the aromatic amino acid (shikimate) biosynthesis pathway, such as aroA and aroC. However, although such attenuated LBVs are well tolerated in mice, these cause undesirable feverish symptoms and prolonged bacterial shedding in humans (Garmory et al. 2002, FEMS Microbiol. Rev. 26: 339-353). Conversely when a host bacterium is sufficiently attenuated for human use to deliver a heterologous antigen, the efficacy of the LBV is invariably disappointing (Garmory et al. 2002, FEMS Microbiol. Rev. 26: 339-353). This is because an improved LBV safety profile correlates with reduced immunogenicity. For example, extensive transposon-mediated insertional mutagenesis of the Salmonella enterica serovar Typhimurium genome failed to identify a mutant that was sufficiently immunogenic yet unable to cause disease in immunocompromised mice (Grant et al. 2016, Infect. Immun. 84: 989-997).

Therefore, there is a need to develop new methods for generating LBVs to improve their safety profile but maintain sufficient immunogenicity to act as effective vaccine carriers. Summary of the Invention

The present invention is based on the surprising discovery that a host bacterium genetically modified to conditionally repress cell division can attenuate the bacterium under those conditions without simultaneously suppressing cell elongation or the ability of the bacterium to act as an effective vaccine carrier.

Thus, in a first aspect of the invention, there is provided a genetically engineered non-natural bacterium comprising one or more of the following genetic modifications: (i) one or more heterologous genes encoding factors that prevent cell division, the expression of which are under the control of an inducible promoter activated when the bacterium is internalised into the intracellular vesicle of an animal cell; (ii) one or more endogenous factors that promote cell division, the expression of which are genetically modified to be repressed when the bacterium is internalised into the intracellular vesicle of an animal cell; and/or (iii) one or more endogenous factors that repress cell division, the expression of which are genetically modified to be under the control of an inducible promoter activated when the bacterium is internalised into the intracellular vesicle of an animal cell.

In a second aspect of the invention, there is provided a non-natural bacterium according to the first aspect of the invention, for use in the prevention or treatment of diseases, including cancer.

In a third aspect of the invention, there is provided the use of the bacterium of the first aspect of the invention in the vaccination of a subject against a disease.

In a fourth aspect of the invention, there is provided a method of manufacturing a non-natural bacterium according to the first aspect of the invention comprising introducing into the bacterium any of the genetic modifications of the first aspect of the invention. Description of the Figures

The invention is illustrated by the following drawings, in which:

Figure 1 shows a plasmid comprising a gene that inhibits cell division (inhibitor) under the control of a phagosomal promoter P. The gene is therefore not transcribed under standard laboratory conditions or during the initial stages of invasion of a subject to be treated. When the non-natural bacterium comprising the plasmid is internalised into a phagosome, the expression of the inhibitor is activated to prevent cell division and induce bacterial filamentation.

Figure 2 shows a plasmid comprising a gene encoding a site-specific recombinase gene under the control of a phagosomal promoter P. The site specific recombination sites targeted and cleaved by the recombinase are engineered to flank a chromosomal gene encoding a factor essential for cell division. When the non-natural bacterium comprising the plasmid is internalised into a phagosome, the expression of the recombinase gene is activated, and the gene encoding the factor essential for cell division is excised, thereby inducing bacterial filamentation.

Figure 3 shows a plasmid comprising a gene encoding a transcriptional repressor protein under the control of a phagosomal promoter P. The native promoter of a chromosomal gene encoding a factor essential for correct cell division is replaced with the cognate inducible promoter bound by the repressor protein. When the host bacterium comprising the plasmid is internalised into a phagosome, the expression of the repressor protein is activated, thus repressing expression of the gene essential for cell division, resulting in bacterial filamentation.

Figure 4 shows A) a plasmid carrying a gene encoding a sRNA that binds to and represses the expression of a mRNA encoding an endogenous factor essential for cell division. The sRNA gene is under the control of a phagosomal promoter P, and is thus only expressed when the host bacterium is internalised into a phagosome. B) The sRNA has a complementary antisense sequence to the 5’ UTR of the target mRNA. The sRNA is bound and stabilised by the Hfq protein, which stabilises the binding of the sRNA to the target mRNA, thus blocking ribosome binding and expression of the target mRNA to prevent cell division, thereby inducing bacterial filamentation (adapted from Yoo et al. 2013, Nature Protocols 8: 1694-1707).

Figure 5 shows a safety mechanism whereby the host bacterium is attenuated by the deletion of a chromosomally-encoded essential gene such as aroC or ssaV. The essential gene is complemented by the introduction of a heterologous functional copy of the gene on a plasmid, thus restoring the viability of the host. One of the genes described in Figures 1 -3 is also cloned into the plasmid. Thus in the event of plasmid loss resulting in the loss of the filamentous phenotype, the heterologous essential gene is also lost, thus maintaining attenuation of the host bacterium.

Figure 6 shows A) Salmonella Typhimurium SL3261 containing the negative control plasmid pSCT4 (containing no genes) growing in PCN medium. After six hours of growth, the bacteria maintain the regular rod shape. B) S. Typhimurium SL3261 containing the plasmid pSCT4minCOX, with the P _SSaG promoter (SEQ ID NO: 35) controlling expression of the minC gene (SEQ ID NO: 1 ). The bacterial morphology was initially the same as the negative control strain, but after 6 hours of growth, minC over-expression arrested cell division and filamentous bacteria were produced.

Figure 7 shows S. Typhimurium SL3261 (pSCT4minCOX) infecting RAW264.7 macrophage cells under 1 ) immunofluorescence and 2) bright-field microscopy. A) The bacterial morphology was rod-shaped two hours post-infection. B) However, after sixteen hours of growth, MinC over-expression resulted in a filamentous bacterial phenotype.

Figure 8 shows S. Typhimurium strains containing plasmid pBRTI RFP infecting RAW 264.7 cells at 16h post-infection under 1 ) fluorescence and 2) phase- contrast microscopy. Micrographs show A. MD58(pBRT1 RFP); B. MD58(pSCT4minCOX)(pBRT 1 RFP); C. MD58(pSCT4minCOX)(pBRT1 RFP) at x1000 magnification; D. WT05(pBRT1 RFP); E.

WT05(pSCT4minCOX)(pBRT1 RFP). Cells containing pBRTI RFP retained their normal rod shape, but those that additionally contained pSCT4minCOX were filamentous.

Figure 9 shows phase contrast micrographs of bacterial cells grown for 7 hours in PCN medium. A. S. Typhimurium MD58 negative control strain with no plasmid; B. MD58(pSCT4FtsZ-1/314); C. MD58(pSCT4FtsZ-D45A); D. MD58(pSCT4FtsZ-E250K-D253K), E. MD58(pSCT4queE). Control strain MD58 maintains its rod-shaped morphology, whereas MD58 expressing mutants of FtsZ or QueE develop a filamentous phenotype.

Figure 10 shows a plasmid map of pSCT4

Figure 11 shows a plasmid map of pBRT 1 RFP

Figure 12 shows a plasmid map of pSCT4minCOX

Figure 13 shows a plasmid map of pSCT4FtsZ-1/314

Figure 14 shows a plasmid map of pSCT4FtsZ-D45A

Figure 15 shows a plasmid map of pSCT4FtsZ-E250K-D253K

Figure 16 shows a plasmid map of pSCT4queE

Detailed description of the invention

The invention is predicated on the surprising discovery that genetically modifying a host bacterium to conditionally repress cell division when the bacterium is internalised into an animal cell attenuates the bacterium without supressing its ability to act as a vaccine delivery vehicle. As used herein,“conditional repression” refers to the engineering of a genetic modification within the host bacterium to be activated only under certain environmental conditions. In this context, “activated” refers to promoting the phenotype of reduced cell division. The activated modification also causes bacterial filamentation.

Conditional repression is activated when the genetically modified host bacterium (also referred to as the“non-natural bacterium”) is internalised into an internal vesicle of an animal cell, such as an endocytic or phagocytic vesicle. The environmental conditions within the internal vesicle activate the one or more genetic modifications that prevent the bacterium from undergoing cell division. This conditional repression has the surprising benefit of attenuating the bacterium, but only within the internal vesicle of the animal cell, without reducing immunogenicity. In fact, the inventors have found that the conditional repression of cell division increases immunogenicity by promoting bacterial filamentation. Without wishing to be bound by theory, filamentation is thought to increase immunogenicity by increasing the surface area upon which heterologous antigen presentation can take place. Thus, the non-natural bacterium of the present invention can be used as an efficacious heterologous antigen vaccine delivery vehicle. Conditional repression has the added benefit of enabling the propagation of the non-natural bacterium under standard laboratory conditions, thus allowing significant quantities of the bacterium to be produced, and furthermore to only attenuate the bacterium when it resides in the most appropriate cellular location for inducing an effective immune response.

Internalisation of bacteria into cells forming part of the body of an animal, especially cells forming the immune system, can increase efficacy of a vaccine when the bacteria are being used to vaccinate an animal against a disease. Likewise, internalisation into cancer cells can form a part of an efficacious immunotherapy against cancer. With regards to cells of the immune system, efficacy of vaccination is particularly increased when the immune cell is an Antigen Presenting Cell (APC), a type of cell which displays on its surface antigen complexed with major histocompatibility complexes. When the bacteria are internalised by an immune cell or by a cancer cell, the cellular location is typically the phagosome.

Therefore as used herein,“animal cell” can refer to any cell, including cancer cells, or fragments of cells, which either forms part of the body of an animal such as a human or other mammal, or derives ancestrally from such a cell. Preferably however,“animal cell” refers to an Antigen Presenting Cell (APC).

Plasmid loss is a well-known problem associated with live bacterial vectors, in which cell division generates plasmid-free daughter cells which do not produce recombinant protein, and therefore have a lighter metabolic burden and come to dominate a culture. It results in a significant reduction in the delivery of the desired non-natural protein. In addition to its main effects, the current invention addresses this problem by reducing cell division and therefore the incidence of plasmid-free daughter cells.

The non-natural bacterium of the invention may be genetically modified or “engineered” to comprise one or more heterologous genes encoding factors that prevent cell division, the expression of which are under the control of an inducible promoter activated when the bacterium is internalised into an animal cell.

As used herein“heterologous gene” refers to a gene that has been introduced into the bacterium, i.e. the introduction of a gene that was not previously present. The gene may be endogenous or exogenous to the bacterium, whereby these terms have their normal meaning in the art. For an endogenous gene, this may comprise introducing an additional copy or copies of said one or more endogenous genes in a heterologous manner. The endogenous gene or genes may also comprise introducing dominant variants of said gene or genes into the host bacterium, whereby“dominant” refers to the ability of the heterologous gene to functionally out-compete the naturally-occurring endogenous counterpart.

The description makes reference to various polypeptide and polynucleotide sequences, describing variants of disclosed sequences. The reference to the term“variants” is intended to encompass sequences with significant sequence identity, for example, at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity. The percentage sequence identity can be determined with the algorithm GAP, BESTFIT or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

The reference to“fragments” is intended to cover shortened versions of the specified sequences which nevertheless retain functional equivalence, i.e. can perform the same function. A fragment may comprise at least 99%, 95%, 90%, 85%, 80%, 75% or at least 70% of the specified sequence.

Numerous methods and techniques for genetically engineering bacterial strains will be well known to the person skilled in the art. These techniques include those required for introducing heterologous genes into the bacteria either via chromosomal integration or via the introduction of a stable autosomal self- replicating genetic element. Exemplary methods for genetically modifying (also referred to as "transforming" or “engineering”) bacterial cells include bacteriophage infection, transduction, conjugation, lipofection or electroporation. A general discussion on these and other methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., FlaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999 ); Protein Methods (Bollag et al., John Wiley & Sons 1996 ); which are hereby incorporated by reference.

As used herein“factor” refers to a biological molecule that has an in vivo activity. In the context of the present invention, this activity is the ability to influence the process of cell division, for example by promoting or repressing cell division. A factor may be a polypeptide or an RNA, wherein these terms have their conventional meaning in the art. In general, “factor” refers to the biological molecule that possesses the biological activity rather than the gene or DNA sequence encoding the factor itself. In some embodiments, the one or more heterologous genes encoding factors that prevent cell division are selected from the list consisting of MinC, SulA (Mukherjee et al (1998) PNAS. 95; 2885-90), SlmA (Bernhardt (2005) Mol Cell. 18: 555-64), ZapE (Ortiz et al (2016) FEMS Microbiol Rev. 40: 57-67), QueE (Yadavalli et al (2016) Nature Comm. 7: 12340), DicF (Balasubramanian et al (2016) Mol Biol & Physiol. 1 : e00021 -15), wild type FtsZ, mutant FtsZ (Strieker (2003) J Bacteriol. 185: 4796-4805; Erickson et al (2001 ) BMC Microbiology. 1 : 7; De Boer et al (2000) J. Bacteriol. 182: 5153-66), ClpXP (Camberg et al (2009) PNAS. 106: 10614-19), mutant YeeUV, or a combination thereof. In preferred embodiments, said one or more heterologous genes comprise MinC.

In some embodiments, MinC comprises the sequence encoded by SEQ ID NO:1 or a functional variant or fragment thereof. In some embodiments, SulA comprises the sequence encoded by SEQ ID NO:3 or a functional variant or fragment thereof. In some embodiments, SlmA comprises the sequence encoded by SEQ ID NO:4 or a functional variant or fragment thereof. In some embodiments, ZapE comprises the sequence encoded by SEQ ID NO:5 or a functional variant or fragment thereof. In some embodiments, QueE comprises the sequence encoded by SEQ ID NO:6 or a functional variant or fragment thereof. In some embodiments, DicF comprises the sequence encoded by SEQ ID NO:7 or a functional variant or fragment thereof. In some embodiments, mutant FtsZ comprises the sequence encoded by any one of SEQ ID NOs:8 - 10 or functional variants or fragments thereof. In some embodiments, ClpXP comprises the sequence encoded by SEQ ID NOs:1 1 or 12 or functional variants or fragments thereof.

Each of these factors prevents cell division and/or induces filamentation of the non-natural bacterium by interfering with the bacterial Min system or a downstream process of septum-production such as formation or attachment of the FtsZ ring. The Min system is required for effective cell division (Rowlett & Margolin (2013) Current Biol. 23: R553-6). For example, MinC, which along with MinD and MinE comprises one of major components of the Min system, is used by the bacterium in combination with FtsZ during cell division to identify the midpoint of the cell. Each component of the Min system operates to generate the dynamic oscillation of FtsZ from the two poles of the bacterium to promote Z- ring formation around the midpoint of the cell. The accurate determination of the midpoint of the cell is essential for enabling cell division into two equally sized progeny. The MinD and MinE components of the Min system bind and localise MinC in a negative gradient from the midpoint to the poles of the cell. As MinC is an inhibitor of FtsZ, the negative gradient allows functional FtsZ to concentrate around the midpoint of the cell to form the Z-ring structure. The introduction and expression of heterologous MinC inhibits the formation of the Z-ring by preventing the localisation of FtsZ to the cellular midpoint, thus repressing cell division.

Other factors, such as SulA and SlmA, also inhibit FtsZ polymerization. Others, such as YeeUV, inhibits FtsZ and MreB polymerization. Particular C-terminal truncation mutants of YeeV prevent binding of YeeV to MreB but not FtsZ (Tan et al (2011 ) Mol Microbiol. 79: 109-1 18). These mutants are particularly advantageous as they inhibit cell division but promote the filamentation of the cell. In some embodiments, the YeeV mutant is encoded by the sequence of SEQ ID NO:2.

Other factors, the increased expression of which supresses FtsZ polymerisation include ZapE, DicF and QueE. Other factors, such as ClpXP, degrade essential components of the Min system to prevent cell division. Conditional activation of heterologous ClpXP expression can therefore prevent cell division of the non natural bacterium.

In some embodiments, one or more of the one or more heterologous genes encode small non-coding RNAs (sRNAs) that repress the expression of one or more endogenous factors that promote cell division. The sequence of each sRNA is complementary to a portion of one mRNA encoding an endogenous factor that promotes cell division. The sRNA may have a varying degree of complementarity to the mRNA. For example, the sRNA may comprise 20 to 30 nucleotides complementary to a region of the mRNA which is to be translationally repressed. In some embodiments, the portion of the sRNA complementary to the mRNA sequence is at least 98% complementary to the target sequence of the mRNA, wherein the % complementarity refers to the exact degree of complementary base-pairing across the region of complementarity. In some embodiments, the region of complementarity in the target mRNA comprises the 5’ un-translated region (UTR) of the mRNA. The complementary sequence within the sRNA may be at the 5’ end of the sRNA. In general, base-pairing between the sRNA and the 5’UTR of the target mRNA represses translation, for example, by occluding ribosomal binding at the translation initiation site. In some embodiments, the base pairing between the sRNA and the 5’ UTR of the mRNA is stabilised by Hfq. Hfq binds to a particular nucleotide sequence in the 3’ region of the sRNA to stabilise base-pairing between the sRNA and target mRNA. Thus, in some embodiments, the sRNA will comprise an Hfq binding region 3’ to the sRNA sequence complementary to the mRNA. Principles of design and construction of sRNAs are discussed in more detail in: Yoo et a/ (2013) Nature Protocols. 8: 1694-1707, which is hereby incorporated by reference.

In some embodiments, the one or more heterologous genes are chromosomally integrated or encoded by an autonomous genetic element. Examples of self- replicating autosomal genetic elements (also referred to herein as“vectors” or “replicons”) include, but are not limited to, plasmids, to which the one or more heterologous genes may be attached so as to bring about the replication of the heterologous gene or attached segment in the target cell. Suitable genetic elements include those that enable the“stable” replication of the genetic element during cell division, i.e. stable genetic elements are those that allow the genetic element to be maintained in all or predominantly all progeny during successive rounds of cell division.

Suitable genetic elements will vary depending on the identity of the host bacterium from which the non-natural bacterium is derived. Examples of suitable genetic elements include, but are not limited to, plasmids comprising an origin of replication selected from pMB1 , ColEI, p15A, pSC101 and RK2, wherein the host bacterium is a gram-negative bacterium, or an origin of replication selected from the group consisting of pWV01 or pUB110, wherein the host bacterium is gram-positive bacteria. Other suitable origins of replication will be well known to the person skilled in the art.

The “genetic elements" described herein comprise a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

As used herein, a "promoter" refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. A promoter may also be a regulatory DNA sequence that affects the binding of RNA polymerase at the transcription initiation site. For the purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence may be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.

Promoters can be constitutively active (wherein“active” means transcription is “on”), spatially restricted or inducible. As used herein“spatially restricted” refers to a promoter that is only active in a specific subset of cells or cellular compartment of a multicellular organism. A spatially restricted promoter can thus be used to activate the expression of a nucleic acid in a particular tissue or cell type of a multicellular organism.

As used herein an“inducible promoter” refers to a promoter that enables the temporal and/or spatial activation of transcription in response to external physical or environmental stimuli. Inducible promoters include those activated by the presence of specific small molecules that alleviate transcriptional repression. For example, transcription from such an inducible promoter may be regulated by a“repressor protein”. As used herein,“repressor protein” refers to a polypeptide that binds to and occupies the inducible promoter to prevent transcription initiation. When bound to the promoter, said repressor protein can prevent binding or recruitment of RNA polymerase or associated co-factors to the transcription initiation site to prevent the activation of transcription. However, upon binding its relevant small molecule, or encountering its relevant physical or environmental stimulus, the repressor protein can no longer bind to the promoter sequence, and thus transcriptional repression is relieved. Examples of repressor proteins include lambda repressor cl and variants thereof (e.g. the thermo-labile mutant cl857 (SEQ ID NO:31 ), the arabinose repressor AraC (SEQ ID NO:33) and the tetracycline repressor TetR (SEQ ID NO:35) (Bertram &Hillen (2008) Microbial Biotechnol 1 :2-16; Rosano & Ceccarelli (2014) Frotniers Microbiol 5: 172; Love ef a/ (1996) Gene 176:49-53).

Other types of inducible promoters are those that activate transcription in response to environmental stimuli. Examples of such inducible promoters include P page P nirB P ssaG P sifA P sifB P sseA P sseG and P ssej (Dunstan et al. (1999) Infect. Immun. 67: 5133-5141 ; Xu et al. (2010) Infect. Immun. 78: 4828-4838; Kroger et al. (2013) Cell Host & Microbe 14: 683-695) _. Suitable inducible promoters for use in the invention are those that activate transcription in response to the environmental conditions of phagocytic vesicles within an animal cell, preferably wherein the animal cell is an antigen presenting cell (APC). For example, such suitable inducible promoters may activate expression of their operably linked genes at a pH in the range of pH 3 to pH 6.5, preferably at a pH in the range of pH 5.0 to pH 6.5, more preferably at a pH of about pH 5.8. The exact inducible promoter most relevant to particular embodiments of the invention will depend on the host bacterium from which the non-natural bacterium is derived. A suitable host-specific inducible promoter can be identified by culturing the host bacterium in so called“lnSPI2” conditions, which are fully described in Kroger et al. 2013, Cell Host & Microbe 14: 683-695, incorporated herein by reference. lnSPI2 conditions comprise culturing the bacterium in Phosphate Carbon Nitrogen (PCN) medium supplemented with 0.4 mM inorganic phosphate at pH 5.8. PCN medium is a medium designed to induce phagosome-dependent promoters, as described in Lober et al. 2006, Int. J. Med. Microbiol. 296: 435—447, which is hereby incorporated by reference. The bacterium can be cultured until an OD ₆ oonm of 0.3 is reached. Deep sequencing and RNA-sequencing of mRNA transcripts can then be combined to ascertain which genes are most preferentially expressed under lnSPI2 conditions in the bacterium. When compared with the transcriptome analysis of the bacterium under standard laboratory conditions, such an analysis can be used to reveal inducible promoters most selectively activated under conditions reminiscent of phagocytic vesicles of APCs.

In some embodiments, the one or more heterologous genes may be under the control of an inducible promoter selected from the list consisting of P _pag c, P _nirB , PssaG, PsifA, PsiB, PsseA, P _S seG and P _sseJ or a combination thereof. Transcription from all of P _pagC , PnirB, PssaG, PsifA, PsiB, PsseA, PsseG and P _ssej is active in phagosomes. The invention embodies variants and fragments of said inducible promoters that maintain inducible operability within phagosomes, or under lnSPI2 conditions. In some embodiments, the functional variants include those having similar or modified sequences to P _pagC , PnirB, PssaG, PsifA, Psm, PsseA, PsseG and P _ssej, and similar or substantially identical promoter activity as the wild-type promoter from which the variant is derived, particularly with respect to its ability to induce expression in vivo. Similar modified sequences may include having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type sequence of any of P _pagC , P _nirB , P _ssaG , P _s m, P _s m, P _sseA , P _sseG and P _sseJ .

In some embodiments, one or more genes encoding endogenous factors that promote cell division are genetically modified in the bacterium to have repressed expression when the bacterium is internalised into an animal cell, i.e. said one or more genes are modified to be conditionally repressed when the bacterium is internalised into an animal cell. Preferably, these one or more endogenous factors will comprise MinD and/or MinE, and repression will be via excision of the MinD and/or MinE gene sequence(s) from the genome, as can be effected by a recombinase acting on suitably placed site-specific recombination sites. Conditional repression can be achieved by engineering said one or more endogenous factors to be selectively removed or transcriptionally repressed when the bacterium is internalised into an animal cell, preferably wherein the non-natural bacterium is internalised into a phagocytic vesicle, preferably wherein the animal cell is an APC.

In some embodiments, the expression of the endogenous factor is conditionally repressed by modifying the 5’ and 3’ regions flanking the gene sequence of the one or more endogenous factors to comprise site-specific recombination sites (SSRs). When the engineered 5’ and 3’ SSRs are in the same orientation, in the presence of the cognate recombinase, the intervening sequence between the SSRs will be excised and the two SSRs will recombine, thus resulting in a single SSR in the genome in the absence of the intervening sequence. The principles of site-specific recombination are disclosed in Hallet & Sherratt (1997) FEMS Microbiol. Rev. 21 :157-178, which is hereby incorporated by reference. In this embodiment, the non-natural bacterium is further engineered to comprise a heterologous gene encoding a recombinase that recognises and cleaves the engineered SSRs. The expression of the recombinase is under the control of an inducible promoter activated when the bacterium is internalised into an animal cell such as an immune cell or a cancer cell, preferably wherein the non-natural bacterium is internalised into a phagocytic vesicle, and preferably wherein the animal cell is an APC. The inducible promoter may be any of which are described herein. Preferably, the inducible promoter is selected from the list consisting of P _pagC , PnirB, PssaG, PsitA, PsiB, PsseA, PsseG and Pssej or functional variants thereof. The heterologous gene may be chromosomally integrated or encoded by an autonomous genetic element.

The 5’ and 3’ flanking regions of the gene-encoding sequence may be modified to incorporate SSRs using standard genetic engineering techniques. For example, the CRISPR/Cas system may be used to site-specifi cally cleave the double-stranded DNA at sites immediately adjacent to the region where the SSR is to be inserted. A template strand can be used to guide recombination of the cleaved DNA to insert the desired site-specific recombination sites. The template strand may comprise the SSR flanked by 5’ and 3’ sequences complimentary to the regions either side of the DNA cleavage site, thus allowing the template-directed insertion of the SSR into the DNA cleavage site. Desired recombination sites can be added to target genome sites in any way known to the skilled person, for example through the use of targeted homologous recombination protocols employing antibiotic selection steps, such as the lambda Red homologous recombination system.

The site-specific recombination site may be loxP (SEQ ID NO:27), wherein the heterologous gene encoding the cognate recombinase comprises Cre (SEQ ID NO:26), or a functional variant thereof. Or, the site-specific recombination site may be FRT (SEQ ID NO: 29), wherein the heterologous gene encoding the cognate recombinase comprises Flp (SEQ ID NO:28, or a functional variant thereof.

In some embodiments, the one or more endogenous factors that promote cell division, the expression of which are to be conditionally repressed, are selected from the list consisting of minD, minE, ftsZ, ftsA, zipA, ftsQ, ftsL, ftsl, ftsN. xerC, xerD, ftsK, or a combination thereof, preferably being selected from minD and/or minE. In some embodiments, the one or more endogenous factors to be excised from the bacterium by conditionally activated site-specific recombination comprises the dif site (SEQ ID NO: 22), or a functional fragment thereof. The dif site is a 28 bp DNA sequence in the terminus region of the bacterial chromosome that is the target site of site-specific recombinases XerC and XerD. XerCD recombines dif to segregate sister chromosomes following chromosome replication. Inhibition of Xer recombination or deletion of dif results in filamentation (Kuempel et al (1991 ), New Biol 3: 799-81 1 ).

In some embodiments, the expression of genes encoding said one or more endogenous factors that promote cell division are modified to be under the control of a repressor protein. The endogenous factors may be engineered to be under transcriptional control of the repressor protein by engineering the endogenous transcriptional promoter site to comprise an inducible promoter to which the repressor protein can bind and repress transcription. Said endogenous promoter site can be engineered by using methods conventional in the art such as de novo synthesis of the promoter. The non-natural bacterium is further engineered to comprise a heterologous gene encoding the cognate repressor protein. The expression of said repressor protein is under the control of an inducible promoter activated when the bacterium is internalised into an animal cell such as an immune cell or a cancer cell, preferably wherein the cell is internalised into a phagocytic vesicle, preferably wherein the cell is an APC. In some embodiments, the inducible promoter is selected from the list consisting of Ppagc, PnirB, PssaG, Pam, PsifB, PsseA, PsseG and P _ssej , or functional variants thereof. In some embodiments, the repressor protein is selected from the list consisting of the lambda repressor cl, the thermo-labile lambda repressor mutant cl857, the arabinose repressor AraC, the tetracycline repressor TetR, or functional variants or fragments thereof. In some embodiments, the repressor protein is encoded one of the sequences selected from the list consisting of SEQ ID NO:31 , SEQ ID NO:33 or SEQ ID NO:35. The heterologous gene encoding the repressor protein may be chromosomally integrated or encoded by an autonomous genetic element.

In some embodiments, the non-natural bacterium of the invention is genetically modified such that one or more genes encoding endogenous factors that repress cell division are under the control of an inducible promoter activated when the bacterium is internalised into an animal cell, preferably wherein the bacterium is internalised into a phagocytic vesicle, preferably wherein the animal cell is an APC.

In some embodiments, the one or more endogenous factors that repress cell division are selected from the list consisting of MinC, SulA, SlmA, ZapE, QueE, DicF, ClpXP, or a combination thereof, but preferably comprise MinC. The inducible promoter may be selected from the list consisting of P _pag c, P _nirB , P _ssaG , PsifA, PsitB, PsseA, PsseG and P _ssej, or functional variants thereof.

In some embodiments, the inducible promoter used in any of the forgoing embodiments of the invention comprises SEQ ID NO: 36 or an operable variant thereof. In some embodiments the bacterium is further modified to partially or completely suppress expression of one or more chromosomally-encoded genes, with the effect of attenuating the bacterium. In these embodiments, the activity of the suppressed gene(s) is instead provided by a complementary copy which is encoded on the same autonomous genetic element encoding the one or more other heterologous genes of the invention. Preferably, these other heterologous genes are those encoding the one or more factors that prevent cell division. Preferably, the complementary copy is under the control of its native promoter, i.e. the promoter which activates the transcription of the endogenous essential gene in vivo. Such a strategy ensures that if a bacterium loses the autonomous genetic element suppressing cell division, thereby recommencing cell division, it will be attenuated. In some embodiments the one or more essential genes is selected from the list consisting of aroA, aroC, purA, purE, asd, cya, crp, clpX, galE, guaA, guaB, htrA, msbB, phoP, phoQ, pmi, pur or a combination thereof. The attenuation of the essential gene can be achieved by excising the chromosomal gene, or introducing attenuating mutations into the coding sequence of the essential gene, or by repressing the expression of the essential gene. In some embodiments, the host bacterium will comprise basal attenuating mutations or deletions of the one or more essential genes. In some embodiments, wherein the host bacterium is derived from Salmonella spp. the one or more essential genes may be additionally selected from ssaV, ssaJ, ssaK, ssaU, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssal, ssaC, and ssaH. The essentiality of each of these genes is described in WO 2009/158240, which is hereby incorporated by reference.

In some embodiments, the non-natural bacterium of the invention is further engineered to express a heterologous gene encoding a heterologous antigen. As used herein, an“antigen” refers to a molecule that is capable of inducing an immune response in a host. The heterologous antigen may be under to control of a constitutive, spatially-restricted, or inducible promoter. The inventors have surprisingly found that the conditional repression of cell division coupled with increased filamentation of the non-natural bacterium attenuates the bacterium and improves immunogenicity. Thus, the non-natural bacterium of the invention is an effective vaccine carrier.

In preferred embodiments the non-natural bacterium expresses an antigenic protein encoded by, for example, a plasmid it carries which encodes a prokaryotic promoter operably linked with a heterologous gene. In some embodiments, however, the heterologous gene may represent the delivery system for a DNA vaccine. In these embodiments, the heterologous gene is encoded on a genetic element such as a plasmid, and is operably linked with a promoter which functions in animal cells. Upon uptake of the genetic element by an animal cell such as an immune cell or cancer cell, the animal cell’s native expression machinery internally produces the antigenic protein, and the effects of vaccination are thereby elicited.

The ability of the non-natural bacterium to induce an immune response against the expressed heterologous antigen can be tested by, for example, immunising animal hosts such as mice. The bacterial vaccine can be administered via, for example, the orogastric, intranasal or intraperitoneal routes. The bacteria then invade or are phagocytosed by antigen-presenting cells which induce antigen expression, leading to the generation of an immune response against said antigen by presentation via Major Histocompatibility Complex (MHC) Class I and II pathways. Numerous tests of this process and the concomitant immunity are known to the skilled person.

In some embodiments, the heterologous antigen is derived from a pathogenic organism selected from the group consisting of virus, bacterium, fungus, or other eukaryotic parasite.

Wherein the heterologous antigen is derived from a virus, the heterologous antigen may be derived from a virus selected from the list consisting of Dengue Fever Virus, Ebola Virus, Encephalomyocarditis Virus, Hepatitis A, B and C Viruses, Herpes Simplex Virus, Human Enterovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Human Parvovirus, Human Rhinovirus, Influenza Virus, Lymphocytic Choriomeningitis Virus, Middle East Respiratory Syndrome Virus, Mouse Mammary Tumour Virus, Rabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, Zika Virus or a combination thereof. Conventional heterologous antigens derived each of these viruses capable of inducing immune responses in a host are well known to the skilled person.

Wherein the heterologous antigen is derived from a bacterium, the heterologous antigen may be derived from a bacterium selected from the list consisting of Aerococcus spp., Aeromonas hydrophila, Actinobaculum schaalii, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Alloscardovia omnicolens, Anaplasma phagocytophilum, Bacillus spp. (including B. anthracis and B. cereus ), Bacteroides fragilis, Bartonella spp. (including B. henselae), Bordetella spp. (including B. pertussis), Borrelia spp. (including B. burgdorferi), Brucella spp. (including B. abortus), Burkholderia spp. (including B. cepacia), Campylobacter jejuni, Chlamydia spp. (including C. trachomatis), Chlamydophila pneumoniae, Clostridium spp. (including C. botulinum, C. difficile and C. tetani), Corynebacterium diptheriae, Enterobacter spp. (including E. aerogenes and E. cloacae), Enterococcus spp. (including E. faecalis and E. faecium), Escherichia coli (including ETEC and 0157:H7), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira spp., Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium spp. (including M. avium, M. bovis and M. tuberculosis), Mycoplasma pneumoniae, Neisseria spp. (including N. gonorrhoeae and N. meningitidis), Neoehrlichia mikurensis, Nocardia spp., Proteus mirabilis, Pseudomonas spp. (including P. aeruginosa and P. stutzeri), Rickettsia spp., Salmonella spp. (including S. enterica), Serratia marcescens, Shigella spp. (inclusing S. dysenteriae, S. flexneri and S. sonnei), Staphylococcus spp. (including S. aureus and S. epidermis), Stenotrophomonas maltophilia, Streptococcus spp. (including S. pyogenes), Treponema pallidum, Tropheryma whipplei, Ureaplasma urealyticum, Vibrio cholera, Yersinia spp. (including Y. pestis and Y. pseudotuberculosis) or combinations thereof. Conventional heterologous antigens derived each of these bacteria capable of inducing immune responses in a host are well known to the skilled person. Wherein the heterologous antigen is derived from a fungus, the heterologous antigen may be derived from a fungus selected from the list consisting of Aspergillus spp. (including A. niger), Blastomyces spp., Candida spp. (including C. albicans, C. auris and C. tropical is), Coccidioides immitis, Cryptococcus neoformans, Gibberella moniliformis, Histoplasma capsulatum, Fonsecae pedrosoi, Fusarium oxysporum, Microsporum spp., Pneumocystis jirovecii, Rhizopus oryzae and Trichophyton spp. Other eukaryotic pathogens include Acanthamoeba spp., Ascaris lumbhcoides, Babesia spp., Bruga spp. (including B. malayi and B. timori), Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Leishmania spp. including ( L donovani and L. major), Necator americanus, Parachlamydia acanthamoebae, Plasmodium spp. (including P. falciparum and P. vivax) Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trypanosoma spp. (including T cruzi and T brucei) and Wuchereria bancrofti. Conventional heterologous antigens derived each of these fungi capable of inducing immune responses in a host are well known to the skilled person.

In some embodiments, the heterologous antigen is a cancer vaccine. Exemplary cancer antigens include tumour-specific proteins, tumour-associated proteins, growth factors or angiogenesis factors. In some embodiments, the cancer antigen may be selected from the list consisting of AFP (alpha-fetoprotein), CA- 125 (ovarian cancer antigen 125), CEA (carcinoembryonic antigen), endoglin (CD105; including transforming growth factor-beta receptor: TGF-beta), ETA (epithelial tumour antigen), Fler2/neu (receptor tyrosine-protein kinase erbB-2), FIMW-MAA (human high molecular weight-melanoma-associated antigen), ISG15 (interferon-stimulated gene 15), MAGE (melanoma-associated antigen), MUC1 (mucin 1 , cell surface associated), NY-ESO-1 (New York esophageal squamous cell carcinoma 1 ), p53, p60, PSA (prostate-specific antigen), survivin (an apoptosis protein), TRP2 (tyrosinase-related protein 2), VEGFR2 (vascular endothelial growth factor receptor 2), or antigenic fragments thereof. As defined herein,“antigenic fragment” refers to a portion of the antigen that is capable of inducing an immune response in the host. In some embodiments, the host bacterium from which the non-natural bacterium is derived is selected from the list of genera consisting of Bacillus spp. (including B. subtilis) Bifidobacterium spp., Clostridium spp., Lactobacillus spp., Lactococcus spp., Listeria spp. (including L. monocytogenes ) or Mycobacterium spp. (including M. bovis, M. smegmatis or M. tuberculosis), Bordetella spp. (including B. pertussis), Pseudomonas spp. and members of the family Enterobacteriaceae: Citrobacter spp., Escherichia spp. (including E. coli), Klebsiella spp., Proteus spp., Salmonella spp., Serratia spp. Shigella spp. (including S. dysenteriae, S. flexneri and S. soneii), Vibrio spp (including V. cholerae) and Yersinia spp. In some embodiments, the bacterium is selected from the genus Salmonella, preferably wherein the bacterium is Salmonella enterica serovar Typhimurium, Typhi or Paratyphi. In some embodiments, the host bacterium may be derived from a Salmonella enterica with a basal mutation in aroA, aroC, ssaV, or a combination thereof, whereby“basal” refers to the bacterium already comprising the attenuating mutation within the endogenous gene.

In a second aspect of the invention, there is provided the non-natural bacterium for use in the treatment or prevention of a disease. In some embodiments, the non-natural bacterium is for use in the treatment or prevention of a disease in an animal. In some embodiments, the animal is a human.

In some embodiments, the disease to be treated or prevented is cancer. The cancer may be selected from the list consisting of Adenoid Cystic Carcinoma, Adrenal Gland Tumour, Amyloidosis, Anal Cancer, Ataxia-Telangiectasia, Atypical Mole Syndrome, Beckwith Wiedemann Syndrome, Bile Duct Cancer, Birt Hogg Dube Syndrome, Bladder Cancer, Bone Cancer, Brain Tumour, Breast Cancer, Carcinoid Tumour, Carney Complex, Cervical Cancer, Colorectal Cancer, Ductal Carcinoma, Endometrial Cancer, Oesophageal Cancer, Familial- Adenomatous Polyposis, Gastric Cancer, Gastrointestinal Stromal Tumour, HER2-Positive Breast Cancer, Hereditary Prostate Cancer, Islet Cell Tumour, Juvenile Polyposis Syndrome, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Acute Lymphoblastic Leukaemia, Acute Myeloid Leukaemia, Chronic Lymphocytic Leukaemia, Chronic Myeloid Leukaemia, Acute Lymphocytic Leukaemia, Liver Cancer, Lobular Carcinoma, Lung Cancer, Small Cell Lung Cancer, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Lynch Syndrome, Malignant Glioma, Mastocytosis, Melanoma, Meningioma, Multiple Endocrine Neoplasia Type 1 , Multiple Endocrine Neoplasia Type 2, Multiple Myeloma, Myelodysplastic Syndrome (MDS), Nasopharyngeal Cancer, Neuroendocrine Tumour, Nevoid Basal Cell Carcinoma Syndrome, Oral Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumours, Parathyroid Cancer, Penile Cancer, Peritoneal Cancer, Peutz-Jeghers Syndrome, Pituitary Gland Tumour, Pleuropulmonary Blastoma (Childhood), Polycythemia Vera, Prostate Cancer, Renal Cell Cancer, Retinoblastoma (Childhood), Salivary Gland Cancer, Sarcoma, Sarcoma - Alveolar Soft Part and Cardiac, Sarcoma - Kaposi, Skin Cancer (Non-Melanoma), Small Bowel Cancer, Small Intestine Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymoma, Thyroid Cancer, Turcot Syndrome, Uterine (Endometrial) Cancer, Vaginal Cancer, Von-Hippel-Lindau Syndrome and Wilms' Tumour (Childhood).

In some embodiments, the heterologous gene encoding the tumour antigen is cognate to the cancer to be treated. In other words, the heterologous tumour antigen can be a tumour-specific antigen expressed in the tumour cells of the cancer to be treated. In some embodiments, the heterologous tumour antigen is a tumour-associated antigen (TAA), whereby a“TAA” refers to an antigen that is expressed at elevated levels in the tumour cells.

In another aspect of the invention, there is provided the use of the non-natural bacterium of the first aspect of the invention in the vaccination of a subject against a disease. The disease may be any of those as described in the second aspect of the invention.

In another aspect of the invention, there is provided a method of manufacturing the non-natural bacterium according to any of the previous aspects of the invention, by introducing the genetic modifications disclosed herein.

The invention is illustrated by the following non-limiting examples: Example 1

To construct the low copy number expression plasmid pSCT4minCOX, primers minCOXF and minCOXR were used to amplify the minC gene of SL3261 genomic DNA. The PCR product and expression plasmid pSCT4c were digested using Ndel and Sail and ligated to generate the precursor plasmid pSCT4cminCOX. pSCT4cminCOX has minC under the control of the phagosome-induced SPI-2 promoter P _SS aG, which is not active in nutrient broths such as LB or TB. PCN (Phosphate Carbon Nitrogen minimal medium) with a low concentration of inorganic phosphate was used to induce the Salmonella SPI-2 regulon. An E. coli pepA mutant strain was used for cloning operations. The pSCT4cminCOX plasmid was transformed into S. Typhimurium SL3261 , and transformants were selected on LB agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB broth in the absence of antibiotic. Xer recombination resulted in the deletion of the cat gene to generate chloramphenicol-sensitive colonies of SL3261 (pSCT4minCOX).

To evaluate the effect of minC overexpression in the morphology of bacteria, a single colony of SL3261 (pSCT4minCOX) was inoculated into LB broth and incubated for 16 hours at 37°C and 200 rpm. This starter culture was used to inoculate PCN medium (pH 5.8) which was incubated for 6 hours at 37°C and 200 rpm. A volume of culture equivalent to an optical density of A ₆ oo = 2 was collected and washed in PBS. A small volume of cells were applied on a microscope slide and observed under the microscope (Figure 6).

The results show the repressed cell division and induced filamentation of the non-natural bacterium comprising the MinC expression plasmid after 6 h. The control bacterium does not show this phenotype.

Example 2

The RAW 264.7 murine macrophage cell line was cultured in DMEM containing 2 mM L-glutamine and 10% Foetal Bovine Serum. Cells were seeded at a density of 2 x 10 ⁵ cells per well in 24-well plates containing glass coverslips and grown for approximately 30 hours. RAW 264.7 cells were infected with S. Typhimurium SL3261 (pSCT4minCOX), incubated for 2 or 16 hours, washed with PBS and fixed using 3% formalin. RAW 264.7 cells were permeabilised using 0.1 % Triton X-100 and subsequently blocked using Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) containing 3% Bovine Serum Albumin for 1 hour. Bacterial cells were then stained using an anti-LPS monoclonal antibody (IE6) directly conjugated to Dylight 488 fluorochrome for 30 minutes (Figure 7).

RAW 264.7 cells were also infected with S. Typhimurium strains MD58 ( aroC ) and WT05 ( aroC , ssaV) containing plasmid pBRTI RFP in which Red Fluorescent Protein (RFP, SEQ ID NO: 40) is under the control of the P _ssa G promoter. The gene encoding monomeric red fluorescent protein was codon- optimised for expression in E. coli and synthesised in plasmid pMA-mRFP. The 695bp fragment containing the RFP cistron was cloned into plasmid pBRTI Nc as an Ndel-Sall fragment. S. Typhimurium strains for infection were used in parallel that additionally contained pSCT4minCOX (Figure 8).

Coverslips were mounted on glass microscope slides using Poly-Mount (Polysciences) and visualised using a fluorescent microscope (Zeiss Axiophot) with attached Zeiss Axiocam camera. Images were captured at 400x magnification unless otherwise stated. Fluorescence imaging shows that at the 16 h time point the non-natural bacterium show the induced filamentation phenotype within the RAW 264.7 cells.

Example 3

Low copy number expression plasmids containing sequences encoding queE and three selected mutant variants of ftsZ under the control of phagosome- induced promoter P _SS aG were constructed as follows. A 960 bp DNA fragment containing the sequence encoding 314 N-terminal amino acids of FtsZ was amplified by PCR from Salmonella Typhi ZFI9 genomic DNA using primers ftsZ(1-314)Ndel and ftsZ(1 -314)Xbal. The PCR product was digested with Ndel- Xbal and cloned into pSCT4c to generate plasmid pSCT4cFtsZ-1/314. Plasmids pSCT4cFtsZ-D45A and pSCT4cFtsZ-E250K-D253K contain the DNA sequences encoding for respectively ftsZ mutant cistrons FtsZ(D45A) and FtsZ(E250K- D253K). The DNA fragments encoding two selected ftsZ cistrons were synthesised and cloned in plasmids pUC57-kan-ftsZ-D45A and pUC57-kan-ftsZ- E250K-D253K. The 1 175bp Ndel-Sall inserts were subcloned into pSCT4c to generate final plasmids pSCT4cFtsZ-D45A and pSCT4cFtsZ-E250K-D253K. To construct plasmid pSCT4cqueE, primers queE-Ndel and queE-Sall were used to amplify queE gene from ZFI9 genomic DNA. The 688bp PCR product was digested with Ndel-Sall and cloned into pSCT4c to generate final plasmid pSCT4cqueE. An E. coli pepA mutant strain was used as host in all cloning operations. Salmonella Typhimurium strain MD58 was transformed with the recombinant expression plasmids and transformants were selected on LB-aro mix plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic selection to allow deletion of the chloramphenicol acetyltransferase {cat) gene via Xer recombination and generation of the following final strains: MD58(pSCT4FtsZ-1/314), MD58(pSCT4FtsZ-D45A), MD58(pSCT4FtsZ-E250K- D253K) and MD58(pSCT4queE).

To study the effect on cell division of ftsZ mutants and of queE overexpression, single colonies of the four MD58 derived strains above described were inoculated in LB-aro and incubated for 20 hours at 37°C. MD58 strain with no plasmid was included as a negative control. The overnight LB-aro mix cultures were used to inoculate PCN medium (pH 5.8) for P _ssa G induction. After 7 hours growth at 37°C in PCN medium, 10 mI of bacterial cultures were applied to a microscope slide and visualised in phase contrast using a Zeiss Axiophot microscope at 1000x magnification (Figure 9). Control strain MD58 grew normally in PCN and maintained a rod-shaped morphology. Flowever, expression of all three FtsZ mutants and of QueE resulted in a filamentous cellular morphology.