# Patent

## Title
Site specific Listeria integration vectors and methods for using the same

## Abstract

Site-specific Listeria integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is the source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a polypeptide coding sequence, e.g., for a heterologous antigen. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria vaccines.

## Background
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with Government support under Grant Nos. 1 R37 Al29619 and 1 R01 Al27655 awarded by the National Institute of Health. The Government has certain rights in this invention.


INTRODUCTION
BACKGROUND OF THE INVENTION
The field of this invention is Listeria species, e.g., Listeria monocytogenes, particularly recombinant strains of Listeria species, and methods for their fabrication and use.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Listeria monocytogenes is a Gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women. Severe L. monocytogenes infections in humans are characterized by meningitis, meningoencephalitis, septicemia, and fetal death. L. monocytogenes is ubiquitous in nature and, in addition, can be isolated from a wide variety of warm blooded animals.
Protocols for recombinantly engineering Listeria species are of interest in both research and therapeutic applications. For example, Listeria species transformation protocols find use in the elucidation of the mechanisms responsible for growth and virulence of these types of bacteria. In addition, such protocols also find use in the preparation of live Listeria vaccines, which vaccines find use in a variety of different medical applications.
To date, a variety of different protocols have been employed to transform Listeria species, where such protocols include: homologous recombination, transposon based recombination, etc. While different protocols are currently available for engineering Listeria species, such methods are not entirely satisfactory. Disadvantages currently experienced with one or more of the available protocols include: (1) instability of the expression cassette in the transformed species; (2) variable impact on virulence of the transformed species; (3) size constraints of the expression cassette that can be placed in the transformed species; etc.
As such, despite the variety of different transformation protocols available, there is continued interest in the identification of further transformation protocols that can expand the repertoire of available genetic tools. Of particular interest would be the development of an efficient, site-specific integration vector that was suitable for use with a wide array of different Listeria species, where the vector did not suffer from one or more of the above disadvantages of the currently available protocols.
Relevant Literature
Patents and published patent applications of interest include: U.S. Pat. No. 5,830,702 and published PCT application serial nos.: WO 99/25376 and WO 00/09733.
SUMMARY OF THE INVENTION
Site-specific Listeria species integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a coding sequence, e.g., a coding sequence for a heterologous polypeptide, etc. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria species vaccines.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. (A) Plasmid map of the pPL1 integration vector. Chloramphenicol resistance genes and E. coli origin of replication are shown in grey, RP4 origin of transfer shown in white, integrase gene and L. monocytogenes p60 promoter shown in black. Multiple cloning site is shown at the bottom of the plasmid with unique restriction sites noted below the MCS in a box. pPL24 and pPL25 inserts are shown schematically below the MCS and were cloned as described in Materials and Methods. Final sizes of the plasmid constructs and the restriction sites used in cloning are noted with each of the inserts. (B) Plasmid map of the integration vector pPL2. The color scheme and genes are the same as in FIG. 1A except the PSA integrase and PSAattPP′ sites as noted. The multiple cloning site with 13 unique restriction sites is shown at the bottom of the plasmid.
FIG. 2. Genomic organization of the attachment sites within the comK gene (A and B) and the tRNAArg gene (C and D). (A) Non-lysogenic L. monocytogenes strain, with an intact comK gene. Primers PL60 and PL61 amplify across the bacterial attachment site comK-attBB′. (B) Lysogenic L. monocytogenes strain (with approximately 40 kb of phage DNA inserted into the comK gene) or integrated strain (with pPL1 construct inserted into the comK gene). Primers PL14 and PL61 amplify across the hybrid attachment site comK-attPB′. (C) L. monocytogenes serotype 4b strain non-lysogenic at tRNAArg-attBB′. Primers NC16 and NC17 amplify across the bacterial attachment site tRNAArg-attBB′ in serotype 4b strains. Asterisk indicates primers NC16 and NC17 are substituted with PL102 and PL103 to amplify across the bacterial attachment site tRNAArg-attBB′ in serotype 1/2 strains. (D) Lysogenic L. monocytogenes strain (with approximately 40 kb of phage DNA or 6 kb pPL2 vector DNA inserted at the 3′ end of the tRNAArg gene. Primers NC16 and PL95 amplify across the hybrid attachment site tRNAArg-attBP′ in both serotype 4b and 1/2 strains.
FIG. 3. Expression and functional complementation of ActA in SLCC-5764. (A) Coomassie blue stained SDS-PAGE of SLCC-5764 derived strains grown to late-log phase. ActA is indicated by an arrow. Lane 1: molecular size marker; lane 2: DP-L3780; lane 3: DP-L4083; lane 4: DP-L4086; lane 5: SLCC-5764; lane 6: DP-L4082; lane 7: DP-L4084; lane 8: DP-L4085; lane 9: DP-L4087. Strains are described in Table 1. (B) Actin tail formation and movement of DP-L4087 in Xenopus cell extract. The top panel is a phase image; the bottom panel is a fluorescent image of the same field.
FIG. 4. (A) Clover-leaf diagram of tRNAArg utilized as the PSA attachment site. The arginine anticodon is circled. The region with sequence identity between the tRNA gene and the PSA attPP′ is outlined. The boundaries of the tRNAArg gene and Cove score (82.37) were predicted with tRNAscan-SE (31). (B) Alignment of the tRNAArg-attBB′ region of L. monocytogenes WSLC 1042 (top line) and the attPP′ region of PSA downstream of the integrase gene. The 74 nt tRNAArg gene of L. monocytogenes is boxed and the 17 bp overlapping region of sequence identity (core integration site) is shaded grey. The tRNAArg gene anticodon is shown in bold and underlined. Identical nucleotide residues are indicated by (:). The numbers located at the left indicate the nucleotide position in the DNA sequences of the WSLC 1042 attachment site (AJ314913) and PSA genome (AJ312240).



DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Site-specific Listeria species integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a coding sequence, e.g., for a heterologous polypeptide, etc. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria species vaccines.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference for the purpose of describing various invention components that are described in the publications which might be used in connection with the presently described invention.
In further describing the subject invention, the subject vectors are reviewed first in greater detail, followed by a review of the methods of using the subject vectors, as well as representative applications in which the subject vectors and methods find use.
Vectors
As summarized above, the subject invention provides Listeria site-specific integration vectors, i.e., vectors that integrate into Listeria genomes in a site-specific manner. The subject vectors are characterized in that they stably integrate into a genome of a Listeria species in a site-specific, as opposed to random, manner. The subject vectors are capable of integrating into the genomes of a wide variety of different Listeria species. Additional features of many embodiments of the subject vectors is that they replicate autonomously in a non-Listeria host cell, e.g., E. coli, and are stable and innocuous in such non-Listeria host cells.
The subject integration vectors are typically double-stranded plasmid vectors, where the vectors are generally at least about 3 kb, often at least about 5 kb and may be large as 15 kb, 20 kb, 25 kb, 30 kb or larger, where the vectors sometimes range in size from about 3-6 to about 20 kb. The vectors include a number of structural features that impart to the vectors the above-described functional characteristics.
One structural feature of the subject vectors is a bacteriophage integrase gene, i.e., a nucleic acid coding sequence for a bacteriophage integrase, which is operably linked to a Listeria specific promoter, such that the gene (coding sequence) is expressed in the Listeria cell for which the vector is designed to be employed. In many embodiments, the bacteriophage integrase gene is one obtained from a listeriophage, i.e., it is a listeriophage integrase gene. A variety of different listeriophages are known in the art, where any convenient listeriophage may serve as the source of the integrase gene, i.e., the integrase encoding nucleic acid. Specific integrases of interest include, but are not limited to: the U153 integrase, the PSA integrase; and the like.
As indicated above, the integrase gene is operably linked to a promoter (as well as regulatory and/or signal sequences, if necessary) that drives expression of the integrase gene when the vector is present in the Listeria cell for which the vector is designed and in which integration of the vector is desired. Any convenient promoter may be employed, where in certain embodiments the promoter is a Listeria specific promoter. Representative promoters of interest include, but are not limited to: the Listeria p60 promoter, the Listeria actA promoter, the Listeria plcA promoter, the Listeria mpl promoter, the Listeria plcB promoter, the Listeria inlA promoter; a heat shock promoter; and the like Promoters may also include certain bacteriophage promoters such as the promoters for T7, Qβ, SP6 and the like if the strain of Listeria also expresses the cognate bacteriophage RNA polymerase. In addition to the integrase gene/promoter element described above, the subject vectors also include a phage attachment site, i.e., a sequence or domain of nucleotides that provide for a site-specific integration with a Listeria genome. Any convenient phage attachment site may be employed, where selection of the phage attachment site will depend, at least in part, on the desired integration location for the vector. Representative phage attachment sites of interest include, but are not limited to: the U153 attPP′ attachment site (as described in greater detail in the experimental section below); the PSA attPP′ attachment site (as described in greater detail in the experimental section below); and the like.
In addition, the subject vectors may include an origin of replication that provides for replication of the vector in a non-Listeria host cell, e.g., E. coli. This origin of replication may be any convenient origin of replication or ori site, where a number of ori sites are known in the art, where particular sites of interest include, but are not limited to: p15A; pSC101; ColEl; pUC; pMB9; and the like.
In addition, the subject vectors may include an origin of transfer site or element when convenient, e.g., when the vector is introduced in the target Listeria cell using a conjugation protocol, as described in greater detail below. Any convenient origin of transfer (oriT) may be employed, where representative origins of transfer of interest include, but are not limited to: RP4 oriT; RSF1010 oriT; and the like.
In addition, the subject vectors typically include at least one restriction endonuclease recognized site, i.e., restriction site, which is located on the vector at a location which is amenable to insertion of a heterologous gene/expression cassette. A variety of restriction sites are known in the art and may be present on the vector, where such sites include those recognized by the following restriction enzymes: HindIII, PstI, SalI, AccI, HincII,XbaI, BamHI, SmaI, XmaI, KpnI, SacI, EcoRI, BstXI, EagI, NotI, SpeI and the like. In many embodiments, the vector includes a polylinker or multiple cloning site, i.e., a closely arranged series or array of sites recognized by a plurality of different restriction enzymes, such as those listed above.
In certain embodiments, the subject vectors include at least one coding sequence, e.g., a coding sequence for heterologous polypeptide/protein coding sequence present in the multiple cloning site, e.g., as a result of using a restriction endonuclease site present in the multiple cloning site to insert the coding sequence into the vector, according to well known recombinant technology protocols. By “heterologous” is meant that the coding sequence encodes a product, e.g., a protein, peptide, polypeptide, glycoprotein, lipoprotein, or other macromolecule, that is not normally expressed in Listeria. In many embodiments, this coding sequence is part of an expression cassette, which provides for expression of the coding sequence in the Listeria cell for which the vector is designed. The term “expression cassette” as used herein refers to an expression module or expression construct made up of a recombinant DNA molecule containing at least one desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism, i.e., the Listeria cell for which the vector is designed, such as the promoter/regulatory/signal sequences identified above, where the expression cassette may include coding sequences for two or more different polypeptides, or multiple copies of the same coding sequence, as desired. The size of the coding sequence and/or expression cassette that includes the same may vary, but typically falls within the range of about 25-30 to about 6000 bp, usually from about 50 to about 2000 bp. As such, the size of the encoded product may vary greatly, and a broad spectrum of different products may be encoded by the expression cassettes present in the vectors of this embodiment.
The nature of the coding sequence and other elements of the expression cassette may vary, depending on the particular application of the vector, e.g., to study Listeria species, to produce Listeria species vaccines, for cytosolic delivery of macromolecules, etc. For example, where the vectors are employed in the production of Listeria vaccines, the coding sequence may encode a heterologous antigen, where representative heterologous antigens of interest include, but are not limited to: (a) viral antigens, e.g., influenza np protein, HIV gag protein, HIV env protein or parts thereof, e.g., gp120 and gp41, HIV nef protein, HIV pol proteins, HIV reverse transcriptase, HIV protease, herpes virus proteins, etc., (b) malarial antigens; (c) fungal antigens; (d) bacterial antigens; (e) tumor and tumor related antigens; and the like. Due to the flexibility of the vector system, virtually any coding sequence of interest may be inserted. Where secretion of the product encoded by the expression cassette is desired, the expression cassette may include a coding sequence for a fusion protein of a selected foreign antigen and a protein that directs secretion, e.g., Listeriolysin O or PI-PLC; a signal sequence, such as hemolysin signal sequence, etc. Where the subject vectors are employed in the preparation of Listeria delivery vehicles, e.g., as described in PCT publication no. WO 00/09733 (the priority application of which is herein incorporated by reference); and Dietrich et al., Nature Biotechnology (1998) 16: 181-185, the heterologous polypeptide coding sequence may be a cytolysin, e.g., phospholipase, pore forming toxin, listeriolysin O, streptolysin O, perfringolysin O, acid activated cytolysins, phage lysins, etc. Other coding sequences of interest include, but are not limited to: cytokines, costimulatory molecules, and the like. As indicated above, the vector may include at least one coding sequence, where in certain embodiments the vectors include two or more coding sequences, where the coding sequences may encode products that act concurrently to provide a desired results.
In general, the coding sequence may encode any of a number of different products and may be of a variety of different sizes, where the above discussion merely provides representative coding sequences of interest.
Methods of Preparing the Subject Vectors
The vectors of the subject invention may be produced using any convenient protocol, including by standard methods of restriction enzyme cleavage, ligation and molecular cloning. One protocol for constructing the subject vectors includes the following steps. First, purified nucleic acid fragments containing desired component nucleotide sequences as well as extraneous sequences are cleaved with restriction endonucleases from initial sources. Fragments containing the desired nucleotide sequences are then separated from unwanted fragments of different size using conventional separation methods, e.g., by agarose gel electrophoresis. The desired fragments are excised from the gel and ligated together in the appropriate configuration so that a circular nucleic acid or plasmid containing the desired sequences, e.g. sequences corresponding to the various elements of the subject vectors, as described above is produced. Where desired, the circular molecules so constructed are then amplified in a host, e.g. E. coli. The procedures of cleavage, plasmid construction, cell transformation and plasmid production involved in these steps are well known to one skilled in the art and the enzymes required for restriction and ligation are available commercially. (See, for example, R. Wu, Ed., Methods in Enzymology, Vol. 68, Academic Press, N.Y. (1979); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982); Catalog 1982-83, New England Biolabs, Inc.; Catalog 1982-83, Bethesda Research Laboratories, Inc. An example of how to construct a vector of the present invention is provided in the Experimental section, below.
Methods
Also provided by the subject invention are methods of using the above described Listeria site specific integration vectors to integrate a heterologous nucleic acid into a Listeria genome. In practicing the subject methods, a vector of the subject invention is introduced into a target Listeria cell under conditions sufficient for integration of the vector into the target cell genome to occur. Any convenient protocol for introducing the vector into the target cell may be employed.
Suitable protocols include: calcium phosphate mediated transfection; protoplast fusion, in which protoplasts harboring amplified amounts of vector are fused with the target cell; electroporation, in which a brief high voltage electric pulse is applied to the target cell to render the cell membrane of the target cell permeable to the vector; microinjection, in which the vector is injected directly into the cell, as described in Capechhi et al, Cell (1980) 22: 479; and the like. The above in vitro protocols are well known in the art and are reviewed in greater detail in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press)(1989) pp16.30-16.55.
In certain embodiments, e.g., where direct introduction into the target Listeria cell does not provide optimal results, one representative method that may be employed is a conjugation method, which comprises: (a) introducing the vector into a non-Listeria host cell, e.g., E. coli; followed by (b) transfer of the vector from the non-Listeria host cell to the Listeria host cell, e.g., by conjugation. Introduction into the non-Listeria host cell may be accomplished using any of the protocols described above. For the conjugation step, any convenient protocol may be employed, where suitable protocols typically include combining the donor and acceptor cells in a suitable medium and maintaining under suitable conditions for conjugation and vector transfer to occur. Specific representative conditions are provided in the Experimental section below.
The above methods result in stable integration of the vector and any expression cassette carried thereby, e.g., that encodes a heterologous protein/foreign antigen, into a target Listeria cell genome in a site specific manner. The subject methods find use with a wide variety of different Listeria species.
Utility
The above described vectors and methods of using the same find use in a variety of different applications, where representative applications include, but are not limited to: (a) research applications; (b) vaccine preparation applications; (c) Listerial delivery vehicle preparation applications; and the like.
One type of application in which the subject vectors and methods of using may be employed is in research of Listeria species. For example, the subject vectors and methods allow simple and efficient strain construction and are widely useful in various strains used to study the intracellular life cycle of L. monocytogenes. Additionally, the subject vectors and methods may be employed to produce stable merodiploid strains to allow refined copy number studies and studies of interactions within a protein through multimerization and testing of the dominance or recessive nature of different alleles of a gene in the same bacterial strain.
The subject vectors and methods also find use in vaccine preparation applications. For example, the subject vectors find use in the production of Listeria cultures capable of expressing a heterologous antigen, i.e., Listeria vaccines, where the Listeria cells employed may be attenuated. The attenuated strains employed may be capable of normal invasion of a host cell, but incapable of normal survival or growth in the cell or cell-to-cell spread, or they may have other alterations that preclude normal pathogenicity.
The vaccines produced using vectors of the present invention are administered to a vertebrate by contacting the vertebrate with a sublethal dose of the genetically engineered Listeria vaccine, where contact typically includes administering the vaccine to the host. Thus the present invention provides for vaccines genetically engineered with the integration vector and provided in a pharmaceutically acceptable formulation. Administration can be oral, parenteral, intranasal, intramuscular, intravascular, direct vaccination of lymph nodes, administration by catheter or any one or more of a variety of well-known administration routes. In farm animals, for example, the vaccine may be administered orally by incorporation of the vaccine in feed or liquid (such as water). It may be supplied as a lyophilized powder, as a frozen formulation or as a component of a capsule, or any other convenient, pharmaceutically acceptable formulation that preserves the antigenicity of the vaccine. Any one of a number of well known pharmaceutically acceptable diluents or excipients may be employed in the vaccines of the invention. Suitable diluents include, for example, sterile, distilled water, saline, phosphate buffered solution, and the like. The amount of the diluent may vary widely, as those skilled in the art will recognize. Suitable excipients are also well known to those skilled in the art and may be selected, for example, from A. Wade and P. J. Weller, eds., Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press: London. The dosage administered may be dependent upon the age, health and weight of the patient, the type of patient, and the existence of concurrent treatment, if any. The vaccines can be employed in dosage forms such as capsules, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid for formulations such as solutions or suspensions for parenteral, intranasal intramuscular, or intravascular use. In accordance with the invention, the vaccine may be employed, in combination with a pharmaceutically acceptable diluent, as a vaccine composition, useful in immunizing a patient against infection from a selected organism or virus or with respect to a tumor, etc. Immunizing a patient means providing the patient with at least some degree of therapeutic or prophylactic immunity against selected pathogens, cancerous cells, etc.
The subject vaccines prepared with the subject vectors find use in methods for eliciting or boosting a helper T cell or a cytotoxic T-cell response to a selected agent, e.g., pathogenic organism, tumor, etc., in a vertebrate, where such methods include administering an effective amount of the Listeria vaccine. The subject vaccines prepared with the subject vectors find use in methods for eliciting in a vertebrate an innate immune response that augments the antigen-specific immune response. Furthermore, the vaccines of the present invention may be used for treatment post-exposure or post diagnosis. In general, the use of vaccines for post-exposure treatment would be recognized by one skilled in the art, for example, in the treatment of rabies and tetanus. The same vaccine of the present invention may be used, for example, both for immunization and to boost immunity after exposure. Alternatively, a different vaccine of the present invention may be used for post-exposure treatment, for example, such as one that is specific for antigens expressed in later stages of exposure. As such, the subject vaccines prepared with the subject vectors find use as both prophylactic and therapeutic vaccines to induce immune responses that are specific for antigens that are relevant to various disease conditions.
The patient may be any human and non-human animal susceptible to infection with the selected organism. The subject vaccines will find particular use with vertebrates such as man, and with domestic animals. Domestic animals include domestic fowl, bovine, porcine, ovine, equine, caprine, Leporidate (such as rabbits), or other animal which may be held in captivity.
In general, the subject vectors and methods find use in the production of vaccines as described U.S. Pat. No. 5,830,702, the disclosure of which is herein incorporated by reference; as well as PCT publication no WO 99/25376, the disclosures of the priority applications of which are herein incorporated by reference.
The subject vectors also find use in the production of listerial delivery vehicles for delivery of macromolecules to target cells, e.g., as described in: PCT publication no. WO 00/09733 (the priority application of which is herein incorporated by reference); and Dietrich et al., Nature Biotechnology (1998) 16: 181-185. A variety of different types of macromolecules may be delivered, including, but not limited to: nucleic acids, polypeptides/proteins, etc., as described in these publications.
Kits & Systems
Also provided are kits and systems that find use in preparing the subject vectors and/or preparing recombinant Listeria cells using the subject vectors and methods, as described above. For example, kits and systems for producing the subject vectors may include one or more of: an initial vector with a multiple cloning site; a restriction endonuclease for cleaving a site in the multiple cloning site, a vector including an expression cassette of interest which is to be inserted into the multiple cloning site; etc. Where the kits and systems are designed for the production of the recombinant Listeria cells, the kits and systems may include vectors, or components for making the same, as described above, Listeria target cells, non-Listeria host cells, and the like. The subject kits may further include other components that find use in the subject methods, e.g., reaction buffers, growth mediums, etc.
The various reagent components of the kits may be present in separate containers, or may all be precombined into a reagent mixture for combination with template DNA.
In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
The following examples are offered by way of illustration and not by way of limitation.
Experimental
I. Materials And Methods
A. Construction of pPL1 Integration Vector.
Standard molecular techniques were used in the construction of the 6101 bp integration vector pPL1 (FIG. 1A). It is a low copy plasmid that replicates autonomously in E. coli and integrates in a site-specific manner in L. monocytogenes, and was assembled from 6 independent DNA sources as follows. Restriction sites in the PCR primers used for construction are underlined. All PCR reactions used in cloning steps utilized Vent DNA polymerase (New England Biolabs).
The multiple cloning site (MCS) from pBluescript KS-(1) (bp 1-171) was cloned after PCR with primers 5′-GGGACGTCATTAACCCTCACTAAAGG-3′ and 5′-GGACGTCAATACGACTCACTATAGG-3′ (SEQ ID NOS: 01 & 02). The low copy Gram-negative origin of replication and chloramphenicol acetyltransferase (CAT) gene from pACYC184 (5) (bp 172-2253) were cloned after PCR with primers 5′-GGACGTCGCTATTTAACGACCCTGC-3′ and 5′-GAGCTGCAGGAGAATTACAACTTATATCGTATGGGG-3′ (SEQ ID NOS: 03 & 04). For direct conjugation from E. coli to L. monocytogenes, the RP4 origin of transfer (oriT) (34) (bp 2254-2624) was cloned from plasmid pCTC3 (50) after PCR with primers 5′-GCACTGCAGCCGCTTGCCCTCATCTGTTACGCC-3′ and 5′-CATGCATGCCTCTCGCCTGTCCCCTCAGTTCAG-3′ (SEQ ID NOS: 05 & 06). The listeriophage U153 integrase gene and attachment site (attPP′) (A. Nolte, P. Lauer, and R. Calendar, unpublished; bp 2629-4127), that direct the site-specific integration of the plasmid were cloned after PCR with primers 5′-GTAGATCTTAACTTTCCATGCGAGAGGAG-3′ and 5′-GGGCATGCGATAAAAAGCAATCTATAGAAAAACAGG-3′ (SEQ ID NOS: 07 & 08). For expression of the U153 integrase gene, the L. monocytogenes p60 promoter (Lauer, P., J. A. Theriot, J. Skoble, M. D. Welch, and D. A. Portnoy. 2001. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility. Mol. Microbiol. 42(5):1163-1177) (bp 4134-4563) was PCR amplified with primers 5′-CCTAAGCTTTCGATCATCATAATTCTGTC-3′ and 5′-GGGCATGCAGATCTTTTTTTCAGAAAATCCCAGTACG-3′ (SEQ ID NOS: 09 & 10) and cloned upstream of the integrase gene. Base pairs 4570-6101 are a Hind III-Aat II restriction fragment subcloned from pUC18-Cat (a kind gift from Nancy Freitag), and contain the inducible Gram-positive CAT gene from pC194 (19) (bp 4788-5850).
B. Cloning of the hly and actA Genes into pPL1.
The hly gene was subcloned from the plasmid pDP-906 (Jones, S., and D. A. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect. Immun. 62(12):5608-5613.) by restriction digestion with BamH I and Xba I, gel purifying a 2.9 kb fragment and by ligating it into pPL1 cut with BamH I and Spe I. The resultant plasmid was designated pPL24 (FIG. 1A). The actA gene was PCR amplified from 10403S genomic DNA with primers 5′-GGTCTAGATCAAGCACATACCTAG-3′ and 5′-CGGGATCCTGAAGCTTGGGAAGCAG-3′(SEQ ID NOS:11 & 12). The 2220 bp PCR product was gel purified, cut with BamH I and Xba I, and cloned into pPL1 cut with BamH I and Spe I. The resultant plasmid was designated pPL25 (FIG. 1A).
C. Phage Curing, Conjugation and Molecular Confirmation of Plasmid Integration.
Phage curing was accomplished by adapting historical methodologies (Cohen, D. 1959. A variant of Phage P2 Originating in Escherichia coli, strain B. Virology 7:112-126; Six, E. 1960. Prophage substitution and curing in lysogenic cells superinfected with hetero-immune phage. J. Bacteriol. 80:728-729.). L. monocytogenes 10403S derivatives carrying a prophage at comK-attBB′ (integrated in the comK ORF as described (Loessner, M. J., R. B. Inman, P. Lauer, and R. Calendar. 2000. Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution. Mol. Microbiol. 35(2):324-340.)) were grown in BHI at 37° C. to 108 CFU/ml and infected with listeriophage U153 (Hodgson, D. A. 2000. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes. Mol. Microbiol. 35(2):312-323.) at a multiplicity of infection of 20:1 in the presence of 5 mM CaCl2. Cultures were incubated by shaking at 37° C. for 75 minutes and inhibition of growth was monitored by comparison of the OD600 of the infected culture with an uninfected control culture. The infected culture was diluted 10−2 and 10−4 in BHI, and both dilutions were grown at 37° C. until the 10-2 dilution had increased 100-fold in optical density. The 10−4 fold dilution was then diluted 10−2, and 3 μl were plated on BHI. Fifty colonies were tested for phage release initially by toothpicking colonies into 0.25 ml LB broth and replica plating at 30° C. on a lawn of Mack-4R (DP-L862), a non-lysogenic rough strain of L. monocytogenes particularly susceptible to forming plaques. Candidates that did not plaque were then tested by spotting 10 μl of culture on a lawn of Mack-4R to detect plaque formation. If this second test was negative, the candidate was tested whether it could support plaque formation by the phage from the parent 10403S strain (Φ10403, (Hodgson, D. A. 2000. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes. Mol. Microbiol. 35(2):312-323.)). Curing was confirmed molecularly by PCR with the comK-attBB′ specific primer pair PL60/PL61 (sequences follow) for the absence of a phage at comK-attBB′. Approximately 10% of colonies were cured using this procedure.
Recipient strains of L. monocytogenes (SLCC-5764, DP-L1169 and DP-L1172) were made streptomycin resistant for counter-selection in conjugation experiments by plate selection on BHI supplemented with 200 μg/ml antibiotic.
pPL1 plasmid constructs were electroporated into E. coli strain SM10 (Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1:784-791.) using standard techniques (Sambrook, J., T. Maniatis, and E. F. Fritsch. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Bacterial strains were grown to mid-log (OD600 ̃0.55) shaking at 30° C. E. coli donor strains were grown in LB containing 25 μg/ml of chloramphenicol, L. monocytogenes recipient strains were grown in BHI. 2.5 ml of donor culture was mixed with 1.5 ml of recipient culture and filtered onto pre-washed 47 mm 0.45 μm HA type filers (Millipore). The filter was washed once with 10 ml BHI, transferred to a BHI plate with no antibiotics and incubated for 2 hours at 30° C. The bacterial cells were gently resuspended in 2.5 ml of BHI and 25 μl and 50 μl aliquots were plated in 3 ml of LB top agar on BHI plates supplemented with 7.5 μg/ml chloramphenicol and 200 μg/ml streptomycin. Plates were incubated at 30° C. overnight and shifted to 37° C. for 2-3 days. Individual colonies were picked and screened by PCR for integration at the phage attachment site using the primers PL14 (5′-CTCATGAACTAGAAAAATGTGG-3′) (SEQ ID NO:13), PL60 (5′-TGAAGTAAACCCGCACACGATG-3′)(SEQ ID NO:14) and PL61 (5′-TGTAACATGGAGGTTCTGGCAATC-3′)(SEQ ID NO: 15). PCR reactions were performed on small portions of individual bacterial colonies picked with sterile P200 pipet tips from BHI plates directly into 20 μl PCR reactions. The primer pair PL14/PL61 specifically amplifies attBP′ in a PCR reaction, resulting in a 743 bp product on integrated strains (both prophage and pPL1 derivatives). The primer pair PL60/PL61 specifically amplifies comK-attBB′ in a PCR reaction, resulting in a 417 bp product only on non-lysogenic strains (i.e. DP-L4056). PCR assays were performed in a Hybaid Omn-E thermocycler with an annealing temperature of 55° C. for 30 cycles. Integrants arose at a frequency of approximately 2×10−4 per donor cell.
D. Hemolysis on Blood Plates and Hemolytic Activity Assay.
Hemolysis on blood plates was scored on tryptic soy agar plates supplemented with 5% defimbrinated sheep blood (HemoStat, Davis Calif.). Hemolytic assays were performed essentially as described (Portnoy, D. A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167(4):1459-1471.). Hemolytic activity is expressed as the reciprocal of the dilution of culture supernatant required to lyse 50% of sheep erythrocytes.
E. Plaquing in L2 Cells.
Plaque sizes were determined as previously described (Lauer, P., J. A. Theriot, J. Skoble, M. D. Welch, and D. A. Portnoy. 2001. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility. Mol. Microbiol. 42(5):1163-1177.). Each strain was plaqued in 6 to 8 independent experiments and compared to 10403S in each experiment.
F. SDS-PAGE of Surface Expressed ActA.
Surface expressed ActA protein was prepared from late-log phase bacterial cultures grown in LB broth (OD600 ̃0.7) by resuspending equivalent amounts in SDS-PAGE buffer and boiling for 5 min. which extracts surface-expressed proteins but does not perturb the cell wall. Equivalent amounts were loaded on 7% SDS-PAGE and visualized with Coomassie blue.
G. Xenopus laevis Cell Extract Motility Assays.
X. laevis egg cytoplasmic extract was prepared as described (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts. Cell 76(3):505-517.) and supplemented with tetramethylrhodamine iodoacetamide-labeled actin (Theriot, J. A., and D. C. Fung. 1998. Listeria monocytogenes-based assays for actin assembly factors. Methods Enzymol. 298:114-122.). SLCC-5764-derived strains were grown overnight to stationary phase, washed 1×, added to cell extracts and incubated for 45 minutes before microscopic observation.
H. LD50 Determinations.
Limited LD50 were performed in BALB/c mice as described (Portnoy, D. A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167(4):1459-1471.). Animal experiments were performed in the laboratory of Archie Bouwer at Oregon Health Sciences Center, Portland, Oreg.
I. Identification of the PSA Attachment Site and Construction of pPL2.
The PSA attachment site (tRNAArg-attBB′) DNA sequence was obtained through a combination of inverse PCR and genome walking. Inverse PCR was performed on Sau3 Al-digested DP-L4061 DNA (WSLC 1042 lysogenic for PSA) using the divergent primers PL95 (5′-ACATAATCAGTCCAAAGTAGATGC)(SEQ ID NO:16) and PL97 (5′-ACGAATGTAAATATTGAGCGG) (SEQ ID NO:17) that anneal within the PSA int gene. The resultant DNA sequence was used to design further oligonucleotides and these were used with the Genome Walker kit (Clontech), per the manufacturer's recommendations. DNA sequence and tRNA analysis was done with using MacVector (Accelrys), DNAsis (Hitachi), BLAST algorithm (2), and tRNAscan-SE (Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25(5):955-964.).
pPL1 was modified to utilize a different attachment site on the L. monocytogenes chromosome by replacing the U153 integrase gene and attachment site in the plasmid. The PSA int and attPP′ were PCR amplified from PSA genomic DNA with primers PL100 (5′-GAAGATCTCCAAAAATAAACAGGTGGTGG) (SEQ ID NO:18) and PL101 (5′-CATGCATGCGTGGAGGGAAAGAAGAACGC) (SEQ ID NO:19) with Vent DNA polymerase, digested with Bgl II and Sph I, and ligated to pPL1 that had been digested with the same enzymes. The resultant plasmid was designated pPL2 (FIG. 1B).
The DNA sequence of the PSA tRNAArg-attBB′ from serotype 1/2 L. monocytogenes strains was obtained by a plasmid-trap strategy. DP-L4211 (pPL2 integrated in 10403S) genomic DNA was digested with Nsi I and Nhe I, which do not cleave in the vector, and ligated under dilute conditions to promote self-ligation. The ligations were transformed into XL1-blue and chloramphenicol resistant colonies were selected. The plasmids obtained were sequenced with the convergent primers PL94 (5′-GGAGGGAAAGAAGAACGC) (SEQ ID NO:20) and PL95 (sequence above) for attPB′ and attBP′, respectively, which flank attPP′ in the PSA genomic DNA sequence. Further, because of the divergence between the sequences downstream of the tRNAArg gene among serotypes, a serotype 1/2 specific PCR assay across tRNAArg-attBB′ was developed from the 10403S DNA sequence and used to determine the prophage status of various L. monocytogenes strains. Primers PL102 (5′-TATCAGACCTAACCCAAACCTTCC) (SEQ ID NO:21) and PL103 (5′-AATCGCAAAATAAAAATCTTCTCG) (SEQ ID NO:22) specifically amplify a 533 bp PCR product in non-lysogenic serotype 1/2 strains. The primer pair NC16 (5′-GTCAAAACATACGCTCTTATC)(SEQ ID NO:23) and PL95 specifically amplify a 499 bp PCR product in strains that are either lysogenic or contain an integration vector at tRNAArg-attBB′.
II. Results and Discussion
A. pPL1 forms Stable, Single-Copy Integrants in Various L. monocytogenes Strains.
pPL1 is the first shuttle integration vector that we constructed to facilitate strain construction in L. monocytogenes. In order to test the pPL1 vector, we needed a L. monocytogenes strain that did not have a phage at the comK bacterial attachment site. We adapted historical methods to cure L. monocytogenes strains of their prophages and found after superinfection with phage U153, which has the same attachment site as the endogenous 10403S prophage, we were able to isolate prophage-free strains (see Materials and Methods). The prophage-cured 10403S strain was designated DP-L4056 and was used in subsequent experiments.
Conjugation was chosen as a method for introducing the vector into the target cells, as many Listeria spp. are inefficiently transformed. Conjugation of pPL1 from E. coli into L. monocytogenes was successful. Drug resistant transconjugants arose at a reproducible frequency of  ̃2×10−4 per donor E. coli cell, approximately three-fold lower than conjugation with autonomously replicating plasmids, indicating  ̃30% integration efficiency for strains receiving the plasmid by conjugation. All chloramphenicol resistant colonies were positive with the PCR assay using primers PL14 and PL61 and negative using a PCR assay across attPP′ in pPL1 (PL14 paired with a primer in the RP4 oriT) indicating that they were true integrants and that the full genetic complement of plasmid pPL1 had integrated into the Listeria chromosome. In addition, this experiment demonstrated that pPL1 was not maintained as an episomal plasmid and that the vector did not integrate as a concatamer.
We tested the stability of the integrants under non-selective growth conditions. Three integrant strains, DP-L4074 and the merodiploid strains DP-L4076 and DP-L4078 (described in the following sections) were passed in liquid BHI media for 100 generations and plated for single colonies. Ninety-six colonies were then exposed to 0.1 μg/ml chloramphenicol (to induce CAT gene expression) and patched on plates containing 7.5 μg/ml antibiotic. All colonies retained drug resistance. Thirty colonies from each non-selective growth experiment were assayed with the PL14/PL61 PCR assay and all PCR reactions resulted in the 743 bp product, indicating all transconjugants retained the integrated plasmid.
We further addressed whether the integration vector would be generally useful for any L. monocytogenes strain with an available attachment site. There have been greater than 320 listeriophages isolated, and many have restricted host ranges. It was unclear whether there was a biological barrier to U153 integrase gene function in host strains that do not support U153 infection. We therefore picked three strains that did not contain a prophage at the comK attachment site; two serotype 4b clinical isolates and SLCC-5764, a serotype 1/2a strain that constitutively expresses the known virulence factors in an unregulated manner and has been useful for studying these virulence factors in vitro. Each of these strains was first made streptomycin resistant for counter selection in conjugation experiments (as described in Materials and Methods). Streptomycin resistant derivatives were chosen on the basis of having the same growth rate as the parent strain to avoid experimental complications related to viability. The resultant strains, DP-L4088, DP-L4089, and DP-L4082 all proved suitable recipients for pPL1 integration at a similar frequency to DP-L4056.
Finally, we conducted a survey of L. monocytogenes isolates to identify suitable strains that do not harbor a prophage at the comK attachment site using the PCR assays across comK-attBB′ (primers PL60/PL61) and the hybrid affPB′ (primers PL14/PL61). The results of these experiments (Table 2) indicated many of the strains commonly used to study the biology and pathogenesis of L. monocytogenes including 10403S, L028, and EGDe had a prophage at comK.







TABLE 2







Prophage status of various strains
















PL60/PL61
PL14/PL61


Strain
Description
Source
serotype
comK
attPB′





10403S
wild type
rabbit pellets
1/2a
−a
+


DP-L4056
10403S phage cured
This work
1/2a
+b


DP-L861
SLCC-5764 (Mack)
WT (overexpresser)
1/2a
+
−


DP-L3818
WSLC 1118::A118
Camembert cheese
4b
−
+


DP-L3633
EGDe
WT (1960s human)
1/2a
−
+


DP-L3293
LO28
WT (clinical origin)
1/2c
−
+


DP-L185
F2397
L.A., Jalisco cheese
4b
+
−


DP-L186
ScottA
Massachusettes
4b
+
−




outbreak, milk


DP-L188
ATTC 19113
Denmark, human
3
+
−


DP-L1168
clinical
cole slaw
4b
+
−


DP-L1169
clinical
patient
4b
+
−


DP-L1170
clinical
patient
4b
+
−


DP-L1171
clinical
brie
1/2b
+
−


DP-L1172
clinical
alfalfa tablets
4b
+
−


DP-L1173
clinical
deceased patient
4b
−
+


DP-L1174
clinical
deceased patient
4b
−
+


DP-L3809
1981 Halifax
placenta
4b
+
−


DP-L3810
1981 Halifax
CSF & brain
4b
+
−


DP-L3812
1981 Halifax
coleslaw
4b
+
−


DP-L3813
1996 Halifax
blood
?
−
+


DP-L3814
1981 Halifax
CSF
4b
+
−


DP-L3815
1993 Halifax
CSF
1/2a
+
−


DP-L3816
1995 Halifax
blood
?
+
−


DP-L3817
1993 Halifax
CSF
1/2a
+
−


DP-L3862
1998 Michigan
patient
4b
−
+





a−: negative PCR result for primer pair noted at top of column.


b+: positive PCR result for primer pair noted at top of column. The PL60/PL61 primer pair specifically amplify a 417 bp PCR product in non-lysogenic strains and result in no PCR product in lysogenic strains. The PL14/PL61 primer pair specifically amplify a 743 bp PCR product in lysogenic strains and result in no PCR product in non-lysogenic strains.






B. The Status of comK Did Not Affect the Virulence of L. monocytogenes. 

We next compared DP-L4056 and DP-L4074 to wild-type 10403S in standard virulence assays to determine if the presence of a prophage at comK, lack of prophage, or integration vector altered the virulence phenotypes. These three strains were assayed for LLO activity, ability to form plaques in monolayers of L2 cells, and for virulence in the mouse LD50 assay (Table 3). All were indistinguishable from one another, strongly suggesting that the integrity of the comK ORF and the presence of pPL1 had no measurable impact on virulence.







TABLE 3







Complementation of actA and hly














Hemolysis







on blood
Hemolytic


Strain
Genotype
plates
activitya
Plaque sizeb
LD50c





10403S
wildtype
+
nd
100 (na)
~2 × 104


DP-L4056
10403S phage cured
+
97
101 (1.4)
<1 × 105


DP-L4074
DP-L4056 comK::pPL1
+
98
 99 (1.4)
<1 × 105


DP-L4027
DP-L2161 phage cured, Δhly
−
0
 0 (0)
  1 × 108


DP-L4075
DP-L4027 Δhly, comK::pPL24
+
99
 97 (3.9)
<1 × 105


DP-L4076
DP-L4056 comK::pPL24
+
198 
 96 (2)
nd


DP-L4029
DP-L3078 phage cured, ΔactA
nd
nd
 0 (0)
  2 × 107


DP-L4077
DP-L4029 ΔactA, comK::pPL25
nd
nd
 86 (4)
<1 × 105


DP-L4078
DP-L4056 comK::pPL25
nd
nd
 72 (6.8)
nd





aHemolytic units data shown is from one representative experiment.


nd: not detennined.


bPlaque size is the average of 8 to 10 independent experiments and shown as a percent of wild type (defined as 100%). Standard deviations are shown in parentheses.


na: not applicable.


cLD50s of 10403S and Δhly (DP-L2161) were determined in (37), the LD50 of the ΔactA strain (DP-L1942, a smaller deletion within the actA ORF that does not support actin nucleation at the bacterial surface) was determined in (4).






C. Full Complementation of hly at the Phage Attachment Site.

Listeriolysin-O (LLO), the gene product of hly, is a secreted pore-forming cytolysin that is responsible for escape from the membrane-bound vacuole when L. monocytogenes first enters a host cell. LLO is absolutely required for the intracellular life cycle of L. monocytogenes and virulence. LLO activity can be measured by hemolytic activity on red blood cells. Hly mutants fail to form plaques in monolayers of L2 cells and are 5 logs less virulent in the mouse LD50 assay.
We cloned the hly structural gene into pPL1 and conjugated this plasmid from E. coli to phage-cured wild type and Δhly L. monocytogenes derivatives, resulting in DP-L4076 (an hly merodiploid) and DP-L4075 (hly only at the phage comK-att site). These strains were tested for hemolytic activity on blood plates, for the relative amount of hemolytic units secreted, ability to form a plaque in a monolayer of L2 cells, and virulence in the mouse LD50 assay (Table 3). The quantitative complementation of hly in the deletion strain background and the doubling of hemolytic units produced in the merodiploid strain indicate two things. First, gene expression is not de facto affected by ectopic expression at the comK chromosomal position. Second, the hly promoter is self-contained. Additionally, a two-fold increase in the amount of LLO is not deleterious to the virulence and intracellular life cycle of L. monocytogenes, at least as measured by plaquing.
D. Complementation of actA at the Phage Attachment Site Approaches Wild-Type Expression.
ActA, a second major L. monocytogenes virulence factor, is responsible for commandeering host cell actin-cytoskeletal factors used for intracellular bacterial motility. ActA is also absolutely required for bacterial pathogenesis as mutants in actA are both unable to spread from cell-to-cell and form a plaque in a cell monolayer and are 3 logs less virulent than wild type. Additionally, ActA expression appears to be more complex than that of LLO: there are two promoters that drive acta expression. One is immediately upstream of the actA ORF and the second is in front of the mpl gene upstream of actA.
We constructed several strains to evaluate the complementation of actA at the phage attachment site. The first group included making second-site complemented (DP-L4077) and merodiploid (DP-L4078) strains in the 10403S background. These were assayed for plaque formation in an L2 monolayer (Table 3). Integrated ActA did not fully complement in this assay (plaque size of 86%) and the merodiploid strain formed an even smaller plaque (72% of wild type). We interpret these results to indicate that there may be a small contribution of the second promoter upstream of the mpl gene for optimal actA expression. Additionally, there appears to be a critical concentration of ActA on the surface of intracellular bacteria because two copies of actA (with 3 promoters driving expression) further decreases the ability to spread from cell to cell, presumably because there is too much ActA on the bacterial surface for optimal motility.
We further tested ActA complementation in the virulence gene over-expressing strain SLCC-5764. ActA is effectively expressed in this strain from the comK-attBB′ site (FIG. 3A, lane 9). Considering the plaquing data of 10403S complemented actA strains, it may have been predicted that the merodiploid strain DP-L4085 would make more ActA than the parent strain. However, this was not observed: the parent strain, the complemented strain and the merodiploid strain all expressed similar levels of ActA (FIG. 3A, lanes 5, 8, and 9). This observation was likely due to the complete lack of regulation and high level of constitutive expression of ActA in SLCC-5764. Additionally, DP-L4087 supports actin nucleation at the bacterial surface, actin tail formation and bacterial motility in cell extracts (FIG. 3B). The results of these cell-extract experiments indicate that the integration vector system for complementation will be useful for in vitro studies of L. monocytogenes motility, facilitating strain construction and placing various molecular constructs in different host strains for study in a desired set of assays. In particular, several alleles of actA that have unusual motility phenotypes have been transferred to the SLCC-5764 ΔactA strain using pPL1 and are currently being evaluated in cell extracts. The study of these mutants in the simplified cell-extract system should yield insights into the activities of poorly understood regions of the ActA protein.
The strains referenced above are provided in the following Table 1.











Strain
Relevant genotype or plasmid







E. coli:



SM10
Conjugation donor. F- thi-1 thr-1 leuB6 recA



tonA21 lacY1 supE44 Mu + C l- [RP4-2(Tc::Mu)



Kmr Tra+


XL1-Blue
Plasmid manipulations. recA1 endA1 gyrA96 thi-1



hsdR17 supE44 relA1 lac [F'proAB lac1qZ ΔM15



Tn10 (Tetr)]


DP-E4067
Integration vector pPL1/SM10


DP-E4068
hly integration vector pPL24/SM10


DP-E4069
actA integration vector pPL25/SM10


DP-E4190
Integration vector pPL2/SM10


L. monocytogenes:


10403S
wild type


DP-L4056
10403S phage cured


DP-L4027
DP-L2161 phage cured, Δhly


DP-L4029
DP-L3078 phage cured, ΔactA


DP-L4074
DP-L4056 comK::pPL1


DP-L4075
DP-L4027 Δhly, comK::pPL24


DP-L4076
DP-L4056 comK::pPL24


DP-L4077
DP-L4029 ΔactA, comK::pPL25


DP-L4078
DP-L4056 comK::pPL25


SLCC-5764
Virulence gene over expresser (Mack, DP-L861)


DP-L862
Mack-4R (SLCC-5764 rough isolate)


DP-L4082
SLCC-5764 Strr derivative


DP-L3780
SLCC-5764 ΔactA (deletion of amino acids 7-633)


DP-L4083
DP-L37805 (Strr dervivative)


DP-L4084
DP-L4082, comK::pPL1


DP-L4085
DP-L4082, comK::pPL25


DP-L4086
DP-L4083, comK::pPL1


DP-L4087
DP-L4083 ΔactA, comK::actA


DP-L4088
DP-L1169S 4b strain, Strr


DP-L4089
DP-L1172S 4b strain, Strr


DP-L4090
DP-L4088, comK::pPL1


DP-L4091
DP-L4089, comK::pPL1


DP-L4199
EGDe, Strr derivative


DP-L4026
WSLC 1042, (ATCC 23074)


DP-L4061
WSLC 1042::PSA


DP-L4221
10403S, tRNAArg::pPL2









E. Phage PSA Integrates into a tRNAArg Gene and pPL2 Construction.

pPL1 integration into L. monocytogenes strains that harbour a prophage in the comK attachment site is hindered by the process of first having to cure the prophage from the host strain. To alleviate the need for the phage-curing step, the specificity of pPL1 integration was changed to that of the PSA prophage. PSA, (Phage from ScottA) is the prophage of L. monocytogenes strain ScottA, a serotype 4b strain that was isolated during an epidemic of human listeriosis. Using the PSA genomic DNA sequence, we identified an integrase-like ORF with a contiguous non-coding sequence that we predicted to contain the attPP′ sequences. The PSA integrase sequence was then used to obtain the DNA sequence of PSA-attBB′ from the PSA lysogenic strain DP-L4061 (see Materials and Methods). PSA was found to integrate in a tRNAArg gene that is 88% identical to a tRNAArg gene (trnSL-ARG2) from B. subtilis. The anticodon of the tRNAArg gene is 5′UCU, the most commonly used arginine anticodon in L. monocytogenes. The PSA and bacterial attachment sites share 17 bp of DNA identity, and the tRNAArg-attBB′ contains a short nucleotide sequence that completes the tRNAArg sequence that is interrupted by integration of PSA (FIG. 4). The attachment site tRNAArg gene is present only once in the genome of L. monocytogenes strain EGDe and apparently only once in the serotype 4b. This indicates that not only is the PSA integration site unique, but also that precise reconstitution of the gene upon integration (or excision) is likely required for survival of the cell.
pPL2 was constructed by replacing the U153 listeriophage integrase gene and attachment site in pPL1 with the PSA listeriophage integrase gene and attachment site. pPL2 was tranformed into SM10 and the resulting strain was mated into 10403S, EGDe (carrying a streptomycin resistance mutation) and the serotype 4b strain DP-L4088. Chloramphenicol resistant transconjugants arose from each of these crosses at approximately 2×10−4 per donor cell, the same rate as pPL1 integration. Two recombinants from each cross were restreaked under drug selection and tested by PCR for the presence of PSA-attBP′ using primers NC16 and PL95. The expected 499 bp PCR product was obtained in each of the colonies tested, indicating pPL2 integrates into tRNAArg-attBB′ in both serotype 1/2 and 4b strains. We tested the stability of the integrated pPL2 in both EGDe and DP-L4088 strains with the same non-selective 100-generation experiment described for pPL1. Forty-nine colonies from each of the amplified cultures were tested for chloramphenicol resistance. The EGDe-derived strains retained 100% drug resistant colonies indicating complete stability of the integrants. In the case of the DP-L4088 integrant, two of the 49 colonies were chloramphenicol sensitive, suggesting a low level of excision can occur in this serotype 4b strain. In order to test whether precise excision had occured, we PCR amplified across tRNAArg-attBB′ and sequenced the PCR products. The wild-type DNA sequence was obtained, indicating a precise excision event.
During the course of our PCR experiments, we noted a divergence between the tRNAArg-attPB′ sites from serotype 4b and serotype 1/2 L. monocytogenes. To determine nature of this divergence, we isolated and sequenced the tRNAArg-attBB′ site from 10403S (as described in Materials and Methods). We found that the sequence of attPB′ in 10403S (3′ of the tRNAArg gene) is unrelated to that of the serotype 4b strain WSLC 1042. In contrast to this, the sequence of attBP (5′ of the tRNAArg gene) in 10403S is 96-97% identical to the corresponding regions in L. monocytogenes serotype 4b strain WSLC 1042, the serotype 4b strain sequenced by TIGR, and the serotype 1/2a strain EGDe sequenced by the European Listeria Consortium. Thus, the bacterial attBB′ sequences recognized by the PSA integrase are likely to encompass more of the attB DNA sequence than the attB′ DNA sequence. Additionally, we tested the availability of the tRNAArg-attBB′ in the common laboratory strains of L. monocytogenes with a PCR assay using primers PL102 and PL103. We found the tRNAArg attachment site to be available in strains 10403S, EGEe, and L028 indicating that pPL2 may be readily utilized in these backgrounds for strain construction, complementation, and genetics studies without first curing endogenous prophages.
F. Expression of Aquoria victoria GFP.
A GFP coding sequence as described in U.S. Pat. No. 5,777,079 (the disclosure of which is herein incorporated by reference), is cloned into plasmid pPL1, transferred into the genome of L. monocytogenes. The GFP coding sequence is amplified by polymerase chain reaction (PCR) and the PCR fragment is cloned into the multiple cloning site of pPL1. A suitable promoter, containing appropriate transcriptional elements and a translational leader sequence for expressing the GFP in L. monocytogenes are cloned at the 5′ end of the GFP coding sequence such that they induce the expression of the GFP protein. The modified pPL1 plasmid constructs are electroporated into E. coli strain SM10 using standard techniques, and the modified pPL1 plasmid construct is conjugated into L. monocytogenes as described above. Recombinant L. monocytogenes are selected on BHI plates supplemented with 7.5 μg/ml chloramphenicol and 200 μg/ml streptomycin. Individual colonies are picked and screened by PCR for integration at the phage attachment site using the primers PL14 (5′-CTCATGAACTAGAAAAATGTGG-3′) (SEQ ID NO:13), PL60 (5′-TGAAGTAAACCCGCACACGATG-3′)(SEQ ID NO:14) and PL61 (5′-TGTAACATGGAGGTTCTGGCAATC-3′)(SEQ ID NO:15). Cultures of recombinant L. monocytogenes are grown, prepared and screened for GFP.
III. Utility of pPL1 and pPL2
The construction and characterization of the first single step site specific integration vectors for use in L. monocytogenes furthers the genetic tools available for the study of this pathogen. These vectors allow more facile strain construction than historic methods and are widely useful in various strains used to study the intracellular life cycle of L. monocytogenes. Additionally, stable merodiploid strains can be constructed to allow refined copy number studies and studies of interactions within a protein through multimerization and testing of the dominance or recessive nature of different alleles of a gene in the same bacterial strain.
pPL1 and pPL2 are also useful for vaccine development, e.g., for at least enhancing, including both eliciting and boosting, an immune response to a target cells or cells, e.g., a foreign pathogen, etc. Several recombinant L. monocytogenes systems have been used to elicit cell-mediated immune responses in mice (Frankel, F. R., S. Hegde, J. Lieberman, and Y. Paterson. 1995. Induction of cell-mediated immune responses to human immunodeficiency virus type 1 Gag protein by using Listeria monocytogenes as a live vaccine vector. J. Immunol. 155(10):4775-4782.; Goossens, P. L., G. Milon, P. Cossart, and M. F. Saron. 1995. Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus. Int. Immunol. 7(5):797-805; Ikonomidis, G., Y. Paterson, F. J. Kos, and D. A. Portnoy. 1994. Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes. J. Exp. Med. 180(6):2209-2218.; Shen, H., M. K. Slifka, M. Matloubian, E. R. Jensen, R. Ahmed, and J. F. Miller. 1995. Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity. Proc. Natl. Acad. Sci. USA 92(9):3987-3991.). One limitation with plasmid-based expression of recombinant proteins in L. monocytogenes is the stability of the plasmids in vivo (i.e. in the host animal) without selection. Additionally, chromosomal construction of strains expressing foreign antigens is time consuming. pPL1 and pPL2 alleviate both of these concerns.
It is evident from the above results and discussion that subject invention provides a number of advantages. The construction and characterization of the first single step site specific integration vectors for use in L. monocytogenes as described herein furthers the genetic tools available for the study of this pathogen. For example, the subject vectors and methods allow more facile strain construction than historic methods and are widely useful in various strains used to study the intracellular life cycle of L. monocytogenes. Furthermore, the subject invention provides important new tools for the production of vaccine preparations. One limitation with plasmid-based expression of recombinant proteins in L. monocytogenes is the stability of the plasmids in vivo (i.e. in the host animal) without selection. Additionally, chromosomal construction of strains expressing foreign antigens is time consuming. The subject vectors and methods of use alleviate both of these concerns. As such, the present invention represents a significant contribution to the art.
All publications and patent application cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

## Claims

1. A recombinant integration vector capable of integrase mediated site-specific Listeria genome integration, wherein said vector is a plasmid, said vector comprising;
a listeriophage integrase attachment (attPP) site and a cloning site containing a heterologous coding sequence, wherein said attPP site is capable of integrase mediated recombination with a listeriophage integrase bacterial attachment (attBB) site; and
a nucleic acid sequence encoding a listeriophage integrase capable of catalyzing attPP′-mediated integration of the vector at an attBB′ site.

2. The integration vector according to claim 1, wherein said attachment site provides for integration at an integration site selected from the group consisting of: a comK integration site and a tRNAArg integration site.

3. The integration vector according to claim 1, wherein said integration vector further includes a multiple cloning site.

4. The integration vector according to claim 3, wherein said integration vector further includes a coding sequence.

5. The integration vector according to claim 1, wherein said coding sequence encodes a polypeptide.

6. The integration vector according to claim 5, wherein said polypeptide is an antigen.

7. A method of transforming a Listeria, said method comprising: contacting said Listeria with an integration vector according to claim 1 under conditions sufficient for said integration vector to integrate into said Listeria's genome.

8. A Listeria transformed with a vector according to claim 1.

9. A strain of Listeria cells according to claim 8, wherein said heterologous coding sequence encodes a heterologous antigen and said Listeria cells express said heterologous antigen encoded by said coding sequence.

10. The strain according to claim 9, wherein said Listeria cells are attenuated.

11. A recombinant culture of Listeria cells according to claim 8.

12. The recombinant culture according to claim 11, wherein said Listeria cells are attenuated.

13. A kit comprising:
a vector according to claim 3; and
at least one nuclease that cuts said vector at a multiple cloning site.

14. The kit according to claim 13, wherein said kit further comprises a host cell.

15. A kit comprising:
a vector according to claim 3;
at least one nuclease that cuts said vector at a multiple cloning site; and
a Listeria cell.

16. A system comprising:
a vector according to claim 3;
at least one nuclease that cuts said vector at a multiple cloning site;
said heterologous coding sequence; and
Listeria cells.

17. The integration vector according to claim 1, wherein said listeriophage integrase attachment site is a PSA bacteriophage integrase attachment site.

18. The integration vector according to claim 17, wherein said attachment site is at least 17 nucleotides in length.

19. The integration vector according to claim 1, wherein said listeriophage integrase attachment site is selected from a U153 attPP attachment site and a PSA attPP attachment site.

20. A recombinant plasmid comprising:
a listeriophage integrase U153 attachment site (U153 attPP′ site), wherein said U153 attPP′ site is capable of integrase mediated integration with a comK listerial genome attachment site (comK attBB′ site) and a nucleic acid sequence encoding a U153 integrase; or
a listeriophage integrase PSA attachment site (PSA attPP′ site), wherein said PSA attPP′ site is capable of integrase mediated integration with a tRNAArg listerial genome attachment site (attBB′ site) and a nucleic acid sequence encoding a PSA integrase,
wherein said plasmid further comprises a cloning site for a nucleic acid encoding a heterologous coding sequence.

21. The plasmid of claim 20, wherein the plasmid comprises a U153 attPP′ site and a nucleic acid encoding U153 integrase, or a PSA attPP′ site and nucleic acid encoding PSA integrase, wherein the nucleic acid encoding the integrase is operably linked with a listerial promoter.

22. The plasmid of claim 21, wherein the nucleic acid encoding the integrase is operably linked with a listerial promoter from p60, actA, plcA, mpl, plcB, or inlA.

23. The plasmid of claim 20, wherein the plasmid comprises a non-listerial bacterial origin of replication.

24. The plasmid of claim 20, further comprising a nucleic acid encoding an origin of transfer.

25. The plasmid of claim 20, wherein the cloning site is a multiple cloning site.

26. The plasmid according to claim 20, wherein said plasmid further comprises a nucleic acid encoding a heterologous coding sequence.

27. The plasmid of claim 26, wherein the heterologous coding sequence encodes a polypeptide.

28. The plasmid of claim 26, wherein the heterologous coding sequence encodes an antigen.

29. The plasmid of claim 26, wherein the antigen is a tumor antigen.

30. A Listeria bacterium comprising the plasmid of claim 20.

31. The Listeria bacterium of claim 30, wherein the plasmid is genomically integrated.

32. The Listeria bacterium of claim 30, wherein the Listeria is attenuated.