Attenuated Salmonella gallinarum strain and uses thereof

Abstract

The present invention relates to an attenuated Salmonella gallinarum strain and its use. Specifically, the attenuated strain contains a gene encoding guanosine tetraphosphate (ppGpp) synthase and a gene inducing the function of the type III secretion system (T3SS) ( ssrAB ), and Gifsy 2 prophage The present invention relates to a novel attenuated Salmonella strain in which all genes have been deleted, and a composition for tumor therapy or diagnosis using the same.

Description

[Technical Field]

[0001] The present invention relates to an attenuated Salmonella gallinarum strain and its uses. Specifically, the attenuated strain contains a gene encoding guanosine tetraphosphate (ppGpp) synthase, a gene inducing the function of the type III secretion system (T3SS), and a gene encoding the enzyme ( ssrA , ssrB , or ssrAB ), and Gifsy 2 prophage The present invention relates to a novel attenuated Salmonella strain in which all genes have been deleted, and a composition for tumor therapy or diagnosis using the same. [Background technology]

[0002] A total of 81,203 people died from cancer in 2019, accounting for 27.5% of all deaths. As a result, cancer is considered the most common cause of death, and according to the Korea Cancer Information Center, the incidence rate has been steadily increasing since 1999, when nationwide cancer statistics began to be compiled.

[0003] Factors contributing to the increased incidence of cancer include environmental factors such as increased air pollution and other environmental pollutants, the consumption of high-fat foods due to Westernized diets, and personal factors such as drinking and smoking. Therefore, the development of anticancer materials for early cancer prevention and treatment is becoming increasingly important. Furthermore, for intractable cancers for which no clear treatment exists, the incidence rate is low but the mortality rate is high, making the development of therapeutic agents urgent. However, compound-based anticancer drugs often exhibit nonspecificity, acting not only on cancer cells but also throughout the body, raising concerns about side effects.

[0004] Meanwhile, since reports that bacterial infections have anti-cancer effects, research into the development of anti-tumor bacterial strains has surged. The anti-tumor bacteria that have been studied in the last 20 years are Bifidobacterium ( Bifidobacterium ), Clostridium ( Clostridium ), Lactococcus ( Lactococcus ), Shigella (Shigella ), Vibrio ( Vibrio ), Listeria ( Listeria ), Escherichia ( Escherichia ) and Salmonella ( Salmonella However, the mechanisms of cancer treatment using these strains have not yet been fully elucidated.

[0005] Among the various strains proposed above, the previously developed attenuated Salmonella strains distribute not only in cancer cells but also in various normal organs, resulting in low cancer cell targeting rates and side effects caused by the strain itself. In particular, patients with weakened immune systems and the elderly can develop fatal sepsis even with attenuated Salmonella strains, so improvements are needed to ensure therapeutic efficacy. Therefore, there is a need to develop new strains that have exceptionally high tumor targeting ability and in vivo stability and can be effectively used in cancer treatment.

[0006] Therefore, the present inventors conducted research to develop an attenuated Salmonella gallinarum strain that has high in vivo safety, excellent tumor targeting ability, survival rate, and therapeutic ability, but very low targeting ability and survival rate in normal organs, and have completed the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent No. 10-2015573 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a novel attenuated Salmonella gallinarum strain that is deleted for the genes encoding guanosine tetraphosphate (ppGpp) synthase, the genes that induce the function of the type III secretion system (T3SS), and the Gifsy 2 prophage gene.

[0009] The present invention also provides a gene encoding a guanosine tetraphosphate (ppGpp) synthase, a gene inducing the function of a type III secretion system (T3SS), Gifsy 2, and a gene encoding a guanosine tetraphosphate (ppGpp) synthase. prophage Genes and glmS A novel attenuated Salmonella gallinarum strain is provided, in which genes are deleted.

[0010] Specifically, the present invention provides the Salmonella gallinarum SG4044 strain (Microorganism Accession No.: KCTC14541BP) ​​or SG4048 strain (Microorganism Accession No.: KCTC14542BP), which has excellent antitumor effects.

[0011] Compositions for tumor therapy or diagnosis comprising the strains of the present invention are provided.

[0012] The present invention provides a method for providing information for tumor diagnosis, comprising the steps of administering the bacterial strain of the present invention to a subject; detecting the bioluminescence of the bacterial strain; and determining that a tumor is present in the subject if bioluminescence is detected. [Means for solving the problem]

[0013] The present invention relates to a gene encoding guanosine tetraphosphate (ppGpp) synthase, a gene inducing the function of a type III secretion system (T3SS), and Gifsy 2. prophage The present invention relates to an attenuated Salmonella gallinarum strain characterized by a gene deletion.

[0014] The present invention also provides a gene encoding a guanosine tetraphosphate (ppGpp) synthase, a gene inducing the function of a type III secretion system (T3SS), Gifsy 2, and a gene encoding a guanosine tetraphosphate (ppGpp) synthase. prophage Genes and glmS The present invention relates to a novel attenuated Salmonella gallinarum strain in which genes are deleted.

[0015] The strains of the present invention comprise a glmS The present invention relates to an attenuated Salmonella gallinarum strain transformed with a plasmid containing the gene.

[0016] The gene encoding the guanosine tetraphosphate (ppGpp) synthase is relA or spoT It may be.

[0017] The genes that induce the function of the type III secretion system (T3SS) are arranged contiguously in the genome. ssrA , or ssrB , or ssrAB is.

[0018] The present invention relates to the Salmonella gallinarum SG4021 strain (Microorganism Accession Number: KCTC13985BP). relA , spoT , ssrAB and Gifsy 2 prophage The present invention relates to an attenuated Salmonella gallinarum strain in which a gene has been deleted.

[0019] The present invention relates to the Salmonella gallinarum SG4021 strain (Microorganism Accession Number: KCTC13985BP). relA , spoT , ssrAB , Gifsy 2 prophage and glmSThe present invention relates to an attenuated Salmonella gallinarum strain in which a gene has been deleted.

[0020] In one embodiment, the attenuated Salmonella gallinarum strain of the present invention may be Salmonella gallinarum strain SG4044 (Microorganism Accession Number: KCTC14541BP).

[0021] In one embodiment, the attenuated Salmonella gallinarum strain of the present invention may be Salmonella gallinarum strain SG4048 (Microorganism Accession Number: KCTC14542BP).

[0022] The strain of the present invention may further comprise a light-emitting gene.

[0023] The luminescence gene is luxCDABE It may be.

[0024] The present invention relates to a pharmaceutical composition for diagnosing or treating tumors, comprising the strain. The tumor may be a solid cancer or adenocarcinoma, preferably, but not limited to, colon cancer, pancreatic cancer, lung cancer, skin cancer, or breast cancer.

[0025] The present invention relates to a method for providing information for tumor diagnosis, which includes the steps of administering the bacterial strain to a subject; detecting bioluminescence of the bacterial strain; and determining that a tumor is present in the subject if bioluminescence is detected. [Effects of the Invention]

[0026] The present invention relates to a method for producing guanosine tetraphosphate (ppGpp)-deficient, a loss of function of the type III secretion system (T3SS), and Gifsy 2. prophageAttenuated Salmonella gallinarum strains constructed by gene deletion can specifically target tumors and have excellent tumor growth inhibitory activity while minimizing damage to normal tissues other than the tumor, making them useful for tumor improvement, treatment, and imaging. [Brief explanation of the drawings]

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[0036] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily understand the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0037] Throughout this specification, when a part is described as "comprising" a certain element, this means that it may further include other elements, rather than excluding other elements, unless otherwise specified.

[0038] The present invention relates to a gene encoding guanosine tetraphosphate (ppGpp) synthase, a gene inducing the function of a type III secretion system (T3SS), and Gifsy 2. prophage The present invention relates to an attenuated Salmonella gallinarum strain in which a gene has been deleted.

[0039] Traditionally, the Salmonella strain most frequently used in bacterial anti-cancer targeted therapy research is Salmonella Typhimurium, and due to the pathogenicity of Salmonella Typhimurium infection, it is always attenuated before use in research (Forbes, NS (2010). Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer, 10(11), 785-794.). The Salmonella genus is so diverse that it is classified into approximately 2,500 species using serological classification methods, and some of these strains have been reported to possess host-specific pathogenicity (Porwollik, S; Boyd, EF; Choy, C; Cheng, P; Florea, L; Proctor, E; McClelland, M (September 2004)). For example, Salmonella Typhi and Salmonella paratyphi possess pathogenic mechanisms that target humans as hosts, while Salmonella Typhimurium has been reported to infect and cause disease in humans as well as cattle, pigs, sheep, horses, and rodents. On the other hand, Salmonella gallinarum is known to specifically infect and exhibit pathogenicity only in birds. Therefore, the attenuated Salmonella gallinarum strain of the present invention is safest for humans and can be effectively used for tumor diagnosis or treatment.

[0040] The term "attenuation" as used herein refers to a modification of a bacterial strain to reduce its pathogenicity. By attenuating a bacterial strain, cytotoxicity and other side effects that may occur due to the pathogenicity of the strain in normal cells other than tumor cells can be prevented. Attenuated bacterial strains can be obtained by various methods known in the art. For example, attenuation can be achieved by deleting or disrupting virulence factors that enable the strain to survive in host cells. pab , proBC , [[ID= , ​ , ​ , ​ , ​, ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ and ​ Genes, and ​ , ​ , ​ , ​ , ​ , ​ This may be achieved by deletion of genes encoded in the salmonella pathogenicity island (SPI), including, but not limited to, the following:

[0041] The term "ppGpp" used in the present invention refers to guanosine tetraphosphate, which may be alternatively referred to as guanosine 5'-diphosphate 3'-diphosphate or guanosine 3'5'-bispyrophosphate. ppGpp is an intracellular signaling substance that induces the expression of genes that cause the virulence of Salmonella gallinarum strains, particularly genes encoded in the Salmonella Pathogenicity Island (SPI). Furthermore, in the present invention, the gene encoding guanosine tetraphosphate (ppGpp) synthase is ​ and ​ It can mean genes.

[0042] ​ and ​ Concomitant deletion of the gene results in impairment of gene transcription or translation and the activity of the gene product. ​ and ​ This may be achieved by genetic modifications, which may involve inactivation of the ppGpp synthase coding sequence (CDS) as well as its promoter.

[0043] Specific inactivation of only a target gene on the genome of a Salmonella gallinarum strain may be achieved by mutation of the entire gene coding region or one or more partial regions thereof through substitution, insertion, deletion, or a combination thereof. For example, gene deletion and insertion of a heterologous sequence into a gene may result in gene truncation, nonsense mutation, frameshift mutation, missense mutation, etc. Such specific inactivation of a gene may be performed by methods commonly used in the art. Meanwhile, gene deletion may be performed by various mutagenesis methods known in the art. For example, ​ and ​ Gene deletions may be performed by PCR mutagenesis and cassette mutagenesis.

[0044] The term "type III secretion system (T3SS)" used in the present invention refers to a gene that is continuously arranged on a gene. ​ and ​ It is regulated by genes. ​ is a transcriptional regulator and membrane protein ​ Therefore, the above-mentioned ​ and / or ​Loss of function due to deletion of SPI2 can result in the inactivation of entire operons and genes contained in Salmonella pathogenicity island 2 (SPI2). After infection, Salmonella distributes the type III secretion system (T3SS) encoded by SPI2 to modify host cell functions and propagate within the host cells.

[0045] In the present invention, Gifsy1 and 2 refer to viruses (bacteriophages) that target bacteria. Based on the results of genome analysis of wild-type Salmonella gallinarum, the inventors have identified Gifsy 2 as a ​ It has been confirmed that the entire gene sequence encoding the gene is contained, and the pathogenicity of Salmonella can be reduced by deleting the gene inserted into the Salmonella genome.

[0046] Genetic removal of bacteriophages from bacterial hosts reduces their pathogenicity and makes them attenuated.

[0047] therefore, ​ , ​ , ​ Gene and Gifsy 2 ​ An attenuated Salmonella gallinarum strain in which the entire gene encoding the .alpha.-glucan has been deleted has a virulence attenuated by more than a million times compared to wild-type Salmonella gallinarum, enabling effective attenuation of Salmonella gallinarum strains.

[0048] ​ Salmonella gallinarum strains lacking the gene can be lysed in animals due to a lack of D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components of peptidoglycan synthesis, and therefore can be used as antibiotic resistance genes instead of antibiotic resistance genes. ​ The gene can be used as a selective determinant for Salmonella gallinarum strains.

[0049] The balanced-lethal host-vector system is a chromosomal ​ To complement deletion mutations ​ It may be constructed by transformation of a plasmid containing the gene. ​ Attenuated Salmonella gallinarum strains successfully transformed with a plasmid containing the gene are capable of biosynthesis of GlcN or GlcNAc, and therefore can survive in environments where GlcN or GlcNAc is lacking, and can act as a selectable marker.

[0050] The plasmid of the present invention may be a plasmid used in the art, for example, a plasmid selected from the group consisting of the pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, but is not limited thereto.

[0051] The plasmid of the present invention may further include one or more selectable markers. The marker is typically a nucleic acid sequence having a property that allows selection by chemical methods, such as a gene that allows transformed cells to be distinguished from non-transformed cells. Examples of selectable markers include, but are not limited to, herbicide resistance genes such as glyphosate, glufosinate ammonium, or phosphinothricin, and antibiotic resistance genes such as ampicillin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol.

[0052] The plasmid of the present invention can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes generally known in the art.

[0053] The method for introducing the plasmid of the present invention into a Salmonella gallinarum strain for transformation may be a method commonly used in the art for introducing nucleic acids into cells, and may be selected from standard techniques known in the art, such as, but not limited to, electroporation, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-Textlan method, cationic liposome method, and lithium acetate-DMSO method.

[0054] The term "operably linked" as used herein refers to a functional connection between a gene expression regulatory sequence and another nucleotide sequence. The gene expression regulatory sequence may be one or more selected from the group consisting of a replication origin, a promoter, and a transcription termination sequence. The transcription termination sequence may be a polyadenylation sequence (pA), and the replication origin may be, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adenovirus replication origin, an AAV replication origin, or a BBV replication origin.

[0055] As used herein, the term "promoter" refers to a region of DNA upstream from a structural gene, and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

[0056] According to one embodiment of the present invention, a promoter is one of the transcriptional regulatory sequences that regulates the initiation of transcription of a specific gene, and may be a polynucleotide fragment having a length of about 100 bp to about 2500 bp. For example, the promoter may be ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter,​ Promoter, ​ Promoter, T7 Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ ( ​ ) promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoter, ​ Promoters and ​ The promoter may be selected from the group consisting of, but not limited to, a promoter and a synthetic promoter.

[0057] The term "luminescence gene" as used herein refers to a gene encoding a protein capable of in vivo or ex vivo (in vitro) imaging, and may include bioluminescence genes (e.g., luciferase genes), chemiluminescence genes, and fluorescent genes of various origins. Devices for imaging the luminescence phenomenon expressed by such genes are known in the art and may be appropriately selected by those skilled in the art.

[0058] The Salmonella gallinarum strain transformed with a plasmid containing the luminescence gene of the present invention targets tumors, allowing visual monitoring of tumors, thereby improving the accuracy and efficacy of tumor treatment. Imaging equipment that can be used with the luminescence gene of the present invention may be appropriately selected by those skilled in the art depending on the type of luminescence gene used.

[0059] In the present invention, ​ " is the gene encoding luciferase ( ​ and ​ a heterodimer of ) and a gene encoding luciferin (Tetradecanal), which is a substrate of the luciferase (​ , ​ and ​ ) is a DNA fragment that contains all of the luminescent substances. Luciferin, a luminescent substance, is activated by ATP within the cell and converted into active luciferin, which is then converted into oxidized luciferin by the action of luciferase, a luminescent enzyme, converting chemical energy into light energy and producing light.

[0060] Tumor cells targeted by the Salmonella gallinarum strain transformed with the luminescence gene of the present invention can be detected by the luminescence gene, and information can be obtained regarding the presence or absence of a tumor in a subject, the location and size of the tumor present in the subject, and the changes in the tumor present in the subject over time.

[0061] In the present invention, the targeting and anticancer activity of the attenuated Salmonella gallinarum strain against colon cancer, pancreatic cancer, lung cancer, skin cancer, and breast cancer cell lines was confirmed, and the excellent anticancer effect of the attenuated Salmonella gallinarum strain of the present invention was confirmed in a CT26 tumor mouse model, which is an adenocarcinoma.

[0062] The attenuated Salmonella gallinarum strain of the present invention can exhibit targeting and tumor suppression activity against various adenocarcinomas or solid cancers, such as colon cancer, pancreatic cancer, lung cancer, skin cancer, and breast cancer.

[0063] Adenocarcinoma is a type of cancer that occurs specifically in cells that make up the glands, and refers to cancer that occurs in the glandular tissue or excretory organs of the stomach, intestines, bronchi, uterus, gallbladder, etc., as well as the prostate, thyroid, and pancreas.

[0064] The present invention will be described in more detail below with reference to examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0065] [Example 1] Preparation of attenuated Salmonella gallinarum strains and methods for evaluating their tumor-suppressing activity

[0066] 1-1.Growth of Salmonella strains in LB medium (Difco Laboratories) containing 1% NaCl. ​ The cells were grown at 37°C under vigorous aeration. Solid support medium was prepared containing 1.5% granular agar (Difco Laboratories), and antibiotics were purchased from Sigma Chemical. When necessary, the antibiotics ampicillin (Amp), kanamycin (Km), and chloramphenicol (Cm) were added at concentrations of 100 μg / ml, 50 μg / ml, and 15 μg / ml, respectively, and N-acetyl-D-glucosamine (GlcNAc) was added at a concentration of 100 mg / ml. The bacterial count in the liquid medium was measured using a hemocytometer.

[0067] 1-2. Salmonella strain lineage As shown in Table 1 below, serovar Gallinarum ( ​ The mutants were derived from SG4021, a clinical isolate isolated from the liver of a chicken infected with fowl typhoid at a Korean poultry farm. The SG4021 strain was deposited at the Korea Institute of Bioscience and Biotechnology (KITB) on October 8, 2019 (microorganism accession number: KCTC13985BP). The SG4044 and SG4048 strains were deposited at the Korea Institute of Bioscience and Biotechnology (KITB) on April 20, 2021 (microorganism accession numbers: KCTC14541BP and KCTC14542BP).

[0068] [Table 1]

[0069] All Salmonella strains were generated by the method developed by Datsenko and Wanner (Proceedings of the National Academy of Sciences 97.12(2000):6640-6645), and Salmonella strain attenuation was achieved by using the ppGpp, ssrAB, and Gifsy 2 genes. ​ Deletions of ΔppGpp, ΔssrAB, Δ Induced by Gifsy 2 prophage.

[0070] The ppGpp deletion mutation was introduced into the genome of SG4021 by the relA::kan R and spot::cm R The ssrAB deletion mutation was induced by sequentially introducing ssrAB::kan into the genome of SG4021. R To create the ppGpp and ssrAB deletion mutants, we first inactivated the antibiotic resistance gene introduced to create ppGpp (kan R and commercials R ), ssrAB::kan R The SG3005 strain, which contained the gene encoding ssrAB, was transduced with P22 bacteriophage to transform the ssrAB::kan R Bacteriophages containing the gene were produced. Inactivation of the antibiotic resistance gene was achieved by the introduction of pCP20 (Helper plasmid), which induces expression of FLP recombinase, through transformation. The antibiotic resistance gene deletion method for all mutant Salmonella gallinarum strains constructed for this invention was the same.

[0071] ssrAB::kan inactivated ppGpp deletion mutant R Transduction of bacteriophage containing the genes ΔrelA, ΔspoT, ssrAB::kan R The mutation was generated (SG4041). The bacteriophage (Gifsy 2) of wild-type Salmonella gallinarum (clinical isolate) ​The presence or absence of the bacteriophage genome (Gifsy 2) was confirmed by whole genome sequencing of wild-type Salmonella gallinarum. ​ For attenuation, the prophage gene was replaced with a chloramphenicol (cm) resistance gene in the SG4021 genome using the Lambda-Red homologous recombination method (Gifsy 2 prophage::cm). R ) and Gifsy 2 ​ A gene deletion mutant Salmonella gallinarum was generated (SG4042).

[0072] ppGpp, ssrAB, Gifsy 2 ​ Deletion mutant Salmonella gallinarum was generated using Gifsy 2 prophage::cm R A bacteriophage (P22, SG4042 strain conjugated) containing the ΔrelA, ΔspoT, ssrAB::kan gene was first generated and then transduced into the SG4041 strain (ΔrelA, ΔspoT, ssrAB::kan). R ,Gifsy 2 prophage::cm R SG4043). In order to avoid the problem of excessive antibiotic resistance in the development of anticancer drugs from attenuated Salmonella gallinarum, all antibiotic resistance genes were removed from the SG4043 strain, resulting in SG4044 (Δ ​ , Δ ​ , Δ ​ , ΔGifsy 2 ​ In order to evaluate the efficacy and value of the attenuated Salmonella strains prepared in this invention, a strain carrying only the chloramphenicol resistance gene was prepared (Δ ​ , Δ ​ , Δ ​ ,Gifsy 2 prophage::cm R ;SG4046).

[0073] To develop strain SG4046, the kanamycin resistance gene was deleted from SG4045 (Δ​ , Δ ​ , Δ ​ ) strain was created and utilized. ​ The kanamycin resistance gene (kan R The gene carrying the pKD13 gene was generated by polymerase chain reaction (PCR) using a pair of 60-nt primers, each containing a 40-nt homology extension and a 20-nt priming sequence, using pKD13 as a template. The primer sequences and SEQ ID NOs are shown in Table 2 below. The template used in PCR was wild-type Salmonella gallinarum bacterial chromosomal DNA.

[0074] [Table 2]

[0075] The purified PCR product was transformed by electrophoresis into attenuated Salmonella (Δ ​ , Δ ​ ;SG4023), which ​ A gene-deleted attenuated Salmonella gallinarum strain was generated (Δ ​ , Δ ​ , glmS::kan R SG4030). Attenuated Salmonella gallinarum (Δ ​ , Δ ​ , Δ ​ , ΔGifsy 2 ​ , glmS::kan R ) for the construction of glmS::kan R The P22 phage containing ​ , Δ ​ , Δ ​ , ΔGifsy 2 ​) prepared glmS::kan R Transduction of P22 phage containing Δ ​ , Δ ​ , Δ ​ , ΔGifsy 2 ​ , glmS::kan R (SG4047) mutant Salmonella gallinarum strain was generated.

[0076] To avoid the problem of excessive antibiotic resistance in the development of anticancer drugs from attenuated Salmonella gallinarum, the kanamycin resistance gene of SG4047 was removed by the same method as above, and the final strain, Δ ​ , Δ ​ , Δ ​ , ΔGifsy 2 ​ , Δ ​ A Salmonella gallinarum strain was generated (SG4048).

[0077] It is not possible to exploit antibiotic resistance genes for selective determinants, since Salmonella strains are particularly prone to releasing plasmids containing and carrying reporter genes that are not necessary for survival in animals. Therefore, they are lysed in animal systems due to a lack of D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components of peptidoglycan synthesis, as essential nutrients for growth. ​ The deletion mutant phenotype was utilized.

[0078] 1-3.GlmS + pLux plasmid Evaluation of tumor targeting ability by bioluminescence and chromosomal ​ To complement the gene deletion, Salmonella gallinarum strain ​ A balanced lethal host vector system was constructed to integrate the gene.

[0079] Specifically, the lux operon of Photobacterium leiognathi ( ​pLux, containing the fragment (approximately 9.5 kbp), was inserted into the pUC19 plasmid backbone. ​ The Lux operon cassette and ​ To construct a plasmid containing all of the above, a Salmonella gallinarum strain was used. ​ The gene was amplified with specific primers, the base sequences and SEQ ID NOs of which are shown in Table 3 below. The template used in the PCR reaction was bacterial chromosomal DNA of wild-type Salmonella gallinarum (SG4021).

[0080] [Table 3]

[0081] The pLux vector and the amplified 1.8 kbp fragment were cut at both ends with Sal I restriction enzyme, and then joined with T4 DNA ligase to form GlmS. + pLux was generated. + Transformed with pLux ​ Deletion mutant Salmonella gallinarum strains can survive in environments lacking D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc) and exhibit bioluminescence, allowing analysis by optical bioluminescence imaging.

[0082] 1-4. Tumor cell lines Murine-derived CT26 (colon tumor cell line), 4T-1 (breast tumor cell line), B16F10 (melanoma tumor cell line), and LL2 (LLC-1, lung tumor cell line) cells were purchased from the American Type Culture Collection, and Murine-derived Panc02 (pancreatic tumor cell line) cells were purchased from the National Cancer Institute, Division of Cancer Treatment & Diagnosis (DCTD). CT26, 4T-1, LLC-1, and Panc02 cell lines were grown in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, while B16F10 cell line was grown in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0083] 1-5. Plasmid stability measurement Overnight subcultures (1 / 1000) were made in Flesh LB medium replenished every 12 hours. Samples were taken and diluted every 24 hours. Appropriate volumes were spread in triplicate on LB plates supplemented with GlcNAc, with or without ampicillin. Colony counts were used to calculate total viable cells (colony-forming units, CFU) and the proportion of Salmonella strains carrying the plasmid.

[0084] 1-6. Mouse model creation Mouse tumor models were created by xenografting various cultured tumor cell lines into the right thigh of mice. Five- to eight-week-old female mice weighing 20–30 kg were purchased from Samtako Company (Korea). All animal care, experiments, and euthanasia were performed according to approved protocols.

[0085] Subcutaneous tumor-bearing mice were generated as follows. The tumor cell lines of Examples 1-4 cultured in vitro were collected and suspended in 30 ml of PBS. CT26, 4T-1, LLC-1, and B16F10 cell lines were then cultured at 1 × 106 1 × 10 for Panc02 cell line 7 The cells were subcutaneously injected into the right thigh of mice. CT26 and 4T-1 cell lines were transplanted into Balb / C mice, and LLC-1, Panc02, and B16F10 cell lines were transplanted into C57 / BL6 mice.

[0086] After tumor cell transplantation, the tumor size was 60 mm 3 When the GlmS of Examples 1-3 is reached, + transformed with pLux, ​ Approximately 5 × 10 deletion mutant attenuated Salmonella gallinarum (SG4050) 8 CFU, and ppGpp, ssrAB, Gifsy 2 of Examples 1-2 were injected intravenously. ​ Deletion of Salmonella gallinarum strains (SG4044 or SG4046, 5 × 10 8 CFU) or ppGpp-deficient Salmonella typhimurium (SMR2130, 1 × 10 7 CFU) strains were administered via the tail vein.

[0087] 1-7. Evaluation of Salmonella strain distribution in mouse internal organs To assess the viability of Salmonella strains, groups of mice (n = 5) were injected with approximately 1 × 10 7 CFU ΔppGpp Salmonella typhimurium (SMR2130) and approximately 5 × 10 8 CFU ΔppGpp, Δ ​ , Gifsy 2 ​ After intravenous administration of Salmonella gallinarum (SG4046), mice were sacrificed on days 1, 3, 5, 10, and 15, and organs were collected. The organ tissues were homogenized in sterile PBS containing 0.05% Tween-20 using a homogenizer. Salmonella strains were recovered from the homogenates and quantified by spreading them on agar plates containing 50 μg / ml kanamycin and 15 μg / ml chloramphenicol.

[0088] 1-8. Tumor size and mouse survival rate analysis Tumor volume was calculated using the following formula: [Calculation formula] Tumor volume (mm 3) = (tumor length × tumor height × tumor width) / 2

[0089] All animal experiments were approved by the Jungnam National University Animal Experiment Ethics Committee (NO. CNUIACUC-H-2016-15) and were performed in accordance with the guidelines. 3 The mice were sacrificed and the survival rate of the mice was evaluated by the Gehan-Breslow-Wlicoxon test.

[0090] 1-9. Optical bioluminescence imaging Bioluminescence imaging of Salmonella strains was used as an indicator for tumor targeting. For tumor targeting, mice were anesthetized with 2% isoplulan and placed in a light-tight chamber equipped with an IVIS100 (Caliper, Hopkinton, MA, USA) equipped with a cooled CCD (charged couple detector) camera. Photons emitted from luciferase-expressing Salmonella gallinarum strains were collected and integrated for 1 min. Pseudocolor images showing photon counts were overlaid on the mouse photograph using Living Image software v.2.25 (Xenogen-Caliper, Hopkinton, MA).

[0091] 1-10.Statistical analysis Statistical analysis was performed using the SPSS 18.0 statistical package (SPSS Inc., Chicago, IL, USA). Two-tailed Student's t-test was used to determine statistical significance of tumor growth between the control and treatment groups. A P value of 0.05 or less was considered statistically significant, and all data are presented as mean ± SD.

[0092] [Example 2] Confirmation of in vivo toxicity and plasmid stability of attenuated Salmonella gallinarum strains 2-1. Confirmation of amino acid requirements of Salmonella gallinarum strains

[0093] As shown in Figure 1, wild-type Salmonella gallinarum grew only in minimal medium supplemented with leucine (Leu), whereas its growth was improved by the addition of arginine (Arg) and phenylalanine (Phe). The ppGpp-deficient attenuated Salmonella gallinarum also grew in minimal medium supplemented with isoleucine (Ile), lysine (Lys), serine (Ser), and valine (Val). Therefore, we confirmed that the ppGpp-deficient Salmonella gallinarum required fewer amino acids than the ppGpp-deficient Salmonella typhimurium (LT2) (Tedin and Norel, J Bacteriol, 2001, 183:6184-6196).

[0094] 2-2. In vivo toxicity evaluation of Salmonella gallinarum strains To apply Salmonella gallinarum strains to bacterial cancer therapy, we first injected wild-type Salmonella gallinarum strains intravenously (IV) and then analyzed the survival rate of mice.

[0095] As shown in Figure 2, when wild-type Salmonella gallinarum strain (SG4021) was intravenously administered to mice, typhoid fever was induced only in poultry fowls, but approximately 10 5 All mice died when administered more than CFU.

[0096] 2-3. Confirmation of dosage by in vivo toxicity evaluation of attenuated Salmonella gallinarum strain In Example 2-2, it was confirmed that the wild-type Salmonella gallinarum strain possesses in vivo toxicity in mice, and ppGpp, ssrAB, Gifsy 2 ​ A Salmonella gallinarum strain was attenuated by deletion.

[0097] Specifically, ΔppGpp, Δ ​ , Gifsy 2 ​ Salmonella gallinarum strain (SG4046) and ΔppGpp Salmonella Typhimurium strain (SMR2130) were intravenously administered to Blab / C mice, and the survival rate of the mice was analyzed.

[0098] To accurately confirm the number of bacteria when examining the distribution of bacterial strains by organ, SG4046 containing antibiotic markers was used.

[0099] As shown in Figs. 3 and 4, ΔppGpp, Δ ​ , Gifsy 2 ​ For Salmonella gallinarum strain (SG4046), approximately 5 × 10 8 The mice survived until the CFU reached approximately 1 × 10 7 Mice were confirmed to have survived to CFU.

[0100] From the experimental results of attenuated Salmonella typhimurium (A1-R), which confirmed tumor targeting ability and antitumor effect, modified lipopolysaccharide, ​ Deletion mutation (VNP20009) and Δ ​ / Δ ​ The strain carrying the double mutation had approximately 1 × 10 6 ~Approx. 1×10 7 It was confirmed that a therapeutic range was formed in the range of CFU. Therefore, the attenuated Salmonella gallinarum of the present invention has superior stability, as its in vivo stability is about 15 times higher than that of the attenuated Salmonella typhimurium.

[0101] In addition, the effective dose of Escherichia coli (Escherichia coli) that has been shown to ensure biostability is approximately 1 x 10 8 CFU (administration route: intravein, IV), it is clear that the attenuated Salmonella gallinarum of the present invention has significantly excellent in vivo stability.

[0102] 2-4. Plasmid stability confirmation To confirm the stability of the transformed plasmid, the same procedure as in Examples 1-5 was repeated to evaluate whether the plasmid was lost.

[0103] As shown in Figure 5 , 99% of the plasmid carried by the wild-type Salmonella strain (SG4021) was lost by day 4, whereas ​ It was confirmed that the plasmid carried by the deleted Salmonella gallinarum strain (SG4050) was fully maintained.

[0104] In subsequent experiments, the GlmS assay described in Examples 1-3 was used to visualize Salmonella gallinarum strains in tumor-bearing mice. + Transformed with pLux, a mutation on the chromosome ​ A Salmonella gallinarum strain (SG4050) carrying the gene was used.

[0105] [Example 3] Validation of tumor targeting by attenuated Salmonella gallinarum strains ΔppGpp, Δ ​ , ΔGifsy 2 ​ To confirm tumor targeting of Salmonella gallinarum strains, Examples 1-9 were performed to image the bioluminescence signals of attenuated Salmonella gallinarum strains.

[0106] As shown in Figure 6, ΔppGpp, Δ ​ , ΔGifsy 2 progeny , Δ glmS Immediately after injection of the Salmonella gallinarum strain (SG4050) (20 min, 0 days post-injection (dpi)), bioluminescence signals were detected primarily in the endothelial organs (liver and spleen), but 3 days post-injection (dpi), bioluminescence signals were observed only in the implanted tumor tissue. In particular, the attenuated Salmonella gallinarum strain was confirmed to specifically target all tumors implanted in four individual mouse lines.

[0107] Therefore, ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny , Δ glmS It has been found that the Salmonella gallinarum strain (SG4048) exhibits excellent tumor targeting effect.

[0108] [Example 4] ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny Confirmation of the anti-cancer effect of Salmonella gallinarum strain

[0109] 4-1. Confirmation of the antitumor effect and survival period extension of attenuated Salmonella gallinarum Approximately 5 × 10 CT26 cells were transplanted into Blab / C mice. 8 CFU ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny Salmonella gallinarum strain (SG4044) was intravenously administered to the mice. As a control group, a PBS-treated group and a ΔppGpp Salmonella Typhimurium strain (1 × 10 7 CFU, SMR2130) were used to detect ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny The in vivo antitumor activity of Salmonella gallinarum strain was analyzed according to Examples 1-8 and 1-10.

[0110] As shown in Figure 7, approximately 5 × 10 8 CFU ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny When Salmonella gallinarum strain (SG4044) was administered to mice bearing colon tumors, tumor growth was significantly delayed compared to the control group.

[0111] As shown in Figure 8, ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny The survival period of the group treated with Salmonella gallinarum (SG4044) was confirmed to be approximately two times longer than that of the control group. The measured life extension period was approximately 24 days in the ΔppGpp Salmonella Typhimurium strain-treated group and 15 days in the PBS-treated group. ssrAB , ΔGifsy 2 progeny The mean survival time for the group treated with Salmonella gallinarum was 38 days.

[0112] 4-2. Confirmation of Salmonella strain distribution pattern in mice To confirm the distribution pattern of Salmonella gallinarum strain in mice, the above Examples 1-7 were carried out to evaluate the distribution of Salmonella counts in each mouse organ over time.

[0113] As shown in Figure 9, ΔppGpp, Δ ssrAB ,Gifsy 2 progeny Salmonella gallinarum strain (5 × 10 8 , SG4046) and the ΔppGpp Salmonella typhimurium strain (1 × 10 7 In both groups administered 100 mg / kg of 100% ethanol (SMR2130), the most abundant Salmonella strain was observed in the tumor 3 days after injection (3 dpi), and the bacterial count remained at a similar level in the tumor for 10 days, but gradually decreased after 10 days.

[0114] In addition, attenuated Salmonella typhimurium (SMR2130) produced 10 serotypes in the liver 1 day after injection (1 dpi). 6 CFU / g, 10 in spleen 7 CFU / g, 10 in lungs, kidneys and heart 4 CFU / g, 10 in serum 2 The bacterial counts in normal tissues were confirmed to be at CFU / g, and the same levels were detected in other tissues except for blood samples throughout the experiment. Meanwhile, in the case of attenuated Salmonella gallinarum (SG4046), bacterial counts similar to those of attenuated Salmonella Typhimurium (SMR2130) were detected 1 day post-injection (1 dpi), but then gradually decreased and were completely eliminated by the 15th day.

[0115] Therefore, the ΔppGpp, Δ ssrAB , ΔGifsy 2 progeny Since the Salmonella gallinarum strain specifically targets tumors and exhibits antitumor effects, it can be used for antitumor purposes without the risk of side effects on other organs. [Accession number]

[0116] 1. Salmonella gallinarum strain SG4021; Salmonella. chickens(SG4021); Microorganism accession number KCTC13985BP 2. Salmonella gallinarum strain SG4044; S.gallinarum ΔrelA,Δspot,ΔssrAB,ΔGifsy 2 prophage (SG4044); Microorganism accession number KCTC14541BP 3. Salmonella gallinarum strain SG4048; S.gallinarum ΔrelA,ΔspoT,ΔssrAB,ΔGifsy 2 prophage,ΔglmS (SG4048) Microorganism accession number: KCTC14542BP

Claims

1. the gene encoding guanosine tetraphosphate (ppGpp) synthase, the gene inducing the function of the type III secretion system (T3SS), and the Gifsy 2 prophage gene are deleted; The attenuated Salmonella gallinarum strain is characterized in that the gene that induces the function of the type III secretion system (T3SS) is ssrA, ssrB, or ssrAB.

2. 2. The attenuated Salmonella gallinarum strain according to claim 1, characterized in that the glmS gene is deleted.

3. The attenuated Salmonella gallinarum strain according to claim 1, wherein the gene encoding the guanosine tetraphosphate (ppGpp) synthase is relA or spoT.

4. The attenuated Salmonella gallinarum strain described in claim 1, characterized in that the relA, spoT, ssrAB and Gifsy 2 prophage genes are deleted in the Salmonella gallinarum SG4021 strain (microorganism accession number: KCTC13985BP).

5. The attenuated Salmonella gallinarum strain described in claim 1, characterized in that the relA, spoT, ssrAB, Gifsy 2 prophage and glmS genes are deleted in the Salmonella gallinarum SG4021 strain (microorganism accession number: KCTC13985BP).

6. The attenuated Salmonella gallinarum strain described in claim 1, characterized in that the strain is Salmonella gallinarum SG4044 strain (microorganism accession number: KCTC14541BP).

7. The attenuated Salmonella gallinarum strain described in claim 1, characterized in that the strain is Salmonella gallinarum SG4048 strain (microorganism accession number: KCTC14542BP).

8. The attenuated Salmonella gallinarum strain of claim 2, wherein the strain is transformed with a plasmid containing the glmS gene operably linked to a promoter.

9. An attenuated Salmonella gallinarum strain as described in claim 1, characterized in that the strain further contains a luminescence gene.

10. The attenuated Salmonella gallinarum strain described in claim 9, characterized in that the luminescence gene is luxCDABE.

11. A pharmaceutical composition for diagnosing or treating tumors, comprising the strain of any one of claims 1 to 10.

12. A pharmaceutical composition for diagnosing or treating a tumor as described in claim 11, characterized in that the tumor is a solid cancer.

13. A pharmaceutical composition for diagnosing or treating a tumor as described in claim 11, characterized in that the tumor is an adenocarcinoma.

14. A pharmaceutical composition for diagnosing or treating a tumor as described in claim 11, characterized in that the tumor is any one cancer selected from the group consisting of colon cancer, pancreatic cancer, lung cancer, skin cancer and breast cancer.

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