Nutritional requirement strains of Staphylococcus bacteria

Abstract

To provide recombinant Staphylococcus bacteria (e.g., S. epidermidis) that are dependent on D-alanine for growth. [Solution] In one aspect, the Disclosure features recombinant Staphylococcus bacteria comprising two inactivated alanine racemase genes (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In another aspect, the Disclosure features a method for producing recombinant Staphylococcus bacteria. In another aspect, the Disclosure provides a method for treating or preventing a rash in a subject, the method comprising the step of administering to the subject an amount effective to treat or prevent a rash in the subject, which is one of the aspects or embodiments described herein.

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 768,485, filed Nov. 16, 2018, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

Background Art

[0002] Background of the Invention Alanine racemase is a pyridoxal phosphate-containing homodimeric enzyme that catalyzes the conversion of L-alanine, an important building block in the biosynthesis of the peptidoglycan layer in bacterial cell walls, to D-alanine. Alanine racemase is typically not present in eukaryotes but is widely distributed among prokaryotes, which makes this enzyme an attractive target for the development of novel antibacterial agents.

[0003] D-alanine is essential for bacterial cell wall formation, but it has been found to be more complex to determine which genes are extremely important in the D-alanine biosynthetic pathway. Bacteria contain either one or two alanine racemase genes. In species with two genes, one is constitutively expressed and assimilatory, while the other is inducible and catabolic (Strych, U. et al., 2007. BMC Microbiol. 7:40; Strych U. et al., Curr. Microbiol. 41:290-294; Strych U. et al., FEMS Microbiol. Lett. 196:93-98). These genes supply the D-alanine required for cell wall biosynthesis, and knockout studies in some of these bacteria have established that the alanine racemase enzyme is essential for growth in the absence of exogenous D-alanine (Franklin, F. C., and W. A. Venables. 1976. Mol. Gen. Genet. 149:229-237; Hols, P. et al., J. Bacteriol. 179:3804-3807; Palumbo, E. et al., FEMS Microbiol. Lett. 233:131-138; Steen, A. et al., J. Bacteriol. 187:114-124; Wijsman, HJ 1972. Genet. Res. 20:269-277).

[0004] We previously developed a double alanine racemase gene knockout strain of S. epidemidis (SEΔalr1Δalr2). However, this double knockout strain did not exhibit D-alanine nutritional requirements, in contrast to Bacillus subtilis, Escherichia coli, and several other bacterial species. Therefore, this disclosure relates to Staphylococcus, which is dependent on D-alanine for growth. Address the need for bacteria. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Strych, U. et al., 2007. BMC Microbiol. 7:40 [Non-Patent Document 2] Strych U. et al., Curr. Microbiol. 41:290-294 [Non-Patent Document 3] Strych U. et al., FEMS Microbiol. Lett. 196:93-98 [Non-Patent Document 4] Franklin, FC, and WA Venables. 1976. Mol. Gen. Genet. 149:229-237 [Non-Patent Document 5] Hols, P. et al., J. Bacteriol. 179:3804-3807 [Overview of the project] [Means for solving the problem]

[0006] Summary of the Invention This disclosure relates to recombinant Staphylococcus bacteria that depend on D-alanine for growth.

[0007] In one aspect, the present disclosure features recombinant Staphylococcus bacteria comprising two inactivated alanine racemase genes (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In one embodiment, the Staphylococcus bacteria are D-alanine dependent for growth. In another embodiment, the Staphylococcus bacteria are Staphylococcus epidermidis (S. epidermidis) and its subspecies. In one embodiment, the Staphylococcus bacteria further comprise one or more further mutations.

[0008] In another aspect, the present disclosure provides a method for producing recombinant Staphylococcus bacteria, the method comprising: (i) transforming competent cells of Staphylococcus strain (SEΔalr1Δalr2) with a plasmid containing D-alanine aminotransferase (dat) knockout; (ii) detecting the presence of the knockout plasmid in the transformed cells; (iii) incubating the transformed cells identified in step (ii); and (iv) purifying the isolated colonies. In one embodiment, the method further comprises testing the isolated colonies for D-alanine nutritional requirements. In another embodiment, the presence of the knockout plasmid in the transformants is detected using polymerase chain reaction (PCR). In yet another embodiment, the recombinant Staphylococcus bacteria are Staphylococcus epidermidis (S. epidermidis) and its subspecies. In one embodiment, the recombinant Staphylococcus bacteria are produced by the method described above.

[0009] In another aspect, the present disclosure features a kit comprising recombinant Staphylococcus bacteria in any one of the aspects or embodiments described herein.

[0010] In another aspect, the present disclosure provides a method for treating or preventing a rash in a subject, the method comprising administering to the subject a population of recombinant Staphylococcus bacteria, one of the aspects or embodiments described herein, in an amount effective to treat or prevent a rash in the subject. [Brief explanation of the drawing]

[0011] [Figure 1]Figure 1 shows the observation of D-alanine nutritional requirements in a S. epidermidis strain with triple gene knockout (SEΔalr1Δalr2Δdat). After transformation with the SE1423 knockout plasmid, plasmid integration, and removal of the plasmid backbone, cells were plated for colonies. 25 colonies were patched onto two different plates, and the plates were incubated overnight at 30°C. Left: TSA plate; Right: TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL). Only three colonies (#7, #12, and #18; highlighted with red circles) were able to grow on TSA supplemented with D-alanine.

[0012] [Figure 2-1] Figures 2A and 2B show the results of PCR testing of the triple knockout strain (SEΔalr1Δalr2Δdat). Cells from patches on plates containing TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL) were used as templates in the PCR reaction: clone #7; KO clone #12; KO clone #18; wild-type SE; SE1423 KO plasmid DNA (vector, as control). Figure 2A: PCR was performed using primers 1423-5F and 1423-3R to distinguish between the wild-type SE1423 locus (2.3 Kb PCR product) and the SE1423 knockout (1.5 Kb PCR product). Figure 2B: PCR was performed using primers 1423-F and 1423-R to detect a 0.7 Kb PCR product specific to the wild-type SE1423 locus. As predicted, the PCR product was not generated from the SE1423 knockout plasmid or the putative SE1423 knockout SE clone. The results confirmed successful SE1423 deletion in clones #7, #12, and #18. [Figure 2-2]Figures 2A and 2B show the results of PCR testing of the triple knockout strain (SEΔalr1Δalr2Δdat). Cells from patches on plates containing TSA + anhydrotetracycline (2 μg / mL) + D-alanine (40 μg / mL) were used as templates in the PCR reaction: clone #7; KO clone #12; KO clone #18; wild-type SE; SE1423 KO plasmid DNA (vector, as control). Figure 2A: PCR was performed using primers 1423-5F and 1423-3R to distinguish between the wild-type SE1423 locus (2.3 Kb PCR product) and the SE1423 knockout (1.5 Kb PCR product). Figure 2B: PCR was performed using primers 1423-F and 1423-R to detect a 0.7 Kb PCR product specific to the wild-type SE1423 locus. As predicted, the PCR product was not generated from the SE1423 knockout plasmid or the putative SE1423 knockout SE clone. The results confirmed successful SE1423 deletion in clones #7, #12, and #18. [Modes for carrying out the invention]

[0013] Detailed description of the invention I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide to one of ordinary skill in the art general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless specifically identified otherwise.

[0014] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0015] The term “comprising” is used herein to mean “including, but not limited to,” and is used interchangeably with that phrase.

[0016] The term “or” is used herein to mean “and / or” and is used interchangeably with that term unless the context clearly indicates otherwise.

[0017] The term “such as” is used herein to mean “including, but not limited to,” and is used interchangeably with that phrase.

[0018] As used herein, the terms “nutrient-dependent” or “nutrient-dependent” refer to the inability of an organism to synthesize certain organic compounds necessary for its growth. A nutrient-dependent strain is an organism that exhibits this characteristic.

[0019] As used herein, the terms “alrA” and “alr” refer to the D-alanine racemase gene, including the normal allele of the alrA gene.

[0020] As used herein, the terms “dat” and “SE1423” refer to the D-alanine aminotransferase gene, including the normal allele of the dat gene.

[0021] As used herein, the terms “polypeptide” or “protein” refer to biological molecules, or macromolecules composed of amino acid residues bound together in a chain. The definition of polypeptide as used herein is intended to encompass proteins (generally higher molecular weight) composed of one or more long chains of amino acid residues and smaller peptides (generally lower molecular weight) composed of several amino acids. In other embodiments, a single amino acid, while not technically a polypeptide, may also be considered within the scope of the invention.

[0022] In relation to the purposes of this invention, the term "isolated" refers to a biological substance (cell, nucleic acid, or protein) that has been removed from its original environment (the environment in which it naturally exists). For example, a polynucleotide that exists naturally in a plant or animal is not isolated, but the same polynucleotide that has been separated from a naturally occurring adjacent nucleic acid is considered "isolated."

[0023] An "isolated nucleic acid molecule" (e.g., an isolated promoter) is one that has been separated from other nucleic acid molecules present in the natural source of that nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that have been separated from the chromosome to which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid molecule does not contain sequences that are naturally adjacent to it in the genomic DNA of the organism from which it originates.

[0024] As used herein, the term “genetic element” means a polynucleotide containing a polypeptide-coding region in a host cell, or a polynucleotide region that modulates replication, transcription or translation, or other processes important for the expression of that polypeptide, or a polynucleotide containing both a polypeptide-coding region and an expression-modulating region operably linked thereto. Genetic elements may be contained as episomal elements; that is, within vectors that replicate as molecules physically independent of the host cell genome. They may also be contained within plasmids. Genetic elements may also be contained within the host cell genome not in their native state, but rather, among other things, in the form of purified DNA or in vectors, after operations such as isolation, cloning, and introduction into host cells.

[0025] As used herein, “promoter” means a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, close to the transcription start site of the structural gene. If the promoter is an inductive promoter, the rate of transcription increases in response to an inducer. For example, a promoter can be regulated in a tissue-specific manner so that it is active only in transcribing the coding region relevant in a particular tissue type.

[0026] As used herein, the term “host cell” means a cell that is transformed or transfected, or capable of being transformed or transfected with an exogenous polynucleotide sequence.

[0027] As used herein, the term “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. Therefore, as used herein, for example, polynucleotides refer, in particular, to DNA that is single-stranded and double-stranded DNA, single-stranded and double-stranded regions, or a mixture of single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules of DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, as used herein, polynucleotides refer to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The strands in such regions may originate from the same molecule or from different molecules. Such regions may contain all, one, or more of the above molecules, but more typically, only some of the above molecules are involved. One of the molecules in the triple helix region is often an oligonucleotide. As used herein, the term polynucleotide includes DNA or RNA as described above, containing one or more modified bases. Thus, DNA or RNA having a modified backbone in terms of stability or other reasons is a “polynucleotide” as the term intends herein. Furthermore, to give just two examples, DNA or RNA containing an unusual base (e.g., inosine) or a modified base (e.g., tritylated base) is a polynucleotide as the term intends herein. It is recognized that a wide variety of modifications have been made to DNA and RNA that serve many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells (including, among other things, simple and complex cells).The term polynucleotide also includes short polynucleotides, often referred to as oligonucleotides. “Polynucleotide” and “nucleic acid” are often used interchangeably herein.

[0028] As used herein, the term "radiotherapy" means a type of cancer treatment that uses a beam of intense energy to kill cancer cells.

[0029] As used herein, the term "chemotherapy" refers to a type of cancer treatment that uses drugs to kill cancer cells.

[0030] As used herein, the term “rash” refers to any skin-related side effect of radiotherapy or chemotherapy. Rash is typically characterized by mild desquamation, papules, roughness, tightness, and possibly itching and warmth of the skin. This includes, but is not limited to, maculopapular rash (an inflammatory dermatitis presenting as eczematous or spongy), itching, lichenoid reactions, psoriasis, acneiform rash, vitiligo-like lesions, autoimmune skin diseases (e.g., bullous pemphigoid, dermatomyositis, alopecia areata), sarcoidosis, or changes in the nails and oral mucosa.

[0031] II. Composition This disclosure describes a triple knockout Staphylococcus bacterium that is a D-alanine trophication-dependent strain. This disclosure provides an engineered Staphylococcus bacterium (e.g., Staphylococcus epidermidis) that is genetically altered to have a double alanine racemase gene knockout and an alanine aminotransferase gene (dat, SE1423) knockout. This disclosure provides a triple knockout S. epidermidis strain (SEΔalr1Δalr2Δdat) with desired D-alanine trophication-dependent properties.

[0032] D-alanine is an essential component for bacteria with a peptidoglycan layer structure. The essentiality of D-alanine stems from its crucial role in the crosslinking of the peptidoglycan chain by the dipeptide D-alanyl-D-alanine. As described in this disclosure, we previously developed a double alanine racemase gene knockout strain of S. epidemidis (SEΔalr1Δalr2). However, this double knockout strain is related to Bacillus In contrast to subtilis, Escherichia coli, and several other bacterial species, S. epidermidis did not exhibit a D-alanine nutritional requirement. The presence of glutamate racemase (interconverting L-glutamate and D-glutamate) and D-alanine aminotransferase (interconverting D-alanine and D-glutamate) in S. epidermidis was thought to provide a bypass for alanine racemase. Therefore, this disclosure provides a knockout of the alanine aminotransferase gene (dat, SE1423) in a double knockout strain (SEΔalr1Δalr2) exhibiting a D-alanine nutritional requirement.

[0033] This disclosure provides genetically engineered bacterial host cells having a deletion in the dat gene or its homolog such that the activity of D-alanine aminotransferase is reduced, thereby making the cells D-alanine-dependent. In another embodiment, bacterial cells are genetically engineered to include a deletion in another gene or operon affecting the dat operon such that the activity of D-alanine aminotransferase is reduced, thereby making the cells D-alanine-dependent.

[0034] bacterial strain The present invention provides genetically altered microorganisms, such as bacteria. The methods described herein are intended to be carried out by inactivating or knocking out the gene encoding the protein homolog of dat in any Staphylococcus bacterial cell, or by other means inactivating the expression or activity of this protein. Assigning a strain to the genus Staphylococcus requires that it be a Gram-positive cocci that forms clusters, produces catalase, has a suitable cell wall structure (including the presence of peptidoglycan type and teichoic acid), and has a DNA G+C content in the range of 30-40 mol%. Examples include, but are not limited to, the following: S. aureus group (including S. argenteus, S. aureus, S. schweitzeri, and S. simiae); S. auricularis group (including S. auricularis); S. carnosus group (including S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans, and S. simulans); S. epidermidis group (including S. capitis, S. caprae, S. epidermidis, and S. saccharolyticus); S. haemolyticus group (including S. devriesei, S. haemolyticus, and S. hominis); S.hyicus-intermedius group (including S.agnetis, S.chromogenes, S.felis, S.delphini, S.hyicus, S.intermedius, S.lutrae, S.microti, S.muscae, S.pseudintermedius, S.rostri, S.schleiferi); S.lugdunensis group (including S.lugdunensis); S.saprophyticus group (including S.arlettae, S.cohnii, S.equorum, S.gallinarum, S.kloosii, S.leei, S.nepalensis, S.saprophyticus, S.succinus, S.xylosus); S.sciuri group (including S.fleurettii, S.lentus, S.sciuri, S.stepanovicii, S.vitulinus); S.simulans group (including S.simulans); S.warneri group (including S.pasteuri, S.warneri). In one embodiment, the Staphylococcus bacterium is Staphylococcus epidermidis.

[0035] Genetic constructs This invention utilizes standard molecular biology techniques, e.g., those described in Sambrook et al. (2001). pJB38 (Boss et al., 2013) was used as the plasmid backbone of a knockout vector based on pJB38 (allele-exchange E. coli-staphylococcal shuttle vector), with additional design features added to the plasmid to improve functionality (Bose, JL et al., Applied and environmental microbiology. 2013;79(7):2218-2224). Specific primers were designed to produce SE1423 knockout (Table 1). [Table 1-1]

[0036] Plasmids are constructed using standard molecular biology techniques with Top10 E. coli as the cloning host, by cloning PCR products that overlap at the EcoRI-SalI site in pJB38. Clones were selected and screened by PCR using primers 1423-5F and 1423-3R (Table 1) to detect PCR products. Clones of the accurate SE1423 knockout plasmid (pJB-1423KO) were dam - / dcm - The cells were transformed into E. coli strain Gm2163. Plasmid DNA was isolated from two Gm2163 transformant clones using the Qiagen Midi Prep Kit and checked by restriction digestion with EcoRI and SalI as described above.

[0037] Use of recombinant Staphylococcus bacteria The Staphylococcus bacteria of the present invention can be used as is to treat a disease, or they can be modified to express a therapeutic polypeptide. In one example, the Staphylococcus bacteria of the present invention can be used to treat a skin disease or disorder. In another embodiment, the Staphylococcus bacteria of the present invention can be modified to express a therapeutic polypeptide or a fragment thereof to treat a skin disease or disorder.

[0038] Rash is one of the most common side effects of cancer treatment (e.g., radiation therapy or chemotherapy). Studies have shown that the use of chemotherapy drugs (e.g., epidermal growth factor receptor (EGFR) inhibitors or immune checkpoint inhibitors) results in the appearance of rashes in approximately 30–100% of patients treated with these drugs (Fabbrocini et al., Skin Appendage Disord. 2015, 1(1):31-7, and Sibaud et al., Am J Clin Dermatol.). 2018, 19(3):345-361 (as incorporated herein by reference). Examples of EGFR inhibitors include, but are not limited to, the monoclonal antibodies cetuximab (Erbitux®) and panitumumab (Vectibix®), and the small molecule tyrosine kinase inhibitors erlotinib (Tarceva®) and gefitinib (Iressa®). Examples of EGFR inhibitors include, but are not limited to, monoclonal antibodies that target cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed cell death ligand 1 (PD-L1). The rash conditions that may arise from the use of these drugs may affect the quality of life of these patients and may sometimes lead to discontinuation of treatment.

[0039] Therefore, in one aspect, the present disclosure provides a method for treating or preventing a rash in a subject, the method comprising the step of administering to the subject a population of recombinant Staphylococcus bacteria, one of the aspects or embodiments described herein, in an amount effective to treat or prevent a rash in the subject. According to one embodiment, the subject having a rash is undergoing cancer treatment. According to one embodiment, the cancer treatment is radiotherapy. According to one embodiment, the cancer treatment is chemotherapy. According to one embodiment, the chemotherapy includes an epidermal growth factor inhibitor. According to one embodiment, the chemotherapy includes a checkpoint inhibitor.

[0040] formulation Formulations for use according to the present invention are formulated to produce a therapeutically effective amount of the desired polypeptide by any pharmaceutically effective amount of recombinant Staphylococcus bacteria, e.g., genetically engineered microorganisms, e.g., at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0% by bacterial weight. It is further evident that the upper limit may include approximately 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0%, or more, with the upper limit being approximately 90.0% by weight of genetically engineered microorganisms, e.g., bacteria.

[0041] In alternative embodiments, formulations for use according to the present invention may contain, for example, at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5%, or more by weight of recombinant Staphylococcus bacteria.

[0042] III. Method This disclosure features a method for producing recombinant Staphylococcus bacteria comprising the steps of (i) transforming competent cells of a Staphylococcus strain (SEΔalr1Δalr2) with a plasmid containing D-alanine aminotransferase (dat) knockout; (ii) detecting the presence of the knockout plasmid in the transformed cells; (iii) incubating the transformed cells identified in step (ii); and (iv) purifying the isolated colonies. In a preferred embodiment, the presence of the knockout plasmid in the transformants is detected using polymerase chain reaction (PCR). In a particular embodiment, the method further comprises testing the isolated colonies for D-alanine nutritional requirements.

[0043] IV. Kit The present invention also provides a kit. In one aspect, the kit of the present invention comprises (a) recombinant Staphylococcus bacteria of the present invention and (b) instructions for use thereof. The compositions of the present invention are described above. In some embodiments, the compositions of the present invention comprise recombinant Staphylococcus bacteria that are D-alanine dependent with respect to growth.

[0044] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. All drawings and references, patents and published patent applications, and the contents of the drawings all cited throughout this application are expressly incorporated herein by reference. [Examples]

[0045] The following embodiments further describe and illustrate embodiments within the scope of the present invention. These embodiments are provided for illustrative purposes only and should not be construed as limitations of the invention, for many variations are possible without departing from the spirit and scope of the invention.

[0046] This invention describes, in one embodiment, the generation of a Staphylococcus epidermidis (S. epidermidis) expression system, thereby enabling the maintenance of the expression plasmid without the use of antibiotics. This experiment demonstrates a long effort to develop a D-alanine-dependent S. epidermidis strain. A double alanine racemase gene knockout S. epidermidis strain (SEΔalr1Δalr2) was previously developed. However, that double knockout strain is related to Bacillus In contrast to subtilis, Escherichia coli, and several other bacterial species, S. epidermidis did not exhibit a D-alanine nutritional requirement. The presence of glutamate racemase (interconverting L-glutamate and D-glutamate) and D-alanine aminotransferase (interconverting D-alanine and D-glutamate) in S. epidermidis was thought to provide a bypass for alanine racemase, as reported in S. aureus and Listeria monocytogenes. Therefore, this invention describes the knockout of the alanine aminotransferase gene (dat, SE1423) in the double knockout strain (SEΔalr1Δalr2) in order to develop a triple knockout S. epidermidis strain (SEΔalr1Δalr2Δdat) exhibiting a D-alanine nutritional requirement.

[0047] Example 1: Vector for deletion of SE1423 (D-alanine aminotransferase) pJB38 (Boss et al., 2013) was used as the plasmid backbone for the knockout vector. Specific primers were designed to produce SE1423 knockout (Table 1). [Table 1-2]

[0048] PCR products of the 5' and 3' facile regions were generated (0.5Kb and 1.0Kb, respectively). These were then used as templates in duplicate PCR to generate a larger PCR product (1.5Kb) containing both the 5' and 3' facile regions. This duplicate PCR product was cloned at the EcoRI-SalI site in pJB38 using Top10 E. coli as the cloning host. Clones were selected and screened by PCR using primers 1423-5F and 1423-3R to detect 1.5Kb PCR products. Plasmid DNA was also isolated and digested with EcoRI and SalI to detect fragments of both the vector backbone (7.0Kb) and inserts (1.5Kb). Clones of the exact SE1423 knockout plasmid (pJB-1423KO) were selected. - / dcm - The cells were transformed into E. coli strain Gm2163. Plasmid DNA was isolated from two Gm2163 transformant clones using the Qiagen Midi Prep Kit and checked by restriction digestion with EcoRI and SalI (as described above).

[0049] Example 2. Generation of a triple knockout strain (SEΔalr1Δalr2Δdat) The pJB-1423 KO plasmid isolated from Gm2163 was transformed into competent S. epidermidis cells (SEΔalr1Δalr2) using TAS + chloramphenicol (10 μg / mL) plates. The presence of the pJB-1423 KO plasmid in the transformants was confirmed by detecting a 1.5 Kb PCR product using primers 1423-5F (EcoRI) and 1423-3R (SalI). A 1.5 Kb PCR product was observed in all 26 clones tested, while a 2.3 Kb PCR product was observed in a reaction containing cell lysates derived from SE host cells. Cells from the two confirmed clones were streaked on fresh plates of TSA + Cm (10 μg / mL) + D-alanine (40 μg / mL). The plates were incubated at 43°C for 24 hours for plasmid integration via homologous recombination. The isolated colonies were streaked again at 43°C for purification. Four isolated colonies were inoculated into 50 mL of TSB + D-alanine (40 μg / mL) in a 250 mL baffled shaking flask to remove the plasmid backbone via a second homologous recombination. The culture was shaken at 30°C for 24 hours. A 0.5 mL aliquot of the culture was transferred to a flask containing 50 mL of fresh medium. This transfer was repeated three times. Cells from the flask were then sterilized with TSA + anhydrotetracycline (ATC). Plated on 2 μg / mL + D-alanine (DA, 40 μg / mL). After 2 days of incubation at 30°C, approximately 100-200 colonies were found. -5 The colonies were formed on plates plated with 100 μl of culture during dilution. Further analysis of the above colonies is described below.

[0050] Example 3. Test on D-alanine nutritional requirements in the triple knockout strain (SEΔalr1Δalr2Δdat). A total of 25 isolated colonies from the TSA+ATC+DA plate were patched onto TAS plates and TAS+ATC+DA plates. The plates were incubated overnight at 30°C. All clones were fully grown on D-alanine-supplemented plates (TSA+ATC+DA). As shown in Figure 1, three clones (#7, #12, and #18) could not grow on TSA without D-alanine supplementation and exhibited D-alanine nutritional requirements. The phenotype of nutritional requirements was observed again when cells from the patches on the TSA+ATC+DA plate were patched again onto TSA plates. Note that some clones from the TSA+ATC+DA plate were predicted to retain the wild-type SE1423 locus because the second homologous recombination could result in the removal of the plasmid backbone without knocking out SE1423.

[0051] We further analyzed clones that were D-alanine-dependent. These 1423KO clones were analyzed. When SE clones were patched together on TSA+Cm (10 μg / mL), they did not proliferate. This indicates the removal of the plasmid backbone containing the chloramphenicol selection marker during the second homologous recombination. PCR using primers JB-Cm-F and JB-Cm-R (Table 1) also confirmed the loss of antibiotic resistance markers (data not shown). PCR using primers 1423-5F and 1423-3R detected a 1.5 Kb PCR product in these KO clones, while the PCR product derived from the SE host was 2.3 Kb, as expected (Figure 2A). Wild-type SE cells produced a 0.7 Kb PCR product using primers 1423-F and 1423-R (both specific to the SE1423 coding sequence); this PCR product was not detected from the KO plasmid DNA or from the putative KO clones (Figure 2B).

[0052] Therefore, based on all experimental data, it can be concluded that SE1423 (dat, D-alanine aminotransferase) was successfully deleted in the double alanine racemase gene knockout strain, generating a triple knockout S. epidermidis strain (SEΔalr1Δalr2Δdat). Furthermore, the desired D-alanine nutritional requirement was observed in the triple knockout strain.

[0053] D-alanine is required for the synthesis of bacterial cell peptidoglycans. In B. subtilis and E. coli, deletion of the alanine racemase gene was sufficient to address the D-alanine nutritional requirement. However, to establish this phenotype in S. epidermidis, two alanine racemase genes (alr1, alur2) and the D-alanine aminotransferase gene dat(SE1423) must be knocked out. Clearly, the combination of glutamate racemase and D-alanine aminotransferase is associated with S. aureus MRSA132 (Moscoso et al., As reported in 2017 and in Listeria monocytogenes (Thompson et al., 1998), it provides a viable bypass to alanine racemase. The S. epidermidis genome contains a third putative alanine racemase homolog (SE1769), but knocking out this gene is not necessary due to D-alanine nutritional requirements under the experimental conditions used in this study.

[0054] Following the successful development of the D-alanine-dependent S. epidermidis strain, the next step is to transform the strain using an expression vector containing the alanine racemase gene as a selection marker. Transformants are selected by plasmid complementation of the D-alanine host trophicity.

[0055] Equal portions Those skilled in the art will recognize, or can confirm by simply using conventional experimental methods, many equivalents to specific embodiments of the inventions described herein. Such equivalents are intended to be encompassed by the following claims. References [ka] The present invention provides, for example, the following items: (Item 1) Recombinant Staphylococcus bacteria, Two inactivated alanine racemase genes (Δalr1Δalr2); and Inactivated D-alanine aminotransferase (dat) gene, Recombinant Staphylococcus bacteria, including (Item 2) The Staphylococcus bacteria described above are recombinant Staphylococcus bacteria as described in item 1, which are D-alanine dependent for growth. (Item 3) The Staphylococcus bacteria mentioned above are Staphylococcus epidermidis (S. epidermidis) and its subspecies, which are recombinant Staphylococcus bacteria as described in item 1. (Item 4) The Staphylococcus bacteria described herein further comprises one or more further mutations, wherein the recombinant Staphylococcus bacteria described in item 1. (Item 5) A method for producing recombinant Staphylococcus bacteria, wherein the method is (i) A step of transforming competent cells of the Staphylococcus strain (SEΔalr1Δalr2) with a plasmid containing D-alanine aminotransferase (dat) knockout; (ii) A step of detecting the presence of the knockout plasmid in transformed cells; (iii) A step of incubating the transformed cells identified in step (ii); and (iv) A step to purify the isolated colonies, A method of including. (Item 6) The method according to item 5, further comprising the step of testing the isolated colonies for D-alanine nutritional requirements. (Item 7) The presence of a knockout plasmid in the transformant is detected using polymerase chain reaction (PCR) as described in item 5. (Item 8) The recombinant Staphylococcus bacteria are Staphylococcus epidermidis (S. epidermidis) and its subspecies, as described in item 5. (Item 9) Recombinant Staphylococcus bacteria produced by the method described in item 5. (Item 10) A kit containing recombinant Staphylococcus bacteria as described in any one of items 1-4 or 9. (Item 11) A method for treating or preventing a rash in a subject, the method comprising administering to the subject a population of recombinant Staphylococcus bacteria described in any one of items 1 to 4 or 9 in an amount effective for treating or preventing a rash in the subject. (Item 12) The subject having a rash is undergoing cancer treatment, as described in item 11. (Item 13) The cancer treatment is radiotherapy, as described in item 12. (Item 14) The cancer treatment is chemotherapy, as described in item 12. (Item 15) The chemotherapy is the method described in item 14, comprising an epidermal growth factor inhibitor. (Item 16) The chemotherapy described above is the method described in item 14, comprising a checkpoint inhibitor.

Claims

[Claim 1] The invention as shown in the drawings.

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