BR-122026002200-A2 - Auxotrophic strains of Staphylococcus bacteria, their use, production method and kit.

Abstract

The present invention relates to recombinant Staphylococcus bacteria (e.g., S. epidermidis) that are dependent on D-alanine for growth. According to one aspect, the invention relates to a recombinant Staphylococcus bacterium comprising two inactivated alanine racemase genes (Δalr1Δalr2) and one inactivated D-alanine aminotransferase (dat) gene. According to another aspect, the invention relates to a method for producing recombinant Staphylococcus bacteria.

Inventors

• TRAVIS MICHAEL WHITFILL
• Ming-De Deng
• DAVID RICHARD DODDS

Assignees

• AZITRA INC

Dates

Publication Date

20260310

Application Date

20190104

Priority Date

20180105

Claims (10)

1. 1. Recombinant Staphylococcus bacterium, characterized in that it comprises: two inactivated genes for alanine racemase (alr1 and alr2); and one inactivated gene for D-alanine aminotransferase (dat).
2. 2. Recombinant Staphylococcus bacterium, according to claim 1, characterized in that: (i) the Staphylococcus bacterium is dependent on D-alanine for growth; and/or (ii) the Staphylococcus bacterium is Staphylococcus epidermidis (S. epidermidis) and its subspecies; and/or (iii) the Staphylococcus bacterium additionally comprises one or more additional mutations, optionally wherein the additional mutations comprise the gene for inactivated glutamic acid racemase, Murl.
3. 3. Recombinant Staphylococcus bacteria, according to claim 1 or 2, characterized in that the bacteria is transformed with a vector based on pUBTR114, optionally wherein the vector based on pUBTR114 is pUBTR119*-Sal-GFP.
4. 4. Recombinant Staphylococcus bacteria, according to any one of claims 1 to 3, characterized in that it additionally comprises a polynucleotide encoding a protein with therapeutic properties.
5. 5. Method for producing a recombinant Staphylococcus bacterium, characterized in that it comprises: (i) transforming a plasmid comprising D-alanine aminotransferase (dat) knockout into competent cells of a Staphylococcus strain, wherein the Staphylococcus strain comprises inactive alrl and alr2 genes for alanine racemase (SEΔalr1Δalr2); (ii) detecting the presence of the knockout plasmid in transformed cells; (iii) incubating the transformed cells identified in step (ii); and (iv) purifying the isolated colonies.
6. 6. Method according to claim 5, characterized in that: (i) it further comprises testing the isolated colonies for D-alanine auxotrophy; and/or (ii) the presence of the knockout plasmid in the transformants is detected using Polymerase Chain Reaction (PCR); and/or (iii) the recombinant Staphylococcus bacterium is Staphylococcus epidermidis (S. epidermis) and its subspecies; and/or (iv) it further comprises transforming the recombinant Staphylococcus bacterium with a pUBTR114-based vector, optionally wherein the pUBTR114-based vector is pUBTR119*-Sal-GFP.
7. 7. Recombinant Staphylococcus bacteria, characterized in that it is produced by the method as defined in claim 5.
8. 8. Kit, characterized in that it comprises recombinant Staphylococcus bacteria as defined in any one of claims 1 to 4 or 17, optionally further comprising a vector based on pU-BTR114.
9. 9. Use of a recombinant Staphylococcus bacterium, comprising: two inactivated alanine racemase genes (alr1 and alr2); one inactivated D-alanine aminotransferase (dat) gene; and a polynucleotide encoding a protein with therapeutic properties, characterized by the fact that it is used in the manufacture of a medicament to treat a disease or disorder.
10. 10. Use according to claim 9, characterized in that the disease or disorder is a skin disease or disorder.

Description

Related Orders [0001] This application claims priority to U.S. Provisional Patent Application No. 62/614,096, filed January 5, 2018, the contents of which are incorporated herein by reference for all purposes. Background of the Invention [0002] Many bacteria use the two amino acids D-alanine and D-glutamic acid in the biosynthesis of the peptidoglycan layer, necessary for the construction of a functional cell wall in these bacteria. Gram-positive bacteria, including species of the genus Staphylococcus, use D-alanine and D-glutamic acid for the synthesis of the peptidoglycan layer in their cell walls. [0003] Genetic codes provide codons for 20 proteogenic amino acids, 19 of which have chirality and are the L isomer. These are considered the "natural" or "standard" amino acids. Amino acids that have the opposite chirality, that is, the D isomer, are considered unnatural and are generally not present in the environment. If an organism, such as a bacterium, requires a D amino acid, then an enzyme or enzymes to produce these unnatural amino acids must be present in the bacterium or deliberately supplied to the bacterium, or it cannot survive. [0004] Alanine racemase is an enzyme that catalyzes the conversion of L-alanine to D-alanine, an essential component in the biosynthesis of the peptidoglycan layer in bacterial cell walls. Alanine racemases are typically absent in eukaryotes but ubiquitous among prokaryotes. [0005] Because D-alanine is essential for the formation of the bacterial cell wall and therefore for the survival of bacteria, bacteria possess an enzyme that can catalyze the production of D-alanine. Since D-alanine is very important for the existence of bacteria, they may possess redundant or multiple enzymes for the biosynthesis of D-alanine. For example, bacteria may contain several genes for alanine racemase. In species with two genes, one may be constitutively expressed and anabolic, 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 provide the D-alanine necessary for cell wall biosynthesis, and knockout studies with several of these bacteria have established that the enzyme alanine racemase 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, H.J. 1972. Genet. Res. 20:269-277). [0006] Removing a microorganism's ability to produce an amino acid necessary for growth results in a microorganism known as an auxotroph. The amino acid necessary for growth must be supplied exogenously if the microorganism's survival and growth are desired. The rearing of auxotrophic microorganisms is well known, especially for E. coli (publicly available on the World Wide Web at cgsc2.biology.yale.edu/Auxotrophs.php; Methods Enzymol. 2015; 565:45-66. doi: 10.1016/bs.mie. 2015.05.012. Epub June 10, 2015. “Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins”. Lin MT, Fukazawa R, Miyajima-Nakano Y, Matsushita S, Choi SK, Iwasaki T, Gennis RB; Nicola Casali, Methods in Molecular Biology, Vol. 235. www.springer.com/gp/book/ 9781588291516, the content of each being incorporated here in its entirety for reference). [0007] Staphylococcus aureus D-alanine auxotrophs were produced with the aim of producing vaccines against methicillin-resistant Staphylococcus aureus strains (Moscoso M et al. 27a EC-CMID 22-25 April 2017, The Congress of ESCMID (P0473); Moscoso et al., Virulence (2018) Vol. 9 (1):604-620, the content of each being incorporated here in full by reference). In this case, it was necessary not only to deactivate the two alanine racemases alr1 and alr2, but also a third enzyme. [0008] If a bacterium is to be introduced into a target environment, it is desirable to be able to control the introduced bacteria after introduction into the target environment, for example, controlling the growth of the introduced bacteria relative to the growth of bacterial populations already present in the target environment. [0009] This control can be imposed by the use of antibiotics, which are selectively toxic to the bacteria being introduced, but are not toxic to the bacterial populations present in the target environment. However, it is often not possible to find antibiotics that have this selectivity. Furthermore, it is often undesirable to use antibiotics, as they can disrupt the target environment in undesirable ways, for example, inducing antibiotic resistance in members of the existing bacterial population or disrupting the target environment, resulting in dysbiosis, or an undesirable situation, for example, diarrhea. [00010] Thus, it is advantageous to use a method to selectively control the growth of