Use of lactic acidification inhibitors in the manufacture of a medicament for treating staphylococcus aureus biofilm-associated infections

Abstract

This invention specifically discloses the application of lactation inhibitors in the preparation of drugs for treating Staphylococcus aureus biofilm-related infections, relating to the field of biomedical technology. This invention provides the application of SarA protein K72 site lactation inhibitors in the preparation of drugs for the prevention and / or treatment of Staphylococcus aureus infection in diabetic patients, revealing that high glucose promotes Staphylococcus aureus biofilm formation through lactation modification of the SarA K72 site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of lactation inhibitors in the preparation of drugs for treating Staphylococcus aureus biofilm-associated infections. Background Technology

[0002] Diabetes is a growing and serious chronic disease worldwide, with high incidence and significant infection risk. Staphylococcus aureus is one of the most common pathogens causing infection in diabetic patients, particularly in those with diabetic foot ulcers, where the infection rate reaches as high as 52.8%. Compared to healthy individuals, infections in diabetic patients often have more severe consequences, frequently leading to treatment failure and prolonged illness. Traditionally, this phenomenon has been attributed to impaired host immune function due to hyperglycemia, resulting in reduced clearance of invading pathogens. However, mounting evidence suggests that the host's pathophysiological environment may directly influence pathogens, reshaping their pathogenic characteristics.

[0003] In the hyperglycemic microenvironment of the host, *Staphylococcus aureus* ingests excessive glucose, which is glycolytically converted to pyruvate, leading to a significant increase in intracellular lactate levels. Lactic acid has long been considered a simple metabolic end product; however, recent studies have revealed that lactate can act as a precursor to a novel post-translational modification—lactation—directly regulating protein function. This mechanism plays a central physiological role in mammals and various prokaryotes. For example, in *Escherichia coli*, YiaC and CobB act as lactate transferase and delactylase, respectively, reshaping the bacterial metabolic network by modifying sugar metabolism enzymes; in *Streptococcus*, lactation is involved in biofilm formation; in *Salmonella*, it broadly affects metabolic enzymes, transport proteins, and translation factors; and in *Staphylococcus aureus* itself, lactation has been shown to enhance the cytolytic activity of α-toxin, thereby increasing its virulence. These findings collectively establish lactate's novel messenger role: lactate is not only a metabolic byproduct but also a key regulator linking metabolic state and bacterial pathogenicity.

[0004] Biofilm formation is a core factor in bacterial pathogenicity, leading to chronic infection and treatment resistance. A biofilm is a structured microbial community formed by bacteria attaching to living or non-living surfaces and secreting extracellular matrix components such as polysaccharides, proteins, and nucleic acids. Once a biofilm forms, bacterial resistance to antibiotics can increase tenfold to a thousandfold, effectively evading clearance by the host's immune system. Studies have found that bacterial biofilm formation in diabetic foot infections is closely related to blood glucose levels. Therefore, exploring new mechanisms of Staphylococcus aureus in diabetic infections from a novel perspective of "host-pathogen interaction," particularly its role in biofilm formation, is crucial for developing new treatment strategies. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide the application of lactation inhibitors in the preparation of drugs for treating Staphylococcus aureus biofilm-associated infections. Sodium oxalate treatment significantly reduced bacterial load in diabetic patients, while simultaneously, bacterial load and biofilm formation on ducts of mice infected with the SarA K72R mutant strain also decreased significantly.

[0006] (II) Technical Solution To achieve the above objectives, this invention provides the application of the SarA protein K72 site as a target in any one or more of the following: (1): Preparation of products for the prevention and / or treatment of Staphylococcus aureus infections; (2): Screening for products for the prevention and / or treatment of Staphylococcus aureus infection.

[0007] In one embodiment, the product includes a pharmaceutical composition, a pharmaceutical preparation, and a medicine box.

[0008] In another aspect, the present invention provides the use of a reagent for detecting the lactation level at the K72 site of the SarA protein in any one or more of the following: (1): Preparation of products for the diagnosis and / or auxiliary diagnosis of Staphylococcus aureus infection; (2): Prepare products for screening Staphylococcus aureus infection; (3): Prepare products for prognostic assessment of Staphylococcus aureus infection.

[0009] In one embodiment, the product includes reagents and kits.

[0010] In another aspect, the present invention also provides the use of an inhibitor of lactation at the K72 site of the SarA protein in the preparation of drugs for the prevention and / or treatment of Staphylococcus aureus infection.

[0011] In one embodiment, the inhibitor includes antibodies, siRNA, shRNA, miRNA, gRNA, sgRNA, antagonists, blockers, and / or antisense oligonucleotides.

[0012] In one embodiment, the inhibitor comprises sodium oxalate.

[0013] In one embodiment, the subject infected with Staphylococcus aureus had diabetes.

[0014] In one embodiment, the subjects include both mammals and non-mammals.

[0015] Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals.

[0016] In one embodiment, the subject is a mouse.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) Therapeutic doses of the inhibitors used in the above applications; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0018] In one embodiment, the pharmaceutical composition further comprises: one or more other active substances for treating Staphylococcus aureus infection and related symptoms and / or signs, wherein the other active substances include: β-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics and / or antituberculosis antibiotics.

[0019] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0020] In another aspect, the present invention also provides the use of the above-described pharmaceutical composition and pharmaceutical preparation in the preparation of drugs for the prevention and / or treatment of Staphylococcus aureus infection.

[0021] (III) Beneficial Effects This invention provides the application of lactation inhibitors in the preparation of drugs for treating Staphylococcus aureus biofilm-related infections. Compared with the prior art, it has the following beneficial effects: 1. Under high glucose and high sodium lactate conditions, biofilm formation and panlactization levels in Staphylococcus aureus were significantly enhanced. Sodium oxalate, a lactate dehydrogenase inhibitor, effectively inhibited the enhanced biofilm formation induced by high glucose, and this effect was consistent with changes in lactate production and panlactization levels.

[0022] 2. Sodium oxalate treatment significantly reduced bacterial load in diabetic patients. At the same time, bacterial load on ducts of mice infected with SarA K72R mutant strain also decreased significantly, suggesting that SarA lactation is also involved in the regulation of high glucose-mediated biofilm formation in vivo.

[0023] 3. After the WT strain adhered to the catheter and was implanted into diabetic mice, a dense biofilm structure was formed on the surface, and bacterial aggregation was obvious. In contrast, the number of bacteria on the catheter surface was reduced in the sodium oxalate treatment group and the SarA K72R mutant infection group, and the biofilm structure was looser. This indicates that intervention with SarA lactation modification can significantly inhibit the formation of Staphylococcus aureus biofilm in vivo under high sugar conditions. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is an analytical diagram showing how high sugar levels promote the formation of Staphylococcus aureus biofilms through lactic acid accumulation.

[0026] Figure 2 This is an analytical diagram showing how high sugar levels upregulate the lactation modification of Staphylococcus aureus proteins through lactic acid accumulation.

[0027] Figure 3 This is an analysis diagram of the K72 site of the SarA protein, a key lactation site mediating high-glucose-induced Staphylococcus aureus biofilm formation.

[0028] Figure 4 This is an analysis of how inhibiting lactate production or SarA K72 lactation can reduce duct-associated Staphylococcus aureus infection in diabetic mice. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Terms and Definitions As used herein, the terms "SarA protein" and "SarA" are used interchangeably and refer to the global transcriptional regulator SarA in Staphylococcus aureus. The SarA protein disclosed in this invention may be a protein encoded by the sequence of SEQ ID NO: 1 (Staphylococcus aureus SarA CDS), or homologous sequences of these proteins that promote biofilm formation after lactation (e.g., homologous sequences of SarA proteins can be obtained from databases or alignment software known in the art), variants, or modified forms. For example, the SarA protein may be selected from: (1): SarA protein having the amino acid sequence shown in SEQ ID NO: 4; or (2): SarA protein homologous to the amino acid sequence shown in SEQ ID NO: 4 or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; or (3): SarA protein derived from (1) or (2) by substitution, deletion or addition of one or more amino acids in the amino acid sequence of (1) or (2).

[0031] The SarA protein disclosed in this invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animals, insects, and mammalian cells) using recombinant technology. Preferably, the SarA protein disclosed in this invention is encoded by the Staphylococcus aureus SarA protein gene or its homologous genes or family genes.

[0032] This invention discloses variant forms of the SarA protein including (but not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the SarA protein.

[0033] As used herein, the terms “SarA gene,” “SarA-encoding gene,” “SarA protein-encoding gene,” or “nucleic acid molecule encoding SarA protein” are used interchangeably and all refer to a nucleotide sequence encoding the SarA protein or polypeptide disclosed in this invention, which may be, for example, the nucleotide sequence shown in SEQ ID NO: 1 (Staphylococcus aureus SarA CDS), a molecule that hybridizes to these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above-mentioned molecules, and the expression function of said gene.

[0034] The SarA gene disclosed in this invention can be selected from: (1) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 1; or (2) A nucleic acid molecule that hybridizes with the sequence defined in (1) under strict conditions and has a function.

[0035] As used herein, the term “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%. For example, the sequence may be a complementary sequence to the sequence defined in (1).

[0036] The full-length nucleotide sequence or fragments of the SarA gene disclosed in this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this disclosure, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0037] It should be understood that the SarA gene disclosed in this invention is preferably obtained from Staphylococcus aureus. Other genes from other animals that are highly homologous to the Staphylococcus aureus gene (e.g., having more than 50%, preferably more than 55%, 60%, 65%, 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity) are also within the scope of this preferred disclosure. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0038] As used herein, the terms “inhibitor” or “inhibitor of SarA lactation” are used interchangeably and refer to substances that can reduce SarA lactation levels.

[0039] The inhibitor disclosed in this invention can inhibit SarA lactation, thereby enabling its further use in the prevention or treatment of diseases and / or related symptoms associated with Staphylococcus aureus infection in diabetes.

[0040] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0041] Methods well known to those skilled in the art can be used to construct expression vectors containing a SarA coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The pBTs and pET28a expression systems are preferred in this invention.

[0042] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0043] Vectors containing the aforementioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells. In this invention, *Escherichia coli* is preferably used as the host cell.

[0044] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0045] In this method, the expression vector can be a viral vector or a non-viral vector. The viral vector can be an adeno-associated virus (AAV) vector, adenovirus vector, alphavirus vector, herpes simplex virus vector, vaccinia virus vector, Sendai virus vector, flavivirus vector, radovirus vector, retrovirus vector, herpesvirus vector, poxvirus vector, or lentivirus vector. The non-viral vector can be a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles, or liposomes. The DNA vector can be a plasmid vector, granular vector, phage vector, or human artificial chromosome.

[0046] The expression vector may contain a suitable regulatory sequence linked to a polynucleotide encoding SarA, enabling SarA expression in *E. coli*, wherein the construct sequence is operatively linked to said polynucleotide. In this case, the integration of the regulatory sequence and the operatively linked SarA-encoding polynucleotide is termed a "gene construct." The gene construct may contain suitable restriction enzyme recognition sites at both ends for cloning in the expression vector.

[0047] As used herein, the term "amplification" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule. Amplification typically involves denaturing the template nucleic acid, annealing the primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending the primers to produce the amplification product. Amplification usually requires the presence of deoxyribonucleoside triphosphates, DNA polymerase (e.g., Platinum Taq), and appropriate buffers and / or cofactors for optimal polymerase activity (e.g., MgCl2 and / or KCl).

[0048] As used herein, the term "primer" is known to those skilled in the art and refers to an oligomeric compound capable of "initiating" DNA synthesis by a template-dependent DNA polymerase, primarily referring to oligonucleotides, but also to modified oligonucleotides, i.e., an oligonucleotide having a free 3'-OH group at its 3' end, to which another "nucleotide" can be attached by a template-dependent DNA polymerase to establish a 3' to 5' phosphodiester bond, thereby using a deoxyribonucleotide triphosphate and thus releasing pyrophosphate.

[0049] As used herein, the term "pharmaceutical composition" refers to a composition comprising an inhibitor of lactation at the K72 site of the SarA protein, formulated together with one or more pharmaceutically acceptable carriers.

[0050] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonade, aromatic water, powders, syrups, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0051] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0052] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0053] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0054] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition, with the remainder being pharmaceutically acceptable carriers and other additives.

[0055] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0056] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0057] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0058] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0059] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0060] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0062] Example 1: High sugar promotes Staphylococcus aureus biofilm formation through lactic acid accumulation: The biofilm formation of *Staphylococcus aureus* (MW2 strain) under different glucose (D-Glucose, Glu, Biosharp) concentrations was quantitatively analyzed using crystal violet staining. A small amount of MW2 glycerol bacterial suspension, frozen at -80°C, was streaked onto TSA (Tryptic Soy Broth Agar) agar plates using an inoculation loop and incubated overnight at 37°C under inverted conditions. Single colonies of *Staphylococcus aureus* from the overnight TSA plates were picked and incubated in 1 ml of TSB (Tryptic Soy Broth) at 37°C with a shaker at 200 rpm for 12-16 hours to obtain bacteria in the logarithmic growth phase. The bacterial suspension was then diluted and adjusted to OD200. 600 =0.1, then diluted 100-fold with TSB medium containing 3% NaCl, and 200 μl was inoculated into each well of a 96-well plate. A TSB blank control group, a 7.5 mM glucose group, and a 15 mM glucose (high glucose) group were set up. The plates were incubated at 37℃ for 16 h. The 96-well plates were removed, the supernatant was discarded, and the plates were washed three times with sterile PBS. Crystal violet staining solution [0.5% (w / v) crystal violet (Biosharp), 10% (v / v) formaldehyde, 50% (v / v) anhydrous ethanol, 0.8% (w / v) NaCl, thoroughly dissolved and filtered through a 0.22 μm filter membrane] was used for staining. After staining, the plates were air-dried and photographed. 200 μl of 33% glacial acetic acid was added to each well to dissolve the crystal violet, and the OD was measured using a microplate reader. 560 Absorbance value at nm.

[0063] The experimental results showed that Staphylococcus aureus biofilm formation was minimal in the TSB basal medium control group. However, biofilm formation was significantly enhanced when the bacteria were cultured in TSB + 7.5 mM glucose and TSB + 15 mM glucose media. Figure 1 In the figure below, 'a' represents the concept of the present invention. This indicates that p < 0.05. This indicates that p < 0.01. (Indicates p < 0.001). Specifically, as glucose concentration increased, the number of blue immobilized patches (i.e., bacterial biofilm stained with crystal violet) at the bottom of the wells gradually increased, indicating that the biofilm-forming ability of Staphylococcus aureus was significantly enhanced under high glucose conditions.

[0064] To establish the core role of lactate in this process, "acquired" and "deficient" functional experiments were conducted. In the acquired experiments, it was found that adding 50 mM and 100 mM sodium lactate (Nala, Sigma) alone could simulate the high glucose effect, significantly promoting biofilm formation, and the effect was concentration-dependent. Figure 1 (b) In the absence of the experiment, the use of the lactate dehydrogenase inhibitor sodium oxalate (Oxamate, Macklin) effectively inhibited the high glucose-induced biomembrane enhancement ( Figure 1 (c in the text)

[0065] The above results were further verified by observing the biofilm using a laser confocal microscope. Figure 1 (d, e): Adjust the overnight activated test bacteria to OD. 600 =0.1, then diluted 1:100 with 3% NaCl TSB medium. 3 ml of the diluted bacterial solution was added to a NEST 35mm glass-bottomed culture dish and incubated at 37℃. After 16 h, the supernatant in the corresponding culture dish was aspirated, and the bacteria were washed with PBS to remove airborne bacteria. 2.5% glutaraldehyde (Macklin) was added for fixation at room temperature for 1.5 h, followed by PBS washing. 2 ml of 15 μM / ml PI (Sigma) was added, and the mixture was incubated at room temperature in the dark for 15 min, followed by PBS washing. 2 ml of 50 μg / ml FITC-ConA (Sigma) was added, and the mixture was incubated at room temperature in the dark for 20 min, followed by PBS washing. Biofilm formation was observed using a CLSM Zeiss LSM710 system. The parameters were set as follows: FITC-ConA excitation wavelength was 488 nm, and emission wavelength was 500-548 nm. PI excitation wavelength was 543 nm, and emission wavelength was 569-719 nm. Confocal images were obtained using the Zeiss ZEN 3.8 software package. Experimental results showed that under high glucose and high sodium lactate conditions, the green fluorescence of extracellular polysaccharides (EPS) in the biofilm significantly increased, indicating increased biofilm formation. However, the addition of 10 mM sodium oxalate reduced the enhanced bacterial biofilm formation caused by high glucose levels.

[0066] To eliminate the potential interference of bacterial growth status on bacterial biofilm formation, the growth curves of each treatment group were monitored: single clonal strains were picked and activated overnight in TSB medium using a shaker. The initial OD of the bacterial culture was adjusted the following day. 600 The value was set to 0.1, and a corresponding treatment group was set. The culture was incubated on a shaker at 37°C and 220 rpm. The OD value was then measured every hour using a spectrophotometer. 600 Numerical values ​​were collected for a total of 16 hours, and bacterial growth curves were plotted. The results showed that both 100 mM sodium lactate and 15 mM glucose exhibited slight inhibitory effects on growth, while the addition of sodium oxalate under high glucose conditions further enhanced the inhibitory effect on bacterial growth. Figure 1(f in the text). The above results indicate that lactic acid production is a necessary condition for high sugar levels to promote Staphylococcus aureus biofilm formation.

[0067] Example 2: High sugar upregulates Staphylococcus aureus protein lactation modification through lactic acid accumulation. Example 1 has demonstrated that lactic acid is a key mediator in the high-sugar promotion of Staphylococcus aureus biofilm formation. Lactic acid accumulated under high-sugar conditions may act as a precursor, driving lactation modification of Staphylococcus aureus proteins, thereby regulating bacterial biofilm formation. To verify this hypothesis, the L-lactic acid levels in bacterial culture supernatant and bacterial cells under different treatment conditions were first detected using an L-lactic acid content assay kit (Solarbio). Figure 2 As shown in a and b, compared with the TSB control group, treatment with 15 mM glucose and 100 mM sodium lactate both significantly increased lactate concentration. Notably, the addition of sodium oxalate, a lactate dehydrogenase inhibitor, effectively inhibited the high-glucose-induced lactate accumulation, confirming that a high-glucose environment indeed promotes bacterial lactate metabolism, leading to lactate production and accumulation. Subsequently, the pan-lactation level of Staphylococcus aureus whole protein was analyzed by Western blotting: protein supernatants from lysed bacteria in different treatment groups were extracted, quantified with BCA, and 100 μg was loaded per well. After 10% gel electrophoresis, membrane transfer and blocking, the membrane was incubated overnight with Anti-Lactyl Lysine Rabbit mAb (PTM) primary antibody, incubated with rabbit secondary antibody at room temperature for 1.5 h, washed with TBST, and exposed with developer. The Western blotting results were completely consistent with the changes in lactate content: both high-glucose and exogenous lactate treatments significantly upregulated the pan-lactation modification level of Staphylococcus aureus proteins. Figure 2 (c) More importantly, the addition of sodium oxalate reversed the increase in pan-lactic acidification levels induced by high sugar. This evidence suggests that lactic acid production is a necessary condition for high sugar environment-induced lactation modification of Staphylococcus aureus proteins.

[0068] Example 3: The SarA protein K72 site is a key lactation site mediating high-glucose-induced Staphylococcus aureus biofilm formation. 1. Purification of SarA protein and preparation of anti-SarA polyclonal antibody using a prokaryotic expression system: Primers were designed based on the published Staphylococcus aureus SarA gene sequence to amplify the coding region from the Staphylococcus aureus genome. BamHI and HindIII restriction enzyme sites were introduced into the primers. The primer DNA sequences are as follows: pET28a-BamHI-SarA-F (SEQ ID NO: 2): CGGGATCCGCAATTACAAAAATCAATGATTGC; pET28a-HindIII-SarA-R (SEQ ID NO: 3): CCCAAGCTTTAGTTCAATTTCGTTGTTTGC. The amplified product was ligated into the multiple cloning site of the pET28a vector with a 6×His tag according to the restriction endonuclease sites, and transformed into Escherichia coli DH5α to obtain the pET28a-SarA expression vector. This vector was then transformed into Escherichia coli BL21, and expression strains were screened. Target protein expression was induced overnight by incubation with 0.5 mM IPTG at 16℃ and 220 rpm. Bacterial lysis was performed by sonication, and the lysate supernatant was prepared. The SarA-his recombinant protein was purified using a His-tagged protein purification kit (Yisheng). The protein was then replaced with PBS, quantified using BCA, and analyzed by SDS-PAGE electrophoresis. The results showed that the purified SarA protein had high purity and good expression levels. Figure 3 In the formula: a: M. protein marker; 1. pET28a-SarA induced by IPTG; 2. Homogenized bacteria after ultrasonic disruption; 3. Supernatant after ultrasonic disruption; 4. Precipitate after ultrasonic disruption; 5. Eluent after protein column attachment; 6. Washing with 50mM imidazole; 7. Washing with 100mM imidazole; 8. Purified protein product (984ng / μl) after elution with 250mM imidazole. 40µg of SarA-his protein was mixed with an equal volume of Freund's adjuvant and immunized New Zealand rabbits via intraperitoneal, intramuscular, and subcutaneous routes. Booster immunizations were performed at 14 and 28 days. Blood was collected from the eyeballs at 35 days to prepare serum, which was filtered through a 0.22µm filter and named anti-SarA polyclonal antibody, then stored at -20℃.

[0069] 2. The expression level and lactation level of SarA under different treatment conditions were detected using anti-SarA polyclonal antibody; the SarA protein expression of Staphylococcus aureus was detected under blank TSB, TSB + high glucose, TSB + high sodium lactate, and TSB + high glucose + sodium oxalate treatments. Western blot results showed that there was no significant difference in SarA protein expression level under different treatments. Figure 3 (b) in the middle.

[0070] 3. Immunoprecipitation (IP) assay to detect the lactation level of SarA protein: Protein supernatant was extracted from the lysed bacteria of different treatment groups. The supernatant was incubated overnight at 4°C with anti-SarA antibody. The next day, Protein / G agarose beads (Abmart) were added and incubated at 4°C for 4 hours. The beads were collected by centrifugation and washed 3-4 times with pre-cooled washing buffer to remove non-specific binding. After boiling with 2× Loading buffer, the immunoprecipitated sample was obtained. The detection of SarA protein lactation modification level was the same as in Example 2. It was found that compared with the blank TSB treatment group, the high glucose and high sodium lactate treatment groups significantly enhanced the lactation modification level of SarA protein. Figure 3 (b) In this context, sodium oxalate can effectively inhibit the lactation modification of SarA caused by high sugar, which proves that SarA is a direct target of lactation modification under high sugar conditions.

[0071] DNA sequence of SarA gene (SEQ ID NO: 1): 5'-ATGGCAATTACAAAAATCAATGATTGCTTTGAGTTGTTATCAATGGTCACTTATGCTGACAAATTAAAAAGTTTAATTAAAAAGGAATTTTCAATTAGCTTTGAAGAATTCGCTGTATTGACATACATCAGCGAAAACAAAGAGAAAGAATACTATTCTAAAGATATTATTAATCATTTAAACTA CAAACAACCACAAGTTGTTAAAGCAGTTAAAATTTTATCTCAAGAAGATTACTTCGATAAAAAACGTAATGAGCATGATGAAAGAACTGTATTAATTCTTGTTAATGCACAACAACGTAAAAAAATCGAATCATTATTGAGTCGAGTAAATAAACGAATCACTGAAGCAAACAACGAAATTGAACTATAA-3'; Amino acid sequence of SarA (SEQ ID NO: 4): MAITKINDCFELLSMVTYADKLKSLIKKEFSISFEEFAVLTYISENKEKEYYLKDIINHLNYKQPQVVKAVKILSQEDYFDKKRNEHDERTVLILVNAQQRKKIESLLSRVNKRITEANNEIEL.

[0072] According to InterPro annotations, the SarA protein belongs to the winged-helix DNA-binding transcription factor family. In its dimer form, it recognizes and binds to DNA through its helix-turn-helix (HTH) structure and wing domain. Based on the residue range shown in InterPro, amino acids 6 to 114 constitute the winged-helix DNA-binding domain of SarA, responsible for binding to the target DNA promoter. The lactation sites (K49, K63, K69, K72, K82) provided by mass spectrometry analysis are all located within this domain. Figure 3 As shown in c and d in the diagram. The lactation modification of lysine residues in this domain may play an important role in the functional regulation of SarA, further affecting its ability to regulate biomembrane formation.

[0073] To investigate the effect of SarA protein lactation modification on its function, five lactation site mutant strains (SarA K49R, SarA K63R, SarA K69R, SarA K72R, and SarA K82R) were constructed in the MW2 strain using homologous recombination. By mutating different lactation sites (K49, K63, K69, K72, and K82) of SarA to arginine (R), the lactation modification at these sites was simulated. Simultaneously, a ΔSarA strain was constructed using homologous recombination as a negative control to explore the effect of lactation modification on Staphylococcus aureus biofilm formation.

[0074] 4. Construction of delactation point mutant strains using homologous recombination: Homologous recombination plasmids were constructed using the thermosensitive Staphylococcus aureus-Escherichia coli shuttle plasmid pBTs, enabling traceless gene mutation. The constructed Staphylococcus aureus MW2 homologous recombination plasmids are: pBTs-ΔSarA, for traceless knockout of the SarA gene; pBTs-SarA K49R, pBTs-SarA K63R, pBTs-SarA K69R, pBTs-SarA K72R, and pBTs-SarA K82R for the construction of SarA point mutant strains. Using MW2 strain genomic DNA as a template, PCR amplification was performed using the high-fidelity DNA polymerase PrimeSTAR® HS DNA Polymerase (TaKaRa). First, approximately 800-1000 bp sequences upstream and downstream of the target mutation site were designed, and homologous arms (up and down fragments) containing the mutation were amplified by PCR. For example, for the SarAK49R mutation, the upstream and downstream fragments upstream and downstream of the SarAK49R point mutation site were amplified using primer pairs SarA-up-F-KpnI / SarA-K49R-up-R and SarA-K49R-down-F / SarA-down-R-SalI, respectively. The resulting fragments were purified by gel extraction and ligated upstream and downstream fragments using overlap PCR. The up-down recombinant fragments and pBTs plasmid were then double-digested with restriction endonucleases (FastDigest), and the digested recombinant fragments and plasmids were ligated using T4 DNA Ligase (Thermo). The constructed plasmid was introduced into *E. coli* DH5α for initial proliferation and plasmid amplification. The successfully recombined plasmid was then transformed into Staphylococcus aureus RN4220 competent cells, and the successfully recombinant plasmid was extracted from RN4220 and introduced into the target strain MW2 competent cells. Utilizing the temperature-sensitive properties of the pBTs plasmid, homologous recombination was induced under specific temperature conditions. Typically, after recombination, the plasmid is inactivated at a suitable temperature, ensuring that the mutation is stably inherited in the genome. Screening on chloramphenicol-containing medium ensured that only strains that successfully underwent homologous recombination were selected. The accuracy of the mutation was confirmed by DNA sequencing. The successfully validated mutant strains were stored at -80°C for long-term preservation and use in subsequent experiments.

[0075] Growth curves of each mutant strain were determined under TSB + high glucose (15 mM glucose) conditions. The results showed that the growth curve of the SarA KTO R mutant strain under high glucose conditions was not significantly different from that of the wild type, indicating that these mutations did not significantly alter the bacterial growth rate. However, the growth rate of the SarA knockout strain (ΔSarA) was significantly reduced, and its growth curve showed a delayed growth trend. Figure 3(e). This result indicates that SarA plays an important role in the growth of Staphylococcus aureus, while delactation mutations did not significantly affect bacterial growth.

[0076] Biofilm formation in each mutant strain under high glucose conditions was detected using crystal violet staining. The results showed that biofilm formation in the SarA K49R, SarA K63R, SarA K69R, and SarA K82R mutants was enhanced to varying degrees, while biofilm formation in the SarA K72R mutant did not increase significantly and remained at a low level, indicating that biofilm formation in this mutant was not enhanced by high glucose conditions. Figure 3 f in the middle.

[0077] At the molecular level, the SarA K72R mutation does not affect the protein expression level of SarA, but it specifically inhibits high glucose-induced SarA lactation. Figure 3 g in (the middle part).

[0078] The above results reveal that the K72 site of the SarA protein is a key lactation site mediating high sugar-induced Staphylococcus aureus biofilm formation.

[0079] The primer nucleotide sequences for constructing the lactic acid mutant strain using the homologous recombination method are as follows: SarA-K49R-up-R (SEQ ID NO: 5): ACAAAGAGAGGGAATACTATTCTAAAGATATTATTAAT; SarA-K49R-down-F (SEQ ID NO: 6): GTATTCCCTCTCTTTGTTTTCGCTGATG; SarA-K63R-up-R (SEQ ID NO: 7): TACCGTCAACCACAAGTTGTTAAAGC; SarA-K63R-down-F (SEQ ID NO: 8): CTTGTGGTTGACGGTAGTTTAAATGATTAATAATATC; SarA-K69R-up-R (SEQ ID NO: 9):ACAAGTTGTTAGAGCAGTTAAAATTTTATCTCAAGAAG; SarA-K69R-down-F (SEQ ID NO: 10): TTTAACTGCTCTAACAACTTGTGGTTGTTTG; SarA-K72R-up-R (SEQ ID NO: 11): TAAAGCAGTTAGAATTTTATCTCAAGAAGATTACTTCG; SarA-K72R-down-F (SEQ ID NO: 12): TTCTTGAGATAAAATTCTAACTGCTTTAACAACTTGT; SarA-K82R-up-R (SEQ ID NO: 13): GATTACTTCGATAGAAAACGTAATGAGCATGATG; SarA-K82R-down-F (SEQ ID NO: 14):CATTACGTTTCTATCGAAGTAATCTTCTTGAGA; SarA-up-F-KpnⅠ (SEQ ID NO: 15): GGGGTACCGGCAAGTCTTCTAAAAGTGAA; SarA-down-R-SalⅠ (SEQ ID NO: 16): GCGTCGACCGTTGATTTGGGTAGTATACT; ΔSarA-up-R (SEQ ID NO: 17): AGGTTTTAAACTTTTGTTTAGCGCAATTTGG; ΔSarA-down-F (SEQ ID NO: 18): CGCTAAACAAAAGTTTAAAACCTCCCTATTTG; pBTs vector primers: pBTs-F (SEQ ID NO: 19): TCACCGACAAACAACAG; pBTs-R (SEQ ID NO: 20): CCAAGCCTATGCCTACA.

[0080] Example 4: Inhibition of lactate production or SarA K72 lactation can reduce duct-associated Staphylococcus aureus infection in diabetic mice: To verify the in vivo correlation between high glucose promoting biofilm formation through the lactate production-SarA lactation pathway, a type 2 diabetes mellitus (T2DM) mouse model was constructed, and catheter implant infection experiments were conducted in vivo.

[0081] The T2DM model was constructed using a high-fat diet (Research Diets) combined with streptozotocin (STZ, Yisheng) induction method. Figure 4 (a) After modeling, the mice exhibited typical diabetic phenotypes, including polydipsia, polyuria, and weight loss. Figure 4 (b) Elevated blood sugar ( Figure 4 (c) In this context, random blood glucose levels >16.7 mM for three consecutive days are considered as the standard for successful model construction.

[0082] The experimental procedure for the catheter infection model is as follows: Four groups were set up: ① control mice + WT strain, ② diabetic mice + WT strain, ③ diabetic mice + WT strain + sodium oxalate treatment, ④ diabetic mice + SarA K72R mutant strain. Staphylococcus aureus was diluted to OD using physiological saline. 600 =0.2, the silicone catheter and bacterial solution were incubated together at 37℃ and 110 rpm for 17 hours to allow bacteria to adhere to the surface of the silicone catheter, and then the catheter was implanted subcutaneously into mice. For the sodium oxalate treatment group, 300 μl of 10 mM sodium oxalate solution was injected subcutaneously near the catheter for three consecutive days starting from the day after implantation, while the other groups were injected with an equal volume of physiological saline. On the fifth day, the catheter was removed and the surface bacterial load was quantitatively analyzed.

[0083] The results showed that, compared with the control mouse + WT strain group, the bacterial load on the duct surface of diabetic mice + WT strain group was significantly increased. Figure 4 d in Figure 4 (e). Sodium oxalate treatment significantly reduced the bacterial load on catheters in diabetic patients. At the same time, the bacterial load on catheters of mice infected with the SarA K72R mutant strain also decreased significantly, suggesting that SarA lactation is also involved in the regulation of high glucose-promoted biofilm formation in vivo.

[0084] Scanning electron microscopy observations further support this result. Figure 4 (f) In diabetic mice, the WT strain formed a dense biofilm structure on the duct surface, with significant bacterial aggregation; while the number of bacteria on the duct surface was reduced in the sodium oxalate treatment group and the K72R mutant infection group, and the biofilm structure was looser, indicating that intervention with SarA lactation modification can significantly inhibit the formation of Staphylococcus aureus biofilm in vivo under high glucose conditions.

[0085] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the SarA protein K72 site as a target in any one or more of the following: (1): Preparation of products for the prevention and / or treatment of Staphylococcus aureus infections; (2): Screening for products for the prevention and / or treatment of Staphylococcus aureus infection.

2. Use according to claim 1, characterized in that, The products include pharmaceutical compositions, pharmaceutical preparations, and medicine boxes.

3. Application of reagents for detecting lactation levels at the K72 site of SarA protein in any one or more of the following: (1): Preparation of products for the diagnosis and / or auxiliary diagnosis of Staphylococcus aureus infection; (2): Prepare products for screening Staphylococcus aureus infection; (3): Prepare products for prognostic assessment of Staphylococcus aureus infection.

4. Use according to claim 3, characterized in that, The products include reagents and kits.

5. Application of inhibitors of lactation at the K72 site of SarA protein in the preparation of drugs for the prevention and / or treatment of Staphylococcus aureus infection.

6. Use according to claim 5, characterized in that, The inhibitors include antibodies, siRNA, shRNA, miRNA, gRNA, sgRNA, antagonists, blockers, and / or antisense oligonucleotides.

7. Use according to claim 6, characterized in that, The inhibitor includes sodium oxalate.

8. The use according to any one of claims 1 to 7, characterized in that, The subjects infected with Staphylococcus aureus had diabetes.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (1) A therapeutically effective amount of the inhibitor used in any one of claims 5-7; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

10. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition of claim 9.

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