Medicine for treating bacterial infection related diseases and application thereof

Abstract

The invention provides a medicine for treating diseases related to bacterial infection and application of the medicine, and particularly relates to the field of medicine. According to the invention, MRSA bacterial calcification can be induced in a targeted manner under the blood calcium ion level with physiological concentration, MRSA death is caused by inhibiting bacterial aerobic respiration and energy metabolism, and bacterial virulence and quorum sensing are inhibited. After in-vivo systemic injection of a mouse, the MRSA site can be efficiently targeted, MRSA calcification sterilization is induced, calcified bacteria can enhance inherent sterilization immunity of a host with a mononuclear macrophage system as a main part, macrophages are promoted to be polarized into an inflammatory macrophage state, the bacterial phagocytic ability and the inflammatory factor secretion ability of the host are enhanced, and the anti-inflammatory effect of the host is improved. Therefore, mouse chronic pulmonary infection and chronic osteomyelitis caused by MRSA are effectively treated, the survival rate of mouse pneumonia and osteomyelitis is remarkably improved, and the bone density of patients with osteomyelitis is improved. The invention has the potential to be developed into a novel non-antibiotic bactericidal drug for treating intractable pathogen infection such as drug-resistant bacteria and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical materials, and particularly relates to a drug for treating diseases related to bacterial infections and its applications. Background Art

[0002] Staphylococcus aureus is one of the most common human bacterial pathogens and is an important cause of skin and soft tissue infections, pneumonia, osteomyelitis, septic arthritis, endocarditis, foreign body infections, and sepsis. Methicillin-resistant Staphylococcus aureus (MRSA) has developed resistance to most antibiotics, so there is a lack of effective treatment methods, seriously threatening human health. For example, chronic osteomyelitis caused by MRSA is a major clinical challenge, which can lead to progressive bone destruction, organ infection, and sepsis. The deep oxygen and nutrient supply in the matrix of bone tissue is relatively low, which also leads to the formation of persistent dormant bacteria, further limiting the use of antibiotics and surgical treatment. Staphylococcus aureus and Pseudomonas aeruginosa are also the most common pathogens in patients with chronic lung infections, including cystic fibrosis and bronchiectasis, and are also common pathogens in patients with chronic obstructive pulmonary disease when they develop infections. In ventilator-associated pneumonia, it is mainly caused by potential multi-drug resistant bacteria, such as MRSA and drug-resistant Pseudomonas aeruginosa.

[0003] MRSA isolates are not limited to healthcare settings such as hospitals. Since the mid-1990s, the number of community-associated MRSA infections has also increased explosively. MRSA can produce a wide range of virulence factor libraries, including secreted toxins (e.g., α, β, γ, δ, exfoliative, enterotoxin), phospholipase C, metalloprotease, capsular polysaccharide, protein A, fatty acid modifying enzymes and lipases, V8 protease, leukocidin, phenol-soluble modulin, golden pigment, and staphylokinase, etc. These factors can promote bacterial colonization in the host, dissolve host tissues, alter host immunity, and produce tissue toxins. In addition to producing specific toxins, the contribution of biofilm formation to antibiotic resistance and chronic infections cannot be ignored. Studies have also found that MRSA colonization patients carrying bacteria for more than 1 year have a very high risk of MRSA infection and death. The high virulence, extensive drug resistance, and biofilm and other characteristics of MRSA can promote bacterial colonization in the body, leading to long-term and repeated chronic infections and relapses, which greatly increases the burden of MRSA infection-related diseases and the mortality rate of the diseases, seriously endangering human health.

[0004] Due to the rich virulence factors, high drug resistance and biofilm formation of MRSA, the treatment of chronic MRSA infections has become increasingly difficult. Since MRSA isolates are resistant to all available penicillins and other β-lactam antibacterial drugs, this poses a major challenge to the treatment of MRSA infections, which also makes Staphylococcus aureus one of the top three bacteria associated with deaths due to bacterial drug resistance. Some studies have shown that although it is possible to temporarily treat MRSA, the treatment effect cannot be maintained in the long term, which may indicate that chronic MRSA infections can currently only be suppressed and are difficult to eradicate. Currently available antibiotics for treating MRSA include fusidic acid, rifampicin, doxycycline, minocycline, ceftobiprole, clindamycin, teicoplanin, TMP-SMX, vancomycin, tigecycline, quinupristin / dalfopristin and linezolid, etc. International guidelines currently recommend vancomycin, teicoplanin or linezolid as first-line treatment drugs. However, since 15-20% of the MRSA genome consists of mobile genetic elements, these elements have the ability to horizontally transfer resistance genes, making it extremely easy for bacteria to acquire drug resistance by transferring mobile genetic elements from other bacteria. Alarmingly, studies have reported a decrease in sensitivity to vancomycin and resistance to linezolid and daptomycin, etc. in MRSA clinical strains. For example, in 1997, a vancomycin-intermediate Staphylococcus aureus (VISA) strain was detected in Japan, and in 2002, a vancomycin-resistant Staphylococcus aureus (VRSA) strain was also found in the United States, which was considered to be caused by the transfer of the vanA gene from vancomycin-resistant Enterococcus faecium. Moreover, MRSA infections usually require high-dose systemic injection of antibiotics for treatment, which may lead to damage to the innate immune system and cause serious adverse reactions, including fever, kidney damage and thrombophlebitis, etc. Therefore, with the increase in bacterial drug resistance, there is an urgent need to find new antibacterial targets for treating chronic infections caused by MRSA.

[0005] Calcification refers to a phenomenon in which calcium salts are deposited in specific organs or tissues of the body. It includes normal calcification (bone and tooth formation) and pathological calcification (dental calculus, vascular calcification, liver calcification, etc.). In recent years, related research has found that targeted induction of tumor cell calcification has the potential to become a new method for treating tumors: A 2013 study showed that inducing cell mineralization through an artificial mineral shell in vitro could reduce the motility of cancer cells and inhibit the migration and invasion ability of cancer cells; and by cutting off nutrient and information exchange, cell viability and related functions would also be inhibited. In 2016, researchers successfully achieved targeted induction of calcification in tumor cells in vivo, and the calcified shell could inhibit the growth and metastasis of tumors without calcifying or damaging healthy tissues and organs. Polysialic acid (PSA) is a macromolecular compound of polysaccharide containing multiple carboxyl groups, and its carboxyl groups can enrich calcium ions to achieve tumor calcification at the physiological concentration of blood calcium level. After researchers cross-linked PSA with folic acid, it could achieve targeted calcification therapy for cervical cancer and breast cancer in vivo, significantly improving the survival rate of cancer-bearing mice. However, currently, the research on induced calcification therapy basically focuses on tumors, and the specific mechanism by which calcification leads to cell death is still poorly understood, which makes it a novel and interesting topic whether targeted induction of bacterial calcification can be used to coat and mask the surface proteins of bacteria and inhibit bacterial energy metabolism to treat chronic infections and explore its further mechanism.

[0006] Studies have found that many intratubular calcium oxalate crystals formed in rats and mice on a high-oxalate diet will eventually disappear, indicating that their kidneys can eliminate these crystals. The calcium phosphate crystals in the renal tubules of mice will also decrease after 5 months. Further research has also found that the elimination of calcification foci in vivo is related to macrophage phagocytosis and degradation of crystals. Microarray analysis of the genes expressed in the renal papilla tissue of patients with kidney stones showed that there was upregulation of genes related to the M1 macrophage phenotype and downregulation of genes related to the M2 macrophage phenotype in kidney stones. Another study also showed that after inducing the differentiation of human monocytes with recombinant human macrophage colony-stimulating factor 1 (CSF1) or granulocyte-macrophage CSF (GM-CSF), and then exposing the cells to calcium oxalate crystals and / or stone fragments, the cells would secrete many chemokines and cytokines crucial for the inflammatory immune response, including CCL2, CCL3, interleukin-1 receptor antagonist (IL-1ra), complement protein C5 and C5a, and IL-8. Primary human monocytes would also differentiate into inflammatory macrophages and produce TNF, IL-1β, IL-8, and IL-10 after being exposed to calcium oxalate crystals in vitro. These all indicate that calcification foci can induce and enhance the immune response mainly based on the body's mononuclear-macrophage system.

[0007] In recent years, due to the irrational use of antibacterial drugs, bacteria are more likely to develop drug resistance, leading to treatment failure. Precision treatment targeting bacteria will help achieve targeted and effective treatment effects. Antibody-drug conjugates (ADCs) are a rising star in the field of targeted therapy. ADCs are complex engineered therapeutic drugs composed of monoclonal antibodies and drugs, connected by chemical linkers. This targeted drug delivery strategy combines the precision of antibody targeting with the effective activity of drugs. Currently, many researchers have precisely delivered chemotherapeutic drugs to tumors with the help of ADCs, and important translational progress has been made. Currently, 4 ADC drugs have been approved in the United States, and more than 60 are in clinical trials. Against Staphylococcus aureus, ADC has been proven to be a new therapy that can effectively kill intracellular Staphylococcus aureus. This ADC is composed of a monoclonal antibody against the cell wall of S. aureus (WTA-mAb) conjugated with a highly effective antibiotic. Wall teichoic acids (WTAs) are pathogen-specific polyanionic glycopolymers connected to the thick peptidoglycan layer in Gram-positive bacteria. The S. aureus WTA-mAb is an antibody cloned and purified from B cells in the peripheral blood of patients recovered from Staphylococcus aureus infection. It can bind efficiently to clinically relevant Staphylococcus aureus strains and the laboratory USA300 strain, and can target and bind to MRSA bacteria in vivo without acting on other normal tissues. Summary of the Invention

[0008] In view of the deficiencies of the prior art, the present invention provides a drug for treating diseases related to bacterial infections and its applications, specifically a novel antibody-PSA conjugate APC, its preparation method and uses. By conjugating polysialic acid (PSA) with the Fab' fragment of the S. aureus WTA monoclonal antibody, a novel bactericidal system (targeted induction of bacterial calcification system) is formed, and an antibody-PSA conjugate with good stability and biocompatibility is synthesized. It not only has specific targeting ability for the site of bacterial infection, but also can inhibit the aerobic respiration and energy metabolism of bacteria by inducing bacterial calcification, cut off bacterial quorum sensing, and down-regulate the secretion of virulence factors, thereby effectively killing bacteria. And the calcified bacteria have the effect of enhancing the host's antibacterial immunity, promoting monocytes-macrophages to become inflammatory macrophage state, and enhancing their ability to phagocytose bacteria, so as to achieve a synergistic bactericidal effect, and can safely and effectively treat chronic MRSA infections.

[0009] To achieve the object of the present invention, the present invention provides the following technical solutions:

[0010] The present invention provides a drug for treating diseases related to bacterial infections, namely a molecule that targets and induces spontaneous calcification of bacteria, and is used for treating chronic Staphylococcus aureus infections, including MRSA infections. The molecule of the drug contains at least two basic units, one of which is a unit with the targeting function for the bacterial infection site, and the other is a unit for inducing bacterial calcification;

[0011] Alternatively, the molecule of the drug contains at least one basic unit, and the basic unit is a unit that can simultaneously have the targeting function for the bacterial infection site and the function for inducing bacterial calcification.

[0012] The novel antibody-PSA conjugate (APC) can achieve precise targeting of Staphylococcus aureus in vivo. Meanwhile, under physiological calcium ion concentration conditions, it can induce bacterial calcification, cover the surface proteins of bacteria, disrupt bacterial communication, thereby resulting in restricted bacterial growth, inhibited respiratory chain, abnormal energy metabolism, decreased virulence, and dysregulation of quorum sensing (QS), and even enhance the immune response of the host.

[0013] Preferably, the unit with the targeting function for the bacterial infection site is the Fab' fragment of the teichoic acid monoclonal antibody of the bacterial cell wall. Preferably, the targeting unit is the Fab' fragment of the WTA monoclonal antibody of Staphylococcus aureus, which can target and bind to Staphylococcus aureus, including MRSA, in vivo and in vitro without acting on normal tissues.

[0014] Preferably, the unit for inducing bacterial calcification is polysialic acid (PSA), which contains a large number of free carboxyl groups and is responsible for enriching calcium and phosphate ions in the microenvironment to generate calcification.

[0015] Preferably, the diseases related to bacterial infections are chronic osteomyelitis or chronic pulmonary infections caused by Staphylococcus aureus infection.

[0016] Preferably, the unit with the targeting function for the bacterial infection site and the unit for inducing bacterial calcification are coupled by a chemical bond to form a conjugate, and the mass ratio range of the unit with the targeting function for the bacterial infection site to the unit for inducing bacterial calcification is 1:100 to 100:1, and the molar ratio range is 1:50 to 200:1.

[0017] In practical applications, the ratio of the targeting functional unit and the calcification induction functional unit can be adjusted according to factors such as the specific condition, bacterial species, infection site, and host immune status. Although the two functional units work synergistically in the drug, they each have a certain degree of independence. As long as there are sufficient targeting functional units to recognize and bind to bacteria, and sufficient calcification induction functional units to induce bacterial calcification, and the drug can exert the expected therapeutic effect, the ratio range of the two functional units in this case is within the protection scope of the present invention.

[0018] Preferably, the particle size range of the novel bactericidal antibody-PSA conjugate APC is 5 - 15 nm, and the average particle size is 7.5 nm.

[0019] Preferably, the antibody-PSA conjugate APC has a maximum absorption peak at a laser wavelength of 280 nm, and the absorption peak does not shift due to changes in the reactant ratio.

[0020] The antibody-PSA conjugate APC can efficiently and rapidly target and bind to the surface of Staphylococcus aureus in vitro. The detection method is as follows: MRSA bacteria co-incubated with a fluorescent antibody (PE-mAb) are detected by flow cytometry. The results show that after incubation with 50 μg / ml of PE-mAb for 10 - 15 minutes, 98.8% of MRSA bacteria can be targeted and bound.

[0021] The antibody-PSA conjugate APC efficiently and rapidly targets the site where bacteria are located and can be completely metabolized and excreted from the body within 48 hours. The detection method is as follows: Mice injected with the fluorescent drug (CY7-APC) via the tail vein are continuously detected by in vivo fluorescence imaging. Within 9 hours after injection of the material, strong fluorescent signals can be detected in the bacterial infection area of the mice, and reach the peak after 10 - 15 minutes. Subsequently, the signals gradually weaken within 1 - 48 hours and disappear from the body after 48 hours.

[0022] The antibody-conjugated polysaccharide has the function of treating pneumonia caused by chronic MRSA. After the treatment course, the survival rate of pneumonia mice has increased from 20 - 40% to 85% - 95%.

[0023] The antibody-conjugated polysaccharide has the function of treating osteomyelitis caused by chronic MRSA. After the treatment course, the survival rate of osteomyelitis mice has increased from 40 - 60% to 85% - 95%. At the same time, the bone density of osteomyelitis has increased from 3200 - 3800 to 3800 - 4400 g / cm.

[0024] Specifically, the preparation method of the drug for treating bacterial infection-related diseases according to the present invention comprises the following steps:

[0025] (1) Dissolve 1000 mg of PSA in deionized water, then add 500 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 300 mg of N-hydroxysuccinimide (NHS), and stir at room temperature to obtain a mixed solution.

[0026] (2) Slowly add the WTA antibody Fab' fragment to the mixed solution in step (1), and continue stirring to obtain the antibody-PSA conjugate APC.

[0027] (3) Put the mixed solution obtained in step (2) into a dialysis bag and dialyze with ultrapure water. After freeze-drying the dialysis product, the product APC is obtained.

[0028] Preferably, in step (1), the reaction temperature is 10 - 40 °C, and the reaction time is not less than 0.5 hours; more preferably, the reaction temperature is 20 °C - 30 °C, and the reaction time is 1 - 2 hours.

[0029] Preferably, in step (2), the reaction temperature is 10 - 30 °C, and the reaction time is not less than 8 hours; preferably, the reaction temperature is 15 °C - 25 °C; more preferably, the reaction temperature is 20 °C; preferably, the reaction time is 8 - 16 hours; more preferably, the reaction time is 12 hours.

[0030] Preferably, in (1), the concentration of PSA in the mixed solution is 5 mg / mL; preferably, in (1), the concentration of EDC in the mixed solution is 2.5 mg / mL; preferably, in (1), the concentration of NHS in the mixed solution is 1.5 mg / mL.

[0031] Preferably, in (2), the final concentration after adding the WTA antibody Fab' fragment is 0.5 mg / mL.

[0032] Preferably, in (3), the molecular weight of the dialysis bag is 1 - 3.5 kDa; more preferably, in (3), the molecular weight of the dialysis bag is 3.5 kDa.

[0033] Preferably, in (3), the dialysis duration is 60 - 84 hours; more preferably, in (3), the dialysis duration is 72 hours.

[0034] Preferably, for the product APC in (3), the mass ratio is mAb:PSA = 1:10.

[0035] The present invention also provides the application of the said drug in the preparation of drugs for treating diseases related to bacterial infections. When applying, the administration route is intravenous administration, tissue lesion interventional administration, oral administration, nasal inhalation administration or intraperitoneal injection administration.

[0036] The bacterial infection-related diseases are chronic osteomyelitis or chronic pulmonary infection caused by Staphylococcus aureus infection.

[0037] The present invention also relates to the use of a novel bactericidal product of APC in any one or more of the fields of medicine, antibacterial and bactericidal.

[0038] Preferably, the material is used as an antibacterial drug / material in vivo and / or in vitro of humans and / or animals.

[0039] Preferably, the material is used as a material for inducing calcification death of bacteria.

[0040] Preferably, the material is used as a quorum sensing inhibitor.

[0041] Preferably, the material is used as a material for regulating the host immune microenvironment.

[0042] The beneficial effects of the present invention are as follows: The present invention designs a bacteria-targeted antibacterial drug antibody-PSA conjugate APC. The drug can rapidly and efficiently target MRSA in vivo and in vitro and induce calcification on the bacterial surface, thereby achieving the effects of killing bacteria, inhibiting the formation of bacterial biofilms and inhibiting quorum sensing; the calcified bacteria can also effectively regulate the host immune microenvironment and enhance the function of the innate immune system mainly composed of the mononuclear-macrophage system. This non-antibiotic synergistic antibacterial effect is not easily resisted by drug-resistant bacteria, so it can effectively inhibit drug-resistant pathogens and can be used to treat diseases caused by drug-resistant bacterial infections. Moreover, after the antibacterial material is treated with cells, the cell viability is greater than 95%. Therefore, the effective bactericidal concentration of the material is far lower than the concentration at which it will produce toxicity to normal tissues, and it has the characteristics of safe antibacterial. In addition, the drug can significantly improve the survival rate of mice with chronic pneumonia and chronic osteomyelitis, reduce the bacterial load at the lesion site, and at the same time restore the bone density of osteomyelitis, and finally cure chronic MRSA infection. The systemic toxicity to other organ tissues during the treatment process can be almost ignored. Therefore, the APC of the present invention can become a very promising new antibacterial drug for treating drug-resistant MRSA infection, and has strong clinical transformation potential. Description of the Drawings

[0043] Figure 1 : APC can target and bind to MRSA bacteria and induce calcification death of bacteria, while inhibiting the formation of bacterial biofilms and destroying the formed biofilms. a is a schematic diagram of the process of APC-induced calcification of MRSA. b is the binding ratio of fluorescently labeled antibody PE-mAb (50 μg / ml) and PE-isotype control antibody (50 μg / ml) to different bacteria. c is the concentration dependence of the bacterial binding ability of PE-mAb. d is the molecular weight distribution curve of APC, mAb, and PSA. e is the ultraviolet-visible spectrum (UV) of mAb, PSA, and APC at 240-400 nm.

[0044] Figure 2 : APC can specifically target and bind to MRSA bacteria, induce bacterial calcification death, inhibit bacterial biofilm formation, and disrupt pre-formed biofilms. a shows the scanning electron microscope (SEM) images of bacteria and the energy-dispersive X-ray spectroscopy (EDX) elemental mapping spectra of calcium elements after treatment with the control group and APC. b shows the transmission electron microscope (TEM) images of bacteria and the selected area electron diffraction (SAED) images after treatment with the control group and APC. c shows the colony-forming unit (CFU) spot counts of bacteria at 0, 12, 24, 36, 48, and 60 h after treatment with different concentrations of APC (0, 25, 50, 100, 200 μg / ml). d shows the CFU spot counts of bacteria in the control (saline solution), PSA (180 μg / ml), mAb (20 μg / ml), mAb (20 μg / ml) + PSA (180 μg / ml), IPC (200 μg / ml), and APC (200 μg / ml) groups at 0, 12, 24, 36, 48, and 60 h after treatment. e shows the viable bacteria plating for inhibiting biofilm formation, the 3D structure of SYTO 9-stained biofilms, the confocal low-magnification integrated images of biofilms under a 10× objective lens, the average fluorescence intensity, and the bacterial quantitative statistics after treatment with saline and APC (500 μg / ml) for 48 h. f shows the viable bacteria plating for disrupting pre-formed biofilms, the 3D structure of SYTO 9-stained biofilms, the confocal low-magnification integrated images of biofilms under a 10× objective lens, the average fluorescence intensity, and the bacterial quantitative statistics after treatment with saline and APC (500 μg / ml) for 48 h.

[0045] Figure 3 : After inducing bacterial calcification, APC can inhibit bacterial energy metabolism and amino acid metabolism, and inhibit bacterial virulence factors. (a) Volcano plot of differentially expressed genes in the APC treatment group and the saline control group by RNA-seq, indicating the top 3 differentially expressed genes. (b) Bubble plot of GO enrichment analysis of differentially expressed genes between groups. (c) Heatmap of differentially expressed genes in pathways related to bacterial energy metabolism. (d) Heatmap of differentially expressed genes in pathways related to bacterial virulence factors, quorum sensing, two-component systems, and peptidoglycan biosynthesis.

[0046] Figure 4: APC-induced bacterial calcification inhibits the expression of bacterial virulence factors and bacterial energy metabolism. (a) Relative levels of mRNA related to virulence factors in bacteria after treatment with normal saline, PSA (180 μg / ml), mAb (20 μg / ml), mAb (20 μg / ml) + PSA (180 μg / ml), IPC (200 μg / ml), or APC (200 μg / ml) for 60 h in MRSA. (b) Expression heatmap of the results of targeted metabolomics detection of metabolites related to energy metabolism in the APC treatment group and the control group. (c-d) ATP levels and NAD + / NADH ratio in bacteria after treatment with normal saline, PSA (180 μg / ml), mAb (20 μg / ml), mAb (20 μg / ml) + PSA (180 μg / ml), IPC (200 μg / ml), or APC (200 μg / ml) for 60 h in MRSA.

[0047] Figure 5 : APC can target MRSA in vivo. a-b, In vivo organ fluorescence imaging and statistics after tail vein injection of Cy7-APC or Cy7-IPC (isotype control antibody-PSA conjugate) in chronic pneumonia mice and healthy non-modeled mice; c-d, Ex vivo organ (lung tissue) fluorescence imaging and statistics after tail vein injection of Cy7-APC or Cy7-IPC (isotype control antibody-PSA conjugate) in chronic pneumonia mice and healthy non-modeled mice; e, Lung tissue sections of GFP-Luc-MRSA chronic pneumonia model mice after tail vein injection of PE-APC or PE-IPC. f, Bacterial imaging of GFP-Luc-MRSA chronic osteomyelitis model mice. g-h, In vivo fluorescence imaging and fluorescence statistical analysis of the tibia in chronic osteomyelitis mice and healthy mice after tail vein injection of Cy7-APC or Cy7-IPC; i-j, Ex vivo fluorescence imaging and statistics of the tibia in chronic osteomyelitis mice and healthy mice after tail vein injection of Cy7-APC or Cy7-IPC.

[0048] Figure 6 : APC can effectively treat MRSA chronic pneumonia and reduce the bacterial content in the lungs. (a) Experimental protocol for the post-infection treatment study in the MRSA chronic pneumonia model. Mice were infected by intratracheal inoculation (i.t.) with MRSA agarose beads. Two days after infection, the mice were treated by intravenous injection (i.v.) with PBS, mAb, PSA, isotype control antibody-PSA-conjugate (IPC), or APC every other day. Lung tissues were collected 28 days after treatment. (b-h) Gross appearance of the lungs (b), percentage of surviving mice (c), core body temperature of the mice (d), and body weight curve of the mice (e), bacterial CFU in lung homogenates (f), dry / wet ratio of the lungs (g) of the mice after treatment with PBS, mAb, PSA, IPC, or APC for 28 days

[0049] Figure 7 : APC can effectively treat chronic MRSA pneumonia, reduce pulmonary inflammatory response, and induce bacterial calcification. (a) HE staining, Gram staining, and alizarin red staining images of lung sections of mice treated with PBS, mAb, PSA, IPC, or APC for 28 days. (b) Statistical chart of pneumonia pathological scores of different treatment groups. (c-d) Fusion map of three-dimensional reconstruction imaging of Micro-CT of the lungs and images of calcified lesions in different treatment groups (c) and statistical chart of the volume of calcified lesions (d). The scale bar in a is 100 μm. The short red arrows in a indicate MRSA embedded in the agarose beads in the trachea. The red dots in c indicate the calcified lesions in the lungs detected by micro-CT.

[0050] Figure 8 : APC can effectively treat chronic MRSA osteomyelitis and reduce the bacterial content in the bone marrow. (a) Experimental protocol studied in the APC treatment of chronic MRSA osteomyelitis model. Mice were infected by injecting (i.o.) MRSA strains into the tibial bone marrow. After 30 days of infection, mice were treated by injecting (i.v.) PBS, mAb, PSA, or APC intravenously once every other day. The tibias were harvested after 28 days of treatment. (b-e) Gross appearance of the tibias of mice treated with PBS, mAb, PSA, or APC (b), percentage of survival (c), core body temperature (d), and body weight curve (e). (f) Dynamic change curve of the Luc bioluminescence intensity of bacteria during the treatment of chronic osteomyelitis mice infected with GFP-Luc-MRSA bacteria. (g) Bacterial CFU in bone marrow homogenates on day 0 and day 28 treated with PBS, mAb, PSA, or APC.

[0051] Figure 9 : APC can effectively treat chronic MRSA osteomyelitis, increase bone density, and reduce the inflammatory response in the bone marrow. (a) 2D images of Micro-CT of the tibias, 3D bone imaging, and (b) statistical chart of bone density in different treatment groups after 28 days. (c-d) HE staining, Gram staining of bone sections in different treatment groups (c), and statistical chart of the number of bacterial abscess masses in the bone (d).

[0052] Figure 10 : APC has good biosafety when applied to cells in vitro. (a-g) Survival rates of HUVECS cells, BEAS-2b cells, MC3T3-E1 cells, primary human PBMCs, primary human neutrophils, primary mouse PBMCs, and primary mouse neutrophils after exposure to gradient concentrations of mAb, PSA, or APC for 24 h.

[0053] Figure 11: APC has good biosafety in mice. (a-b) Survival rate (a) and body weight curve (b) of healthy BALB / c mice 28 days after intravenous injection of 100 μL PBS, mAb (20 mg / kg), PSA (180 mg / kg), or APC (200 mg / kg) every other day. (c-d) Blood routine (white blood cells, red blood cells, platelets, and hemoglobin) (c), liver function indicators (total bilirubin, alanine aminotransferase, aspartate aminotransferase), kidney function indicators (creatinine, blood urea nitrogen), blood calcium level, cardiac function markers (CK-MB, lactate dehydrogenase), and glycolipid metabolism markers (blood glucose, total cholesterol, low-density lipoprotein, and high-density lipoprotein) levels on the 7th day and 28th day after intravenous injection of 100 μL PBS, mAb (20 mg / kg), PSA (180 mg / kg), or APC (200 mg / kg) into healthy mice every other day. The dotted line represents the normal range of blood indicators.

[0054] Figure 12 : APC has good biosafety in mice. (a-b) H&E staining of sections of major organs (heart, liver, spleen, lung, kidney) of mice on the 7th day (a) and 28th day (b) after different drug treatments. Scale bar, 100 μm.

[0055] Figure 13 : Single-cell RNA sequencing of the lungs reveals that calcified MRSA promotes host innate immunity as an immunomodulator. (a) Schematic diagram of the 10×Genomics scRNA-seq procedure for removing the lungs of BALB / c mice 14 days after receiving PBS or APC treatment. Three mice were treated in parallel for each group. (b) tSNE plot of 47,171 cells from 6 lungs, showing 10 cell populations identified based on canonical marker genes. (c) Statistical analysis of the difference in cell numbers between the PBS and APC groups in each cell population. (d) Functional scores of the MPS cell population for anti-inflammatory, pro-inflammatory, inflammatory cell chemotaxis, and inflammatory cytokine release. (e) Top 5 upregulated and downregulated genes among the differentially expressed genes between groups in 13 subpopulations of the MPS cell population.

[0056] Figure 14: Calcitic MRSA promotes host innate immunity mediated by S100A8 and S100A9. (a) Proportion of inflammatory macrophages in wild-type mouse alveolar macrophages (MH-S) co-incubated with MH-S cells with double gene knockout of calbindin S100a8 and S100a9, PBS, fixed MRSA, or fixed calcitic MRSA for 48 hours. (b-d) Local magnified views under a confocal 63× microscope (b), fluorescence intensity of GFP-MRSA in individual MHS cells detected by flow cytometry (c), and proportion of GFP-MRSA fluorescence-positive cells (d) after wild-type and knockout MH-S cells were co-cultured with PBS, fixed MRSA, and fixed calcitic MRSA for 48 hours. (e-f) Levels of S100A8 (e) and S100A9 (f) in the co-culture supernatants of wild-type and knockout MH-S cells with PBS, fixed MRSA, and fixed calcitic MRSA.

[0057] Figure 15 This is a schematic diagram of the drug treatment mechanism of the present invention. Detailed implementation manners

[0058] The invention will be further described below in conjunction with embodiments and drawings; in this embodiment, the preparation of APC antibody-conjugated polysaccharide, antibacterial data in vitro and in vivo, and data on treating chronic MRSA infection in vivo and promoting innate immunity are used to illustrate its antibacterial and immune-regulating effects.

[0059] Experimental materials

[0060] Methicillin-resistant Staphylococcus aureus (MRSA) was obtained from the American Type Culture Collection (ATCC43300). MRSA expressing green fluorescent protein (GFP) and luciferase (MRSA-GFP-Luci) was constructed by Fenghai Biotechnology Co., Ltd. and cultured on TSB 10 cm (TSB with 10 μg / mL chloramphenicol) or TSA 10 cm (TSA agar with 10 μg / mL chloramphenicol) plates. Fetal bovine serum (FBS) was purchased from Gibco. Mouse alveolar macrophages (MH-S), human umbilical vein endothelial cells (HUVECs), human normal lung epithelial cells (BEAS-2B), and mouse embryonic osteoblasts (MC3T3-E1) were obtained from the American Type Culture Collection. TSB broth and TSA broth agar, phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Solarbio Science & Technology Co., Ltd. Double-distilled water (ddH2O) was from a Milli-Q purification system. BALB / c mice were from Shanghai Biotechnology Co., Ltd. The cell counting kit (CCK-8) was purchased from Meilun Biotechnology (Dalian, China).

[0061] All cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin) and placed in an incubator at 37 °C with 95% O2 and 5% CO2. Cells were collected by centrifugation (1000 rpm, 4 minutes) and subcultured with 0.25% trypsin until the confluence reached approximately 80%.

[0062] Characterization instruments

[0063] A transmission electron microscope (TEM) and a scanning electron microscope (SEM) were used to observe the structure and elemental analysis of bacteria after calcification. The fluorescence of drugs in animals was counted by a small animal in vivo imager. A small animal Micro-CT was used to monitor calcification foci and measure bone density in the lungs and tibias of mice.

[0064] Example 1 Preparation of antibacterial drug antibody-PSA conjugate APC

[0065] (1) Preparation of Staphylococcus aureus WTA-monoclonal antibody Fab' fragment:

[0066] Construction of the expression vector for the Fab' fragment of the WTA monoclonal antibody: The amino acid sequence of the Fab' fragment of the WTA monoclonal antibody was downloaded from the RCSB PDB protein database (https: / / www.rcsb.org / structure / 5D6C). The pcDNA3.1 plasmid was constructed based on the amino acid sequence of the Fab' fragment of the WTA antibody, and the recombinant plasmid was amplified by PCR, double digested with EcoRI / BamHI, and the plasmid information was identified by sequencing after transformation of competent Escherichia coli DH5α cells. The plasmid with the correct sequence was obtained by sequencing; the plasmid was amplified for production and transferred downstream for expression.

[0067] Cell culture and protein expression: HEK293 cells were subcultured using 293 serum-free CD medium (product number SMM 293-TI, Beijing Sino Biological Inc.). The plasmid of the target protein for expression was mixed with the transfection reagent TF1 and then added to the cells. The 293 serum-free feeding solution (product number: M293-SUPI-100, Beijing Sino Biological Inc.) was added on the 1st, 3rd, and 5th days after transfection respectively.

[0068] Protein purification: Protein purification was carried out 7 days after cell culture. The expressed protein was purified by affinity chromatography column with protein L antibody fragment to purify the antibody Fab fragment. The purification process is as follows: 1. After centrifugation of the culture medium, the remaining insoluble matter was removed by filtration and then the target protein was purified using an affinity chromatography column. 2. Affinity chromatography purification: After equilibrating the chromatography column with the loading buffer, the supernatant of the culture solution was loaded onto the chromatography column. After the impurity proteins passed through, the target protein was eluted with the elution buffer. After changing the solution of the purified target protein, the protein concentration was calibrated and the purity was detected.

[0069] Quality detection: The obtained protein samples were monitored for the protein expression process and the concentration and purity of the products were detected by ultraviolet absorption OD 280 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0070] (2) Dissolve 1000 mg of PSA (product number: 70431-34-4, Zhongke Hongji) in deionized water, then add 500 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 300 mg of N-hydroxysuccinimide (NHS), and stir at 20-30 °C for 1 hour to obtain a mixed solution. In the mixed solution, the concentration of PSA is 5 mg / mL, the concentration of EDC is 2.5 mg / mL, and the concentration of NHS is 1.5 mg / mL.

[0071] (3) Slowly add 250 mg of WTA antibody Fab' fragment to the mixed solution in step (2). The final concentration after adding the WTA antibody Fab' fragment is 1.25 mg / mL, and continue to stir at 20 °C for 12 hours to obtain a mixed solution containing antibody-PSA conjugate (APC).

[0072] (4) Add the mixed solution obtained in step (3) into a dialysis bag with a molecular weight of 3.5 kDa and dialyze with ultrapure water for 72 hours. After freeze-drying the dialysis product, the product APC was obtained.

[0073] Example 2: APC can specifically bind to MRSA bacteria and induce bacterial calcification

[0074] The ability of the antibody to specifically bind to MRSA was detected by flow cytometry. The Fab' fragment of human cytomegalovirus IgG antibody (RCSB PDB Protein Data Bank (https: / / www.rcsb.org / structure / 4HH9)) was used as an isotype control antibody, and Streptococcus pneumoniae and Pseudomonas aeruginosa were used as bacterial controls. MRSA bacteria, Streptococcus pneumoniae and Pseudomonas aeruginosa (1×10 7(CFU / mL) were respectively suspended in HB buffer, and then the fluorescently labeled antibody (PE-mAb or PE-isotype control antibody) was directly added to the blocking reaction and incubated at room temperature for another 10 - 20 minutes. After washing 3 times with Hank's balanced salt solution, flow cytometry was performed on a CytoFLEX S flow cytometer to detect the proportion of fluorescent bacteria. The molecular weight distributions of APC and PSA, mAb were measured using a mass photometer (Refeyn, Oxford, UK).

[0075] The results showed that 98.8% of MRSA bacteria could bind to the fluorescently labeled antibody PE-mAb, much higher than the isotype control group and the bacterial control group ( Figure 1 b). The proportion of bacteria binding to PE-mAb showed a concentration-dependent increasing trend ( Figure 1 c). The molecular weight distribution curve of APC detected by the mass photometer method showed that the molecular weight of APC was the superposition of the molecular weights of PSA and mAb ( Figure 1 d). The mAb monoclonal antibody had an obvious ultraviolet absorption peak at 280 nm, and after cross-linking PSA, APC also had an obvious ultraviolet absorption around 280 nm ( Figure 1 e). Through scanning electron microscopy SEM ( Figure 2 a) and transmission electron microscopy TEM ( Figure 2 b) for image acquisition of bacteria after APC treatment, it could be seen that there was a tightly wrapped outer shell around the MRSA bacteria. Through SEM-energy dispersive X-ray spectroscopy (EDX) elemental analysis, it could be obtained that there was obvious calcium element enrichment in the outer shell ( Figure 2 a), and transmission electron microscopy (TEM) showed that obvious calcified crystals were formed around MRSA, and selected area electron diffraction (SAED) confirmed the formation of the crystals ( Figure 2 b), while the MRSA in the control group had a smooth surface, no outer shell, and no calcium element enrichment ( Figure 2 a and b). These data indicated that APC could target and bind to MRSA bacteria and induce the formation of a calcified outer shell on the bacterial surface.

[0076] Example 3: Calcification leads to bacterial death, inhibits bacterial biofilm formation, and destroys the formed biofilm.

[0077] The TEM images showed that after APC-induced calcification, the calcified MRSA died and dissolved ( Figure 2 b). To verify the activity of bacteria after induced calcification, we performed CFU plate counting on APC-treated MRSA at different time points. The results showed that at the same calcium and phosphorus concentrations, as the APC concentration increased, the number of colony-forming units (CFUs) of MRSA gradually decreased ( Figure 2 c). After 60 hours, the CFUs in the APC group were significantly less than those in the control group ( Figure 2d). These results together demonstrate that the calcification coating induced by APC promotes bacterial death.

[0078] Biofilms are also crucial in the pathogenic process of MRSA. Therefore, we also examined whether calcification has the efficacy to inhibit bacterial biofilm formation and disrupt pre-formed biofilms. Through confocal three-dimensional imaging and plating counts of viable bacteria in the biofilms, the results showed that the biofilms formed by bacteria treated with APC and the viable bacteria within the biofilms were significantly reduced compared to the control group ( Figure 2 e), and APC treatment could also significantly disrupt pre-formed biofilms and kill the viable bacteria within them ( Figure 2 f).

[0079] Example 4: Calcification kills bacteria by inhibiting normal bacterial physiological metabolism and virulence factors.

[0080] To explore the mechanism by which calcification causes bacterial death, we performed RNA-seq-based transcriptome analysis on APC-treated MRSA. Overall, a total of 3,031 single genes were annotated in this sequencing. Among them, 171 genes had significantly decreased expression in the calcification group, and 28 genes had significantly increased expression ( Figure 3 a). Functional annotation of the obtained differentially expressed genes was performed through the Gene Ontology (GO) functional database, and the results showed that the differentially expressed genes were mainly enriched in ATP metabolic pathways, amino acid metabolism, translation, protein metabolism, pathogenicity, and transmembrane transport, etc. ( Figure 3 b). Then we focused on all the differentially expressed genes in the energy metabolism-related pathways and classified them according to more detailed KEGG functions. The results showed that in the calcification group, except for the differentially expressed genes related to the energy metabolism substrate CoA, which were up-regulated, the differentially expressed genes in other pathways such as oxidative phosphorylation, gluconeogenesis, and fatty acid metabolism were all down-regulated ( Figure 3 c). In addition, we also found that the differentially expressed genes related to bacterial toxicity associated with the pathogenicity of MRSA were all down-regulated, including virulence factors, quorum sensing, two-component systems, etc. ( Figure 3 d). To further verify that calcification can inhibit bacterial virulence factors and energy metabolism, we performed qT-PCR and targeted metabolomics analysis on calcified bacteria to quantify the mRNA levels of genes related to virulence factors and the metabolite levels related to energy metabolism. The results showed that after bacterial calcification, the mRNA expression of genes related to virulence factors was significantly down-regulated ( Figure 4 a); except for cyclic adenosine monophosphate (cAMP), all the differentially expressed metabolites related to energy metabolism in the calcification group were significantly down-regulated ( Figure 4 b). In addition, the intracellular ATP level and NAD in the calcification group +The ratio of [substance] / NADH also decreased significantly compared with the control group ( Figure 4 c-d). These data indicate that calcification-mediated bacterial death is mainly achieved by inhibiting important physiological processes of bacteria, including energy metabolism, amino acid metabolism, and translation. In addition, the calcified shell disrupts bacterial membrane transport, restricting material exchange and bacterial communication. This inhibition not only prevents the entry of exogenous nutrients but also inhibits the efflux of bacterial virulence factors and quorum sensing ( Figure 15 a). In summary, calcification kills bacteria by weakening the overall "vitality" of bacteria.

[0081] Example 5: APC can effectively treat chronic pneumonia and chronic osteomyelitis in mice.

[0082] To verify the efficacy of APC in treating mice, we used 6-8-week-old female BALB / c mice (Shanghai SLAC Laboratory Animal Co., Ltd.) to establish two common chronic infection models of chronic pneumonia and chronic osteomyelitis, and used them to evaluate the drug efficacy. Before the formal experiment, we first verified the ability of APC to target and bind to MRSA in vivo. After systemic injection of Cy7-APC into chronic pneumonia mice and healthy control mice, the distribution of the drug was shown by a small animal imaging system. The results showed that the fluorescence intensity of Cy7-APC targeted and aggregated in the lungs of chronic pneumonia mice was significantly higher than that of the healthy control group and the isotype control antibody group ( Figure 5 a-d), and lung section scanning also showed obvious co-localization of PE-APC and the MRSA strain GFP-Luc-MRSA expressing both green fluorescent protein and luciferase in the lungs, while the isotype control antibody (Fab’ fragment of the above human cytomegalovirus IgG antibody)-PSA conjugate (IPC) had no significant co-localization effect with MRSA bacteria ( Figure 5 e). We also established a chronic osteomyelitis model with GFP-Luc-MRSA. The small animal imaging system showed that after systemic injection of Cy7-APC or Cy7-IPC, the fluorescence of the fluorescent drug aggregated in the tibia on the osteomyelitis lesion side was significantly higher than that of the contralateral healthy tibia, but the fluorescence value after injection of Cy7-APC was higher ( Figure 5 f-j), and combined with the results of lung tissue sections, it can be seen that IPC is non-specifically aggregated at the inflammatory site. In summary, these results indicate that in the mouse models of chronic pneumonia and chronic osteomyelitis, systemically injected APC can accurately target the location of MRSA and tightly bind to the surface of MRSA bacteria. Then, we treated the mice with chronic pneumonia and chronic osteomyelitis by administering 100 μL of PBS, mAb (20 mg / kg), PSA (180 mg / kg), or APC (200 mg / kg) via the mouse tail vein every other day until the 28th day of treatment ( Figure 6 a and Figure 8 a).

[0083] For murine chronic pneumonia, to investigate whether a high-calcium diet could enhance the bactericidal effect of APC, we administered a high-calcium diet (2.5% w / w; calcium dihydrogen phosphate) to mice with chronic pneumonia in the APC + calcium group. The results showed that, in terms of the development of chronic pneumonia, the gross appearance of the lungs of mice treated with APC and APC + calcium tended to be normal, while the control group showed obvious chronic congestion and lung abscesses ( Figure 6 b). The 28-day survival rate of mice in the APC + calcium group was significantly higher than that of the PBS group ( Figure 6 c). Compared with the mAb group, the core body temperature of mice in the APC group and the APC + calcium group was higher at 28 days, and the body weight was also significantly greater than that of the PBS group ( Figure 6 d, e). The CFU value of MRSA in the APC + calcium group was also significantly lower than that of the PBS group ( Figure 6 f). The lung wet / dry weight ratios of the APC group and the APC + calcium group were both significantly lower ( Figure 6 g). Hematoxylin and eosin (H&E) staining of lung tissue showed that the lung tissue structure improved after treatment with APC and APC + calcium, while the other four groups showed obvious chronic inflammation, such as lymphocyte infiltration and alveolar wall thickening ( Figure 7 a). In the APC group and the APC + Ca group, Gram staining showed that the pneumonia pathological score and bacterial activity of agarose beads inoculated into the trachea were significantly lower than those of the other four groups ( Figure 7 a-b). To confirm the formation of calcification in vivo, we performed alizarin red staining and pulmonary micro-computed tomography (micro-CT). The results showed that stained calcification could be detected in the lungs of mice treated with APC and APC + calcium ( Figure 7 c), and the calcification volume increased significantly in the APC group and the APC + calcium group ( Figure 7 c-d). These findings indicate that both APC and APC + Ca treatments induced calcification and death of MRSA in chronic pneumonia and alleviated inflammation. However, the combination of APC and a high-calcium diet significantly reduced the bacterial load and mortality. Therefore, oral calcium supplementation during APC treatment can enhance its therapeutic effect.

[0084] In murine chronic osteomyelitis, all groups were fed a high-calcium diet. The appearance of the tibia of mice after APC treatment was more similar to that of a normal tibia, while obvious swelling, abscesses, and deformities were visible in the tibias of the control group ( Figure 8 b). The 28-day survival rate and the body weight on the 28th day of treatment of mice in the APC group were significantly higher than those of the mAb group and the PBS group ( Figure 8 c and Figure 8 e), and the core body temperature was significantly higher than that of the other three groups ( Figure 8d), in chronic osteomyelitis infected with GFP-Luc-MRSA bacteria, the bacterial fluorescence value in the APC group basically disappeared after 4 weeks of treatment, and the fluorescence value was significantly lower than that in the mAb group ( Figure 8 f). The bacterial concentration in the bone marrow of the APC group was significantly lower than that in the other three groups ( Figure 8 g). The results of tibial Micro-CT scanning showed that the integrity of the affected tibia in the mice after APC treatment was close to that of the non-modeled bone, and the bone density was significantly increased compared with the other three groups ( Figure 9 a-b). Both tibial H&E staining and Gram staining showed that the tissue structure tended to be normal after APC treatment, and the number of bacterial abscess clusters (SACs) in the bone marrow was significantly lower than that in the other three groups ( Figure 9 c-d). The above data indicate that APC treatment can also cause bacterial death in chronic osteomyelitis, reduce the bacterial content in the bone marrow, relieve inflammation, and increase bone density.

[0085] Example 6: APC has good biosafety.

[0086] To comprehensively evaluate the safety of APC, we evaluated the drug toxicity from in vitro and in vivo, short-term and long-term effects respectively. The results showed that there were no significant differences in the survival rates of human umbilical vein endothelial cells (Huvecs), human normal lung epithelial cells (Beas-2b), mouse embryonic osteoblasts (MC3T3), primary human peripheral blood mononuclear cells (PBMC), primary human neutrophils, primary mouse PBMC, and primary mouse neutrophils after treatment with different concentration gradients of mAb, PSA, and APC for 24 hours ( Figure 10 a-g). Then we used healthy BALB / c mice to explore the short-term and long-term toxicity of APC in vivo. Mice were administered 100 μL PBS, mAb (20 mg / kg), PSA (180 mg / kg), or APC (200 mg / kg) via the tail vein every other day (the same as the chronic infection treatment protocol). During the 28-day treatment period, the survival rate of each group of mice was 100% ( Figure 11 a), and the body weight of each group increased steadily during the treatment period without significant differences ( Figure 11 b). Blood was collected from the eyeballs at 7 days and 28 days. It was found that 7 days or 28 days after administration, the white blood cells, red blood cells, platelets, hemoglobin levels, liver function indicators (total bilirubin, alanine aminotransferase, aspartate aminotransferase), kidney function indicators (creatinine, blood urea nitrogen), blood calcium levels, cardiac function markers [creatine kinase isoenzyme (CK-MB), lactate dehydrogenase], and glycolipid metabolism markers (blood glucose, serum cholesterol, low-density lipoprotein, and high-density lipoprotein) of the mice treated with the three drugs were not significantly different from those of the PBS control group ( Figure 11c-d) It shows that APC has no significant effect on the hematopoietic system, liver function, heart and kidney function, and blood calcium ion content in mice. At the same time, the H&E staining results of the sections of important organs such as the heart, liver, spleen, lung, and kidney of mice 7 days or 28 days after drug administration show that no obvious inflammatory reaction occurred in these organs after administration of the three drugs, and the normal tissue structure was maintained. Figure 12 a-b).

[0087] Example 7: APC treatment can enhance the innate immune system of the body and synergistically kill bacteria.

[0088] To comprehensively characterize the impact of induced bacterial calcification on the body's immune microenvironment, we used the 10x Genomics platform to perform single-cell RNA sequencing (scRNA-seq) analysis on all cells from the lungs of mice at the mid-stage of calcification treatment (14 days, when there is both calcification formation and the presence of bacteria and inflammation in the lungs), and used biological replicates to enhance robustness. Figure 13 a). After quality control and data filtering, we merged 47,171 single cells from all 6 lungs into the dataset. Unsupervised clustering and t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction were implemented in the Seurat software package, and 10 major cell populations were identified based on typical marker genes, including endothelial cells, epithelial cells, fibroblasts, neutrophils, T cells, B cells, natural killer cells (NK cells), mononuclear phagocytic system (MPS), red blood cells, and neuronal cells. Figure 13 b). Among the immune cells, the number of neutrophils in the APC treatment group was higher than that in the control group, and the number of NK cells was lower than that in the control group, but there was no significant difference. Figure 13 c).

[0089] MPS plays an important role in the antibacterial innate immunity of the body and has functions such as secreting pro-inflammatory factors, phagocytosing bacteria, and presenting antigens. We performed functional scoring on MPS in the control group and the APC group. The results showed that the pro-inflammatory score of the APC treatment group was significantly higher than that of the control group. In addition, the scores of the treatment group in the inflammatory cell chemotaxis function and inflammatory factor release function were also significantly increased compared with the control group. Figure 13 d). MPS contains monocytes (MNC), macrophages. and dendritic cells (DCs), which have different functions and origins. Therefore, we further explored their subsets by performing secondary clustering analysis on this population. A total of 13 clusters were identified and cell types were distinguished based on marker genes. Among these 13 subsets, we analyzed the differentially expressed genes between APCs and the control group, and found that the two calcium-binding proteins, S100A8 and S100A9, were present in the top 5 genes with significantly increased expression in the APC groups of multiple subsets of macrophages and monocytes ( Figure 13 e).

[0090] To further verify the effect of calcified MRSA on macrophage activation in vitro and to validate the key role of S100a8 and S100a9 in the immunomodulatory function of calcified bacteria. We used mouse alveolar macrophages (MH-S) and S100a8 and S100a9 double-knockout MH-S cells constructed using CRISPR-Cas9 for validation experiments. The knockout nucleotide sequences were sgRNA-A1 (S100a8): AATTGTGGTAGACATCAATG-AGG; sgRNA-B1 (S100a9): CTTCCATCAATACTCTAGGA-AGG. The results showed that co-culture of fixed calcified MRSA with MH-S could significantly increase the proportion of MH-S cells that were double-positive for CD80 and CD86 (surface markers of inflammatory macrophages) ( Figure 14 a). After stimulation with calcified bacteria, the ability of MH-S to phagocytose MRSA also increased significantly ( Figure 14 b-d). The levels of S100A8 and S100A9 in the supernatant of MH-S cells also increased significantly ( Figure 13 e-f). However, knockout of S100a8 and S100a9 could significantly reverse the differences in the proportion of inflammatory macrophages and bacterial phagocytosis ability between cells co-incubated with calcified MRSA and uncalcified MRSA ( Figure 13 d-f). These findings further confirmed that calcified MRSA induces the activation of inflammatory macrophages through S100A8 and S100A9, resulting in increased macrophage phagocytosis ability, increased infiltration of inflammatory cells, increased secretion of inflammatory mediators, and ultimately enhanced host antibacterial immunity ( Figure 15 b).

Claims

1. A drug for treating bacterial infection-related diseases, characterized in that: The drug molecule contains at least two basic units. One of the basic units is a unit that has the function of targeting the site of bacterial infection. Another basic unit is a unit for inducing bacterial calcification; Or the drug molecule contains at least one basic unit, The basic unit is a unit that can simultaneously have the function of targeting the bacterial infection site and being used to induce bacterial calcification.

2. The drug for treating bacterial infection-related diseases according to claim 1, characterized in that: The unit with the function of targeting the bacterial infection site is the Fab' fragment of the monoclonal antibody against the teichoic acid of the infected bacterial wall.

3. The drug for treating bacterial infection-related diseases according to claim 2, characterized in that: The unit with the function of targeting the bacterial infection site is the Fab' fragment of the monoclonal antibody against the wall teichoic acid of Staphylococcus aureus.

4. The drug for treating bacterial infection-related diseases according to claim 3, characterized in that: The unit for inducing bacterial calcification is polysialic acid.

5. The drug for treating bacterial infection-related diseases according to claim 1, characterized in that: The bacterial infection-related disease is chronic osteomyelitis or chronic lung infection caused by Staphylococcus aureus infection.

6. The drug for treating bacterial infection-related diseases according to claim 1, characterized in that: The unit with the function of targeting the bacterial infection site and the unit for inducing bacterial calcification are coupled by chemical bonds to form a conjugate, wherein the mass ratio of the unit with the function of targeting the bacterial infection site and the unit for inducing bacterial calcification ranges from 1:100 to 100:1, and the molar ratio ranges from 1:50 to 200:

1.

7. Use of the drug according to any one of claims 1 to 6 in the preparation of drugs for treating diseases related to bacterial infection.

8. The use according to claim 7, characterized in that: When used, the administration method is intravenous administration, tissue lesion intervention administration, oral administration, nasal inhalation administration or intraperitoneal injection.

9. The use according to claim 7, characterized in that: The bacterial infection-related disease is chronic osteomyelitis or chronic lung infection caused by Staphylococcus aureus infection.

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